Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

MicroRNAs as Potential Biomarkers for Chemoresistance in Adenocarcinomas of the Esophagogastric Junction

MicroRNAs as Potential Biomarkers for Chemoresistance in Adenocarcinomas of the Esophagogastric... Hindawi Journal of Oncology Volume 2019, Article ID 4903152, 11 pages https://doi.org/10.1155/2019/4903152 Research Article MicroRNAs as Potential Biomarkers for Chemoresistance in Adenocarcinomas of the Esophagogastric Junction 1 2 1 3 Christina Just, Juliana Knief , Pamela Lazar-Karsten, Ekaterina Petrova, 3 4 3 2 Richard Hummel, Christoph Ro¨cken , Ulrich Wellner, and Christoph Thorns Department of Pathology, Section of Hematopathology and Endocrine Pathology, University Hospital of Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany Department of Pathology, Marienkrankenhaus Hamburg, Alfredstrasse 9, 22087 Hamburg, Germany Department of Surgery, University Hospital of Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany Department of Pathology, University Hospital of Schleswig-Holstein, Campus Kiel, Arnold-Heller-Strasse 3, 24105 Kiel, Germany Correspondence should be addressed to Juliana Knief; knief.patho@marienkrankenhaus.org Received 14 February 2019; Accepted 15 July 2019; Published 29 July 2019 Guest Editor: Kenneth J. Vega Copyright © 2019 Christina Just et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Concerning adenocarcinomas of the esophagogastric junction, neoadjuvant chemotherapy is regularly implemented, but patients’ response varies greatly, with some cases showing no therapeutic effect, being deemed as chemoresistant. Small, noncoding RNAs (miRNAs) have evolved as key players in biological processes, including malignant diseases, often promoting tumor growth and expansion. In addition, specific miRNAs have been implicated in the development of chemoresistance through evasion of apoptosis, cell cycle alterations, and drug target modification. We performed a retrospective study of 33 patients receiving neoadjuvant chemotherapy by measuring their miRNA expression profiles. Histologic tumor regression was evaluated using resection specimens, while miRNA profiles were prepared using preoperative biopsies without prior therapy. A preselected panel of 96 miRNAs, known to be of importance in various malignancies, was used to test for significant differences between responsive (chemosensitive) and nonresponsive (chemoresistant) cases. *e cohort consisted of 12 nonresponsive and 21 responsive cases with the following 4 miRNAs differentially expressed between both the groups: hsa-let-7f-5p, hsa-miRNA- 221-3p, hsa-miRNA-31-5p, and hsa-miRNA-191-5p. *e former 3 showed upregulation in chemoresistant cases, while the latter showed upregulation in chemosensitive cases. In addition, significant correlation between high expression of hsa- miRNA-194-5p and prolonged survival could be demonstrated (p value <0.0001). In conclusion, we identified a panel of 3 miRNAs predicting chemoresistance and a single miRNA contributing to chemosensitivity. *ese miRNAs might function as prognostic biomarkers and enable clinicians to better predict the effect of one or more reliably select patients benefitting from (neoadjuvant) chemotherapy. increasingly been used not only as diagnostic but also as 1. Introduction prognostic biomarkers, and several studies have suggested Since the discovery of microRNAs (miRNAs), which are the existence of tissue-specific miRNA signatures which small, noncoding RNAs with a length of 19–22 nucleotides might be used to classify different cancer types [3–5]. [1], multiple studies have focused on the importance of their Regarding human cancer in general, miRNAs have been function and participation in human diseases ranging from found to act not only as oncogenes, promoting tumor inflammatory disorders and autoimmune diseases to ma- growth and dissemination, but also as tumor suppressors, lignant tumors including melanoma, various epithelial inhibiting tumor cell proliferation and migration and in- cancers, and hematological malignancies [2]. miRNAs have ducing apoptosis [6]. Sometimes, their function varies in a 2 Journal of Oncology single organ, showing divergent behavior in specific histo- study. All patients received neoadjuvant chemotherapy. logic tumor types (for example, adenocarcinoma vs. squa- Only tumor-containing samples of preoperative biopsies mous cell carcinoma of the esophagus) [7]. (without prior therapy) were used for miRNA analysis, while Concerning carcinomas of the upper gastrointestinal whole-resection specimens (postchemotherapeutic) were tract, especially gastric cancer and adenocarcinoma of the taken to determine the degree of histological regression and esophagogastric junction, a multitude of miRNAs have been thereby treatment response. All cases were collected as part identified as useful biomarkers: for example, upregulation of of routine clinical care at the University Hospital of miRNA-17-5p, miRNA-20a, miRNA-106b, miRNA-150, Schleswig-Holstein, Campus Luebeck, during 1997–2013. and miRNA-93 has been reported to inhibit apoptosis and to All analyses performed were in accordance with the Dec- promote cell cycle progression, whereas downregulation of laration of Helsinki and had been approved by the local miRNA-29 and miRNA-375 has been shown to increase cell Ethics Committee beforehand (reference number 14-242A). growth and migration [4, 8]. Other commonly dysregulated miRNAs include miRNA-21 and miRNA-19a/b which both 2.2. Histologic Examination. Samples were carefully exam- promote lymph node and distant metastases as well as in- ined by two researchers (CJ and JK) with a light microscope vasion of blood vessels when overexpressed [4]. In addition, (Axioskop, Zeiss, Jena, Germany), and histologic tumor a seven-miRNA panel consisting of miRNA-10b, miRNA- types were determined using the current WHO standard 21, miRNA-223, miRNA-338, let-7a, miRNA-30a-5p, and [28]. Regression after chemotherapy was determined using miRNA-126 has been found to reliably predict survival in haematoxylin- and eosin-(H&E-) stained slides and rated gastric cancer patients and is related not only to overall but according to the system devised by Becker et al. [29]. Re- also relapse free survival [9]. Besides, various miRNAs have gression grades 1a and 1b were considered as having been found to be associated with poor survival in both responded to therapy (responder group), while regression gastric carcinomas and adenocarcinomas of the esoph- grade 3 was considered nonresponsive (nonresponder agogastric junction, including miRNA-16, miRNA-21, group). Cases with regression grade 2 were not included in miRNA-29, miRNA-125b, miRNA-130a, miRNA-141, the study as an assignment to either group could not reliably miRNA-203a, miRNA-222, miRNA-302c, and miRNA-451 be undertaken (partial response). [8, 10–15]. Complementing their diagnostic and prognostic sig- nificance, miRNAs have also been found to contribute to 2.3. RNA Isolation and miRNA Profiling. RNA for profiling chemoresistance and/or chemosensitivity via regulation of of miRNA was isolated from FFPE tissue using the apoptosis, DNA damage, and repair mechanisms, and ep- RecoverAll total nucleic acid isolation kit (Applied Bio- ithelial-mesenchymal transition and modulation of drug systems, Carlsbad, California, USA). RNA concentrations targets, drug-metabolizing enzymes, and drug efflux were quantified using the NanoDrop Spectrophotometer transporters [16, 17]. Especially in gastric cancer, the fol- (Nanodrop Technologies, Montchanin, New Castle, Dela- lowing miRNAs (amongst others) have been found to ware, USA). Afterwards, reverse transcription (RT) using substantially contribute to chemoresistance: let-7b, miRNA- amounts of 20 ng of total RNA by applying the miRCURY 106a, miRNA-142, miRNA-143, miRNA-21, miRNA-338, LNA Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, miRNA-340, miRNA-497, miRNA-503, and miRNA- Denmark), containing synthetic RNA Spike Ins, was per- 582—their target genes including PTEN (phosphatase and formed. 5 μl of the RT products was combined with the tension homolog), BCL (B-cell lymphoma 2), and IGF1R PCR master mix and nuclease-free water from the miR- (insulin-like growth factor 1 receptor) [18–25]. For esoph- CURY LNA ExiLENT SYBR Green master mix (Exiqon, ageal (adeno-) carcinoma, miRNA-141, miRNA-148a, Vedbaek, Denmark). After that, 10 μl of the PCR Master miRNA-200c, miRNA-221, miRNA-27a, and miRNA-296 mix-cDNA mix was added to each 384-well plate of the are said to contribute to chemoresistance [26]. *is miR- miRCURY LNA Universal RT miR Ready-to-Use PCR, mediated chemoresistance is mainly directed against com- Cancer focus panel, V4 (Exiqon, Vedbaek, Denmark). Fi- monly used therapeutic agents such as cisplatin, 5-fluoro- nally, qPCR was performed by using the LightCycler 480 uracil, and vincristine [27]. instrument (Roche molecular systems Inc., Mannheim, Following these observations and taking into account Germany). All reactions were carried out according to the that data concerning chemoresistance in carcinomas of the manufacturer’s instructions. Initial data analysis was exe- esophagogastric junction are still scarce and many studies cuted by using the LightCycler 480 Software (Roche mo- have focused on cell lines rather than tissue specimen, our lecular systems Inc., Mannheim, Germany) to obtain raw Ct study aimed to further contribute to the knowledge for this values. Ct values were used to determine the amount of entity by comparing miRNA profiles in chemoresistant and miRNA in a sample (both parameters showing an inverse chemosensitive cancer tissue. correlation). 2. Materials and Methods 2.4. Preprocessing of Data. In order to compensate for 2.1. Selection of Cases. Samples from formalin-fixed, par- variations in quality of extracted RNA, extraction yield, and affin-embedded (FFPE) tissue containing adenocarcinomas efficiency of reverse transcription, normalization of data was of the esophagogastric junction were included in the present carried out using GenEx Software Version 6.1 (Trial Version; Journal of Oncology 3 MultiD Analyses AB, Munich). *e first normalization cases as mucinous adenocarcinoma, and 4 cases as un- method used was the Normfinder algorithm which has been differentiated/unclassifiable according to the current WHO described in detail earlier [30]. In our analysis, a panel of 38 standard [36]. After thorough histologic examination of miRs from the data set and a single miR (hsa-miRNA-103a- whole-resection specimens, the cohort consisted of 12 3p) were selected for preprocessing as hsa-miRNA-103a-3p nonresponders (regression grade 3) and 21 responders was stably expressed throughout the cohort and has been (regression grades 1a and 1b). Tumor regression was de- reported as being highly reliable for normalization of data termined as mentioned above; representative examples of [31]. Secondly, external controls (so-called Spike regression grading are shown in Figure 1. Further charac- Ins—preformulated, commercially available RNAs with teristic features of the study cohort are summarized in defined lengths and binding capacities), which were in- Table 1. cluded in the beginning as potential references, were used to normalize the data [32]. *e last normalization method used 3.2. Correlation with Clinical Characteristics. Correlation was the global mean algorithm which—in three step- between both groups showed that undifferentiated carci- s—reduces nonspecific background noise, calculates the nomas and poorly differentiated carcinomas (differentiation arithmetic mean value for all samples, and then subtracts grade 3) were more common in the nonresponder group, this value from each individual value [33]. *e global mean while responders showed a higher proportion of tubular method is usually applied in cases where large numbers of adenocarcinomas (p values 0.034 and 0.043, respectively). miRNAs are tested and has been reported to be superior to No differences could be detected concerning gender, depth other normalization methods in this particular setting [34]. of infiltration, lymphovascular/perineural invasion, nodal status, or presence of distant metastases (p values between 2.5. Analysis of Subgroups. To test for significant differences 0.087 and 0.443, as shown in Table 1). in miRNA expression profiles between responder and nonresponder groups, the Mann–Whitney-U test for un- 3.3. ;erapy Regimen. Neoadjuvant chemotherapy was ad- paired samples was applied using SPSS Statistics Version 22 ministered in all cases, and 3 patients received radiation (IBM, Ehringen, Germany). Because exploratory data therapy in addition (both belonging to the responder group analysis was performed, adjusting for multiple testing was with irradiation doses of 45 Gy, 59 Gy, and 66 Gy, not required. Afterwards, overlaps of differentially expressed respectively). miRNAs between the applied normalization procedures Concerning the nonresponder group, neoadjuvant described above were compared. chemotherapy consisted in most cases of 5-fluorouracil combined with leucovorin, oxaliplatin, and docetaxel (so- 2.6. Correlation with Clinical Characteristics. To estimate called FLOT regimen; 8/12 patients). *e remaining differences in clinical features (age, gender, differentiation patients received either 5-fluorouracil in combination with cisplatin (2 cases) or epirubicin combined with oxaliplatin (2 grade, nodal status, depth of infiltration, perineural invasion, lymphovascular invasion, and presence of distant metasta- cases). ses) between both groups and between clinical features and In the responder group, a combination of cisplatin and miRNA expression levels, the χ test was applied and a p 5-fluorouracil was administered in most cases (12/21 pa- tients). Only 4 patients received treatment using the FLOT value <0.05 was considered statistically significant. regimen, and 2 were treated with 5-fluorouracil in combi- nation with leucovorin and etoposide. *e remaining three 2.7. Correlation with Overall Survival. To assess the prog- patients received chemotherapy without 5-fluorouracil nostic value of miRNA expression, the median for each containing oxaliplatin, etoposide, and irinotecan. analyzed miRNA was calculated. Cases were then di- chotomized, either showing an expression level above or below the median as described previously [35]. Overall 3.4. miRNA Analysis. Overall, the following 4 miRNAs were survival curves were visualized via Kaplan–Meier estimates differentially expressed between responder and non- using SPSS Statistics Version 22 (IBM, Ehringen, Germany). responder groups: hsa-let-7f-5p, hsa-miRNA-191-5p, hsa- In addition, Cox regression analysis was used to test for miRNA-221-3p, and hsa-miRNA-31-5p. Concerning the different normalisation methods, only independence, taking into consideration gender, depth of infiltration, differentiation grade, nodal status, and presence minor variations were detected: applying the Normfinder algorithm, hsa-let-7f-5p, hsa-miRNA-191-5p, and hsa- or absence of distant metastases. Data were adjusted for multiple testing using the Bon- miRNA-31-5p were differentially expressed (p values 0.03, 0.005, and 0.047). For normalisation with Spike Ins, dif- ferroni procedure; after that, a p value <0.00052 was con- sidered statistically significant for this test. ferences in expression of hsa-miRNA-221-3p and hsa- miRNA-31-5p could be observed (p values 0.022 and 0.02). For normalisation with global mean, hsa-let-7f-5p, hsa- 3. Results miRNA-191-5p, hsa-miRNA-221-3p, and hsa-miRNA-31- 5p were differentially expressed (p values 0.025, 0.014, 0.04, 3.1. Histology. Overall, 24 cases were classified as tubular adenocarcinoma, 3 cases as poorly cohesive carcinoma, 2 and 0.033, respectively). Finally, for normalisation using 4 Journal of Oncology (a) (b) (c) Figure 1: Pictures of histologic tumor regression: (a) regression grade 1a (no vital tumor); (b) regression grade 1b (<10% vital tumor cells); (c) regression grade 3 (>50% vital tumor cells). H&E staining, magnification 200x. hsa-miRNA-103a-3p, hsa-let-7f-5p and hsa-miRNA-31-5p testing, significant differences in survival according to high showed differences in expression (p values 0.038 and 0.036). or low expression of miRNAs were detected only for hsa- *roughout all normalization methods, there was higher miRNA-194-5p with a p value <0.0001. Survival times in expression of hsa-let-7f-5p, hsa-miRNA-221-3p, and hsa- patients with higher expression were nearly three times miRNA-31-5p in the nonresponder group, while hsa- longer than in those with low expression (97.67 months vs. miRNA-191-5p showed higher expression in the responder 32.69 months). Cox regression analysis, however, could not group. *e respective miRNA expression patterns (Ct show independence for prediction of patients’ survival after values) are depicted in Figure 2. taking into consideration gender, depth of infiltration, differentiation grade, nodal status, and presence or absence of distant metastases (p � 0.462; hazard ratio 1.469; 95% 3.5. miRNAs and Tumor Differentiation Grade. Correlation confidence interval 0.527–4.096). In addition, there was no between miRNA expression profiles and tumor differenti- correlation between expression levels of hsa-miRNA-194-5p ation grade found that poorer differentiation (G3) was and drug response (p � 0.456). significantly associated with decreased levels of hsa-miRNA- Appropriate survival curves and mean survival times 200a-3p and elevated levels of hsa-miRNA-21-5p, hsa- including 95% confidence interval are shown in Figure 3 and miRNA-222-3p, hsa-miRNA-25-3p, and hsa-let-7d-5p (p Table 2. values 0.03–0.031). 4. Discussion 3.6. miRNAs and Patients’ Prognosis. Complete survival data *e mechanisms underlying chemotherapy and multidrug were available for 29 patients with a mean follow-up period resistance in human cancer are polymorphic [37]. In this of 44.52 months (range 1–100 months). During follow-up, context, miRNAs have been found to regulate apoptosis, 12 patients (41.38%) died. After adjusting for multiple DNA repair, and epithelial-mesenchymal transition and Journal of Oncology 5 Table 1: Characteristics of adenocarcinoma of the esophagogastric cell cycle regulation has been reported as a key mechanism junction according to the responder or nonresponder status. in tumor progression; in addition, in cervical cancer, increased expression levels are associated with epithelial- Characteristics Responders Nonresponders p value mesenchymal transition, migration, and invasion by Total n 21 12 targeting TWIST2 [41, 42]. For human glioblastomas, Gender cervical and colon carcinoma cells downregulation of Male 18 8 0.198 PTEN and activation of Akt and STAT3—mediated by Female 3 4 increased levels of hsa-miRNA-221-3p—have been shown WHO classification Tubular 17 7 as key players in tumor cell survival and radio- and Poorly cohesive 2 1 chemoresistance [43–46]. In addition, in hepatocellular Mucinous 2 0 0.034 carcinoma, upregulation of hsa-miRNA-221-3p decreases Undifferentiated 0 4 the expression of HDAC6, a tumor suppressor, and Others promotes tumorigenesis [47]. One study focusing solely Differentiation grade on esophageal adenocarcinomas showed that the che- Well (G1) 0 0 moresistance was conveyed through alteration of the Moderately (G2) 19 7 0.043 Wnt/β-catenin pathway and DKK2, CDH1, CD44, MYC, Poor (G3) 2 5 and ABCG2 expression [26]. Concerning hsa-miRNA-31- pT (low) 5p, only few studies have addressed how increased ex- pT0 2 0 pT1a 2 0 pression contributes to chemoresistance: in malignant 0.136 pT1b 5 0 pleural mesothelioma and hepatocellular carcinoma, it pT2 3 1 promotes chemoresistance by targeting OCT1 and ABCB9 pT (high) [48, 49]. In ovarian cancer, chemoresistance is increased pT3 8 10 by modulation of specific calcium-regulated potassium pT4a 1 1 channels [50]. Conversely, an opposite effect has also been pT4b 0 0 reported: overexpression of hsa-miRNA-31-5p decreases pN levels of stathmin 1, a microtubule-depolymerizing pN0 12 6 molecule that leads to reduced chemosensitivity in pN1 3 4 0.203 ovarian cancer [51]. In addition, in osteosarcoma, upre- pN2 6 1 gulation of hsa-miRNA-31-5p inhibits tumor cell mi- pN3 0 1 LVSI gration and invasion by targeting PI3K3C2A [52]. Present 6 5 Regarding hsa-miRNA-191-5p, the few studies conducted 0.443 Absent 15 7 so far show that overexpression promotes chemo- Perineural invasion resistance by modulating p53 and TET1 in chol- Present 1 3 angiocarcinomas [53]. Furthermore, an association with 0.087 Absent 20 9 various estrogen-dependent genes such as ANXA1, Distant metastases PIWIL2, CASP4, ESR1/ESR2, PLAC1, and SOCS2, has Present 4 1 0.409 been shown in breast cancer—however, up to date, no Absent 17 11 such data concerning adenocarcinomas of the esoph- LVSI: lymphovascular space invasion; bold lettering in p values indicates a agogastric junction have been published [54]. statistically significant difference. *e miRNA signature we discovered seems to differ from previously published data; other studies found that to modulate drug targets, drug-metabolizing enzymes, or especially in esophageal carcinoma—in addition to miRNA- drug efflux transporters [16, 17]. In our study, analyzing 221—miRNA-141, miRNA-200c, miRNA-148a, miRNA- adenocarcinomas of the esophagogastric junction, we 296, miRNA-23, miRNA-223, and miRNA-27a are sub- stantially contributing to chemoresistance [26, 55]. Never- identified a panel of four miRNAs which were differen- tially expressed between patients responding or not theless, as some studies focus on plasma-circulating miRNAs or cell lines and not exclusively on expression in tumor responding to neoadjuvant chemotherapy. We found higher expression of hsa-let-7f-5p, hsa-miRNA-221-3p, tissue, results are comparable only to a limited degree [13]. Ours is—to our knowledge—the first study focusing on and hsa-miRNA-31-5p in the nonresponder group, while hsa-miRNA-191-5p showed higher expression in the re- miRNA expression profiles in adenocarcinomas of the esophagogastric junction and their meaning for therapeutic sponder group. *e molecular mechanisms contributing to chemoresistance or chemosensitivity concerning these response based on tissue specimens. four miRNAs are—up to date—not fully understood. Hsa-let-7f-5p is located on the long arm of chromosome 9 Nevertheless, the function and effects of these miRNAs as (9q22.3) and shows involvement in immune cell differenti- stated in the literature might give some clues as to what ation, angiogenesis, and cellular growth arrest [56–58]. It is commonly affected in multiple human cancers, including the underlying mechanisms might be: for hsa-let-7f-5p, a proangiogenic effect has been reported; thus, over- melanoma, lung, and head/neck cancer, with downregulation in the majority of cases [59–61]. Nevertheless, upregulation is expression might contribute to tumor progression via tumor neoangiogenesis [38–40]. For hsa-miRNA-221-3p, also encountered, for instance, in papillary, follicular, and 6 Journal of Oncology 4 2 –1 –1 Non responder Responder Non responder Responder (a) (b) –1 –2 –2 Non responder Responder Non responder Responder (c) (d) Figure 2: Boxplots and Ct values according to differentially expressed miRNAs in responder and nonresponder groups: (a) Ct values for hsa-let-7f-5p; (b) Ct values for hsa-miRNA-191-5p; (c) Ct values for hsa-miRNA-221-3p; (d) Ct values for hsa-miRNA-31-5p. Lower Ct values indicate higher miR expression, while higher Ct values indicate lower miR levels. chemo- and/or radio sensitivity and induces tumor cell anaplastic thyroid cancer as well as breast cancer and ovarian cancer [62–64]. It has been associated not only with che- apoptosis [26, 68, 69]. In addition, in tissue experiments, hsa-miRNA-221-3p has been shown to promote resistance mosensitivity and treatment response in gastric cancer [65] but also with chemotherapy resistance in breast cancer [37]. to a variety of regularly used therapeutic agents including 5- In our study, increased expression of hsa-let-7f-5p showed a fluorouracil, tyrosine kinase inhibitors, and antiandrogens distinctive association with chemoresistance which seems to [37, 70–73]. be in contrast to previously published data concerning car- *ese findings are difficult to compare with our study cinomas of the upper gastrointestinal tract [65]. Still, as data population as both responders and nonresponders had been concerning the association of miR expression levels and treated using a combination chemotherapy containing 5- chemotherapy response are still often controversial and fluorouracil in most cases (28/33 cases). sometimes different functions (either as a tumor suppressor Downregulation of hsa-miRNA-31-5p has been de- scribed in a variety of human cancers, for instance, triple- or as an oncogene) have been reported for different histologic tumor types (adenocarcinoma vs. squamous cell carcinoma) negative breast carcinoma [67] and indicates shortened in a single organ, our data might only mirror a specific effect overall survival in gastric cancer [74] as well as the presence for a defined subset of patients [7]. of locally advanced tumor stages, implicating the function as Hsa-miRNA-221-3p is well characterized, and its a miRNA with tumor suppressor properties. function and involvement in human cancer has been ex- Data concerning its association with therapy response tensively described. It is commonly known as an onco- are more controversial: while some studies report that miRNA, promoting tumor proliferation, invasion, dissem- overexpression promotes chemoresistance in gastric and ination, and metastasis [66, 67]. Multiple analyses have ovarian cancer [50, 75] and breast carcinomas [76], others focused on cell lines where overexpression is commonly could demonstrate that upregulation in gallbladder car- associated with chemoresistance and knockdown restores cinomas leads to increased chemosensitivity [77]. In hsa-miR-221-3p hsa-let-7f-5p hsa-miR-31-5p hsa-miR-191-5p Journal of Oncology 7 Concerning its association with chemotherapy outcome, 1.0 single studies have found hsa-miRNA-191-5p to promote chemoresistance [54], while others could show no influence on chemosensitivity or chemoresistance at all [82]. Overall, 0.8 literature addressing this particular issue is still very sparse. In contrast to the data described above, in our study, high levels of hsa-miRNA-191-5p showed a clear-cut association 0.6 with chemosensitive cases responding to neoadjuvant chemotherapy. It remains to be seen whether these results can be 0.4 reproduced in larger studies or if a similar effect can be shown in other cancer entities. It may be conceivable that our findings reflect—as it is possibly the case with both hsa- 0.2 let-7f-5p and hsa-miRNA-31-5p—an effect which is only discernible in a defined subset of tumors or specific tumor p < 0.0001 entities. 0.0 Due to the small case number in our study and the often limited availability of both pretherapeutic biopsies and re- 0 20.0 40.0 60.0 80.0 100.0 Follow-up (months) section specimen in a single institution, we additionally con- sulted a web database (GEO database) to supplement our data. Expression levels of hsa-miR-194 Here, we could find only one additional study analyzing High expression High expression- chemotherapy response in a very small cohort (8 cases) of censored upper gastrointestinal carcinomas, namely, stomach cancer, Low expression Low expression- proposing a miRNA signature used for predicting chemo- censored therapy outcome [65]. *is signature, however, differed from Figure 3: Kaplan–Meier curve showing survival differences our findings, showing an association of chemoresistance and according to high or low expression of hsa-miRNA-194-5p with a p high expression of let-7g, miR-342, miR-16, miR-181, miR-1, value <0.0001. Survival times are given in months. and miR-34. Whether this might be due to small case numbers in both studies or whether this reflects differences between two Table 2: Average survival times according to high or low ex- cancer entities with a fundamentally different pathophysiology pression of hsa-miRNA-194-5p. remains to be seen. Another study analyzed esophageal ade- nocarcinomas (14 cases) and reported that miRNA-221 con- Average survival (months) SD 95% CI p value tributes to chemoresistance via the Wnt/β-catenin pathway, hsa-miRNA-194-5p supporting our findings [26]. High 97.67 1.91 93.93–101.4 <0.0001 In addition, we correlated the miRNA expression with Low 32.69 7.89 17.22–48.16 patients’ overall survival and could demonstrate that overall survival was significantly correlated with expression levels of hsa-miRNA-194-5p (p value <0.0001) although in- addition, hsa-miRNA-31-5p expression levels seem to influence the therapy response not only in general but also dependence could not be demonstrated applying Cox re- gression analysis (p � 0.462; hazard ratio 1.469; 95% according to different chemotherapeutic agents: in cell line experiments, it has been demonstrated that down- confidence interval 0.527–4.096). In accordance with our regulation can promote resistance to platinum-based results, where higher levels of hsa-miRNA-194-5p corre- therapies and paclitaxel [78], while upregulation corre- sponded to prolonged survival (97.67 months vs. lates with resistance against 5-fluorouracil [79]. Our study 32.69 months), overexpression has been reported to inhibit cell proliferation and to act as a tumor suppressor in la- adds to the current understanding as we could demon- strate increased chemotherapy resistance in cases with ryngeal SCC, prostate cancer, melanoma, bladder cancer, NSCLC, and clear cell renal carcinoma [83–87]. In addition, higher hsa-miRNA-31-5p expression levels. It seems as if the therapeutic response might not only depend on the overexpression has been found to inhibit growth and pro- liferation in gastric cancer cell lines and esophageal squa- tumor subtype analyzed but also on the therapy applied and that hsa-miRNA-31-5p might exhibit both oncogenic mous cell carcinoma [88, 89]. Other miRNAs which are and tumor suppressive functions according to a specific commonly linked to prolonged or shortened overall survival context. in gastric or esophageal carcinomas—for instance, miRNA- *roughout the literature, hsa-miRNA-191-5p is de- 16, miRNA-21, miRNA-29, miRNA-125b, miRNA-130a, scribed as having oncogenic properties, leading to increased miRNA-141, miRNA-203a, miRNA-222, miRNA-302c, and tumor cell proliferation, invasion, and inhibition of apo- miRNA-451—could not be reproduced in our study [4, 8, 9, 11, 14]. *is might well be due to a smaller number of ptosis [80]. *is holds true for various epithelial cancer types such as breast, pancreatic, and hepatocellular carcinomas patients included in our study which limits the statistical reliability as well as the fact that, after adjusting for multiple [81] or cholangiocarcinoma. Here, overexpression is addi- tionally associated with decreased overall survival [53]. testing, only a p value<0.00052 was considered statistically Cumulative survival 8 Journal of Oncology [3] G. Juodzbalys, D. Kasradze, M. Cicciu` et al., “Modern mo- significant which excluded a few otherwise statistically lecular biomarkers of head and neck cancer. Part I. Epigenetic significant miRNAs. diagnostics and prognostics: systematic review,” Cancer Biomarkers: Section A of Disease Markers, vol. 17, no. 4, 5. Conclusion pp. 487–502, 2016. [4] H.-S. Liu, “MicroRNAs as potential biomarkers for gastric Our results imply that miRNAs might play an important role cancer,” World Journal of Gastroenterology, vol. 20, no. 34, in the evolution of chemoresistance and/or chemosensitivity pp. 12007–12017, 2014. in adenocarcinomas of the esophagogastric junction. Nev- [5] M. Zhou, H. Hara, Y. Dai et al., “Circulating organ-specific ertheless, as—due to the restricted availability of both pre- MicroRNAs serve as biomarkers in organ-specific diseases: and posttherapeutic tissue samples in a single institu- implications for organ allo-and xeno-transplantation,” tion—the number of patients in our study was limited, International Journal of Molecular Sciences, vol. 17, no. 8, statistical results should be interpreted with caution. In p. 1232, 2016. addition, only tumor tissue was compared without estab- [6] A. M. Strubberg and B. B. Madison, “MicroRNAs in the lishing baseline levels of miRNA expression in nonneo- etiology of colorectal cancer: pathways and clinical implica- plastic mucosa. tions,” Disease Models & Mechanisms, vol. 10, no. 3, Regardless of the abovementioned limitations, our re- pp. 197–214, 2017. sults contribute to other studies postulating that miRNAs [7] R. Hezova, A. Kovarikova, J. Srovnal et al., “MiR-205 func- might be a pretherapeutic means to predict therapy response tions as a tumor suppressor in adenocarcinoma and an on- cogene in squamous cell carcinoma of esophagus,” Tumor in the future and better stratify patients who benefit from Biology, vol. 37, no. 6, pp. 8007–8018, 2016. neoadjuvant therapy or who might not benefit at all and [8] D. Wang, Z. Fan, F. Liu, and J. Zuo, “Hsa-mir-21 and Hsa-mir- therefore could be spared of adverse effects of noneffective 29 in tissue as potential diagnostic and prognostic biomarkers treatment strategies. for gastric cancer,” Cellular Physiology and Biochemistry, It remains to be seen if the miRNA signature we vol. 37, no. 4, pp. 1454–1462, 2015. established for adenocarcinomas of the esophagogastric [9] X. Li, Y. Zhang, Y. Zhang, J. Ding, K. Wu, and D. Fan, junction can be reproduced in future studies or for different “Survival prediction of gastric cancer by a seven-microRNA tumor entities. signature,” Gut, vol. 59, no. 5, pp. 579–585, 2010. [10] H. Jiang, W.-W. Yu, L.-L. Wang, and Y. Peng, “miR-130a acts Abbreviations as a potential diagnostic biomarker and promotes gastric cancer migration, invasion and proliferation by targeting Ct: Cycle threshold RUNX3,” Oncology Reports, vol. 34, no. 3, pp. 1153–1161, hsa: Homo sapiens miRNA: MicroRNA. [11] W. Liu, Z. Dong, J. Liang et al., “Downregulation of potential tumor suppressor miR-203a by promoter methylation con- Data Availability tributes to the invasiveness of gastric cardia adenocarcinoma,” Cancer Investigation, vol. 34, no. 10, pp. 506–516, 2016. *e data used to support the findings of this study are [12] Y. B. Lu, J. J. Hu, W. J. Sun, X. H. Duan, and X. Chen, available from the corresponding author upon request. “Prognostic value of miR-141 downregulation in gastric cancer,” Genetics and Molecular Research, vol. 14, no. 4, pp. 17305–17311, 2015. Disclosure [13] M. Odenthal, J. Hee, and I. Gockel, “Serum microRNA Part of this study has been presented as a scientific poster at profiles as prognostic/predictive markers in the multimodality the ASCP Annual Meeting 2017 (Chicago, Illinois). therapy of locally advanced adenocarcinomas of the gastro- esophageal junction,” International Journal of Cancer, vol. 137, no. 1, pp. 230–237, 2015. Conflicts of Interest [14] C. Ren, H. Chen, C. Han, D. Fu, D. Wang, and M. Shen, “High expression of miR-16 and miR-451 predicating better prog- *e authors declare that they have no conflicts of interest. nosis in patients with gastric cancer,” Journal of Cancer Re- search and Clinical Oncology, vol. 142, no. 12, pp. 2489–2496, Authors’ Contributions [15] J. G. Wu, J.-J. Wang, X. Jiang et al., “MiR-125b promotes cell Christina Just and Juliana Knief contributed equally to this migration and invasion by targeting PPP1CA-RB signal work. pathways in gastric cancer, resulting in a poor prognosis,” Gastric Cancer: Official Journal of the International Gastric References Cancer Association and the Japanese Gastric Cancer Associ- ation, vol. 18, no. 4, pp. 729–739, 2015. [1] R. C. Lee, R. L. Feinbaum, and V. Ambros, “*e C. elegans [16] G. Xiong, M. Feng, G. Yang et al., “*e underlying mecha- heterochronic gene lin-4 encodes small RNAs with antisense nisms of non-coding RNAs in the chemoresistance of pan- complementarity to lin-14,” Cell, vol. 75, no. 5, pp. 843–854, creatic cancer,” Cancer Letters, vol. 397, pp. 94–102, 2017. [2] J. Song, Y. Ouyang, J. Che et al., “Potential value of miR-221/ [17] T. Zheng, J. Wang, X. Chen, and L. Liu, “Role of microrna in anticancer drug resistance. International journal of cancer,” 222 as diagnostic, prognostic, and therapeutic biomarkers for diseases,” Frontiers in Immunology, vol. 8, p. 56, 2017. International Journal of Cancer, vol. 126, no. 1, pp. 2–10, 2010. Journal of Oncology 9 [18] Y. Fang, H. Shen, H. Li et al., “miR-106a confers cisplatin mean normalization,” in Methods in Molecular Biology, resistance by regulating PTEN/Akt pathway in gastric cancer Springer, New York, NY, USA, 2012. cells,” Acta biochimica et biophysica Sinica, vol. 45, no. 11, [35] D. Calatayud, C. Dehlendorff, M. K. Boisen et al., “Tissue pp. 963–972, 2013. MicroRNA profiles as diagnostic and prognostic biomarkers [19] X. Han, J.-J. Zhang, Z.-Q. Han, H.-B. Zhang, and Z.-A. Wang, in patients with resectable pancreatic ductal adenocarcinoma “Let-7b attenuates cisplatin resistance and tumor growth in and periampullary cancers,” Biomarker Research, vol. 5, no. 1, p. 8, 2017. gastric cancer by targeting AURKB,” Cancer Gene ;erapy, vol. 25, no. 11-12, pp. 300–308, 2018. [36] F. T. Bosman, F. Carneiro, R. H. Hruban, and N. D. *eise, Who Classification of Tumours of the Digestive System, In- [20] X. Liu, H. Cai, W. Sheng, H. Huang, Z. Long, and Y. Wang, “microRNAs expression profile related with response to ternational Agency for Research on Cancer, Lyon, France, 4th preoperative radiochemotherapy in patients with locally ad- edition, 2010. vanced gastric cancer,” BMC Cancer, vol. 18, no. 1, p. 1048, [37] K. R. Kutanzi, O. V. Yurchenko, F. A. Beland, V. F. Checkhun, 2018. and I. P. Pogribny, “MicroRNA-mediated drug resistance in [21] T. Wang, G. Ge, Y. Ding et al., “MiR-503 regulates cisplatin breast cancer,” Clinical Epigenetics, vol. 2, no. 2, pp. 171–185, resistance of human gastric cancer cell lines by targeting 2011. [38] A. Eirin, S. M. Riester, X.-Y. Zhu et al., “MicroRNA and IGF1R and BCL ,” Chinese Medical Journal, vol. 127, no. 12, pp. 2357–2362, 2014. mRNA cargo of extracellular vesicles from porcine adipose tissue-derived mesenchymal stem cells,” Gene, vol. 551, no. 1, [22] S. M. Yang, C. Huang, X.-F. Li, M.-Z. Yu, Y. He, and J. Li, “miR-21 confers cisplatin resistance in gastric cancer cells by pp. 55–64, 2014. regulating PTEN,” Toxicology, vol. 306, pp. 162–168, 2013. [39] X. Qin, F. Chang, Z. Wang, and W. Jiang, “Correlation of [23] Y. Zhang, Q. Lu, and X. Cai, “MicroRNA-106a induces circulating pro-angiogenic mirnas with cardiotoxicity in- multidrug resistance in gastric cancer by targeting RUNX3,” duced by epirubicin/cyclophosphamide followed by docetaxel FEBS Letters, vol. 587, no. 18, pp. 3069–3075, 2013. in patients with breast cancer,” Cancer Biomarkers, vol. 23, [24] W. Zhu, D. Zhu, S. Lu et al., “miR-497 modulates multidrug no. 4, pp. 473–484, 2018. [40] C. Urbich, A. Kuehbacher, and S. Dimmeler, “Role of resistance of human cancer cell lines by targeting BCL ,” Medical Oncology, vol. 29, no. 1, pp. 384–391, 2012. microRNAs in vascular diseases, inflammation, and angio- genesis,” Cardiovasc Research, vol. 79, no. 4, pp. 581–588, [25] M. Zhuang, Q. Shi, X. Zhang et al., “Involvement of miR-143 in cisplatin resistance of gastric cancer cells via targeting 2008. IGF R and BCL ,” Tumour Biology, vol. 36, no. 4, [41] X. Y. He, Z.-L. Tan, Q. Mou et al., “microRNA-221 enhances 1 2 pp. 2737–2745, 2015. MYCN via targeting nemo-like kinase and functions as an [26] Y. Wang, Y. Zhao, A. Herbst et al., “miR-221 mediates oncogene related to poor prognosis in neuroblastoma,” chemoresistance of esophageal adenocarcinoma by direct Clinical Cancer Research, vol. 23, no. 11, pp. 2905–2918, targeting of DKK2 expression,” Annals of Surgery, vol. 264, 2017. [42] W. F. Wei, C.-F. Zhou, X.-G. Wu et al., “MicroRNA-221-3p, a no. 5, pp. 804–814, 2016b. [27] I. Riquelme, P. Letelier, A. Riffo-Campos, P. Brebi, and J. Roa, TWIST2 target, promotes cervical cancer metastasis by di- rectly targeting THBS2,” Cell Death & Disease, vol. 8, no. 12, “Emerging role of miRNAs in the drug resistance of gastric cancer,” International Journal of Molecular Sciences, vol. 17, p. 3220, 2017. no. 3, p. 424, 2016. [43] J. Du, W. LiNa, C. Li et al., “MicroRNA-221 targets PTEN to [28] J.-F. Flejou, “who classification of digestive tumors: the fourth reduce the sensitivity of cervical cancer cells to gefitinib edition,” Annales de Pathologie, vol. 31, no. 5, pp. S27–S31, through the PI K/Akt signaling pathway,” Tumour Biology, 2011. vol. 37, no. 3, pp. 3939–3947, 2016. [29] K. Becker, J. D. Mueller, C. Schulmacher et al., “Histo- [44] W. Li, F. Guo, P. Wang, S. Hong, and C. Zhang, “miR-221/222 morphology and grading of regression in gastric carcinoma confers radioresistance in glioblastoma cells through acti- treated with neoadjuvant chemotherapy,” Cancer, vol. 98, vating Akt independent of PTEN status,” Current Molecular no. 7, pp. 1521–1530, 2003. Medicine, vol. 14, no. 1, pp. 185–195, 2014. [30] C. L. Andersen, J. L. Jensen, and T. F. Ørntoft, “Normalization [45] Y. Ren, M. Yang, R. Ma et al., “Microcystin-LR promotes of real-time quantitative reverse transcription-PCR data: a migration via the cooperation between microRNA-221/PTEN model-based variance estimation approach to identify genes and STAT3 signal pathway in colon cancer cell line DLD-1,” suited for normalization, applied to bladder and colon cancer Ecotoxicology and Environmental Safety, vol. 167, pp. 107–113, data sets,” Cancer Research, vol. 64, no. 15, pp. 5245–5250, 2019. 2004. [46] Q. Xie, Y. Yan, Z. Huang, X. Zhong, and L. Huang, [31] H. J. Peltier and G. J. Latham, “Normalization of microRNA “MicroRNA-221 targeting PI3-K/Akt signaling axis induces expression levels in quantitative RT-PCR assays: identification cell proliferation and BCNU resistance in human glioblas- of suitable reference RNA targets in normal and cancerous toma,” Neuropathology, vol. 34, no. 5, pp. 455–464, 2014. human solid tissues,” RNA, vol. 14, no. 5, pp. 844–852, 2008. [47] H. J. Bae, K. H. Jung, J. W. Eun et al., “MicroRNA-221 governs [32] P. Fardin, S. Moretti, B. Biasotti, A. Ricciardi, S. Bonassi, and tumor suppressor HDAC6 to potentiate malignant progres- L. Varesio, “Normalization of low-density microarray using sion of liver cancer,” Journal of Hepatology, vol. 63, no. 2, external spike-in controls: analysis of macrophage cell lines pp. 408–419, 2015. expression profile,” BMC Genomics, vol. 8, no. 1, p. 17, 2007. [48] H. L. Moody, M. J. Lind, and S. G. Maher, “MicroRNA-31 [33] P. Mestdagh, P. V. Vlierberghe, A. D. Weer et al., “A novel and regulates chemosensitivity in malignant pleural mesotheli- universal method for microRNA RT-qPCR data normaliza- oma,” Molecular ;erapy—Nucleic Acids, vol. 8, pp. 317–329, tion,” Genome Biology, vol. 10, no. 6, p. R64, 2009. 2017. [34] B. D’Haene, P. Mestdagh, J. Hellemans, and J. Vandesompele, [49] K. T. Que, Y. Zhou, Y. You et al., “MicroRNA-31-5p regulates “miRNA expression profiling: from reference genes to global chemosensitivity by preventing the nuclear location of PARP1 10 Journal of Oncology in hepatocellular carcinoma,” Journal of Experimental & [65] C. H. Kim, H. K. Kim, R. L. Rettig et al., “miRNA signature Clinical Cancer Research, vol. 37, no. 1, p. 268, 2018. associated with outcome of gastric cancer patients following chemotherapy,” BMC Medical Genomics, vol. 4, no. 1, p. 79, 2011. [50] P. Samuel, R. C. Pink, D. P. Caley et al., “Over-expression of [66] W. Liu, N. Song, H. Yao, L. Zhao, H. Liu, and G. Li, “miR-221 miR-31 or loss of KCNMA1 leads to increased cisplatin re- and miR-222 simultaneously target reck and regulate growth sistance in ovarian cancer cells,” Tumour Biology, vol. 37, and invasion of gastric cancer cells,” Medical Science Monitor: no. 2, pp. 2565–2573, 2016. International Medical Journal of Experimental and Clinical [51] M. K. Hassan, H. Watari, T. Mitamura et al., “P18/Stathmin1 Research, vol. 21, pp. 2718–2725, 2015. is regulated by miR-31 in ovarian cancer in response to [67] M. Ouyang, Y. Li, S. Ye et al., “MicroRNA profiling implies taxane,” Oncoscience, vol. 2, pp. 294–308, 2015. new markers of chemoresistance of triple-negative breast [52] L. M. Chao, W. Sun, H. Chen, B. Y. Liu, P. F. Li, and cancer,” PLoS One, vol. 9, no. 5, Article ID e96228, 2014. D. W Zhao, “MicroRNA-31 inhibits osteosarcoma cell pro- [68] Z. Chun-Zhi, H. Lei, Z. An-ling et al., “MicroRNA-221 and liferation, migration and invasion by targeting PIK3C2A,” microRNA-222 regulate gastric carcinoma cell proliferation European review for medical and pharmacological sciences, and radioresistance by targeting PTEN,” BMC Cancer, vol. 10, vol. 22, no. 21, pp. 7205–7213, 2018. no. 1, p. 367, 2010. [53] H. Li, Z.-Q. Zhou, Z.-R. Yang et al., “MicroRNA-191 acts as a [69] C. W. Dai, Q.-W. Bai, G.-S. Zhang et al., “MicroRNA let-7f is tumor promoter by modulating the TET1-p53 pathway in down-regulated in patients with refractory acute myeloid intrahepatic cholangiocarcinoma,” Hepatology, vol. 66, no. 1, leukemia and is involved in chemotherapy resistance of pp. 136–151, 2017a. adriamycin-resistant leukemic cells,” Leukemia & Lymphoma, [54] N. Nagpal, H. M. Ahmad, B. Molparia, and R. Kulshreshtha, vol. 55, no. 7, pp. 1645–1648, 2014. “MicroRNA-191, an estrogen-responsive microRNA, func- [70] M. Garofalo and C. M. Croce, “MicroRNAs as therapeutic tions as an oncogenic regulator in human breast cancer,” targets in chemoresistance,” Drug Resistance Updates: Reviews Carcinogenesis, vol. 34, no. 8, pp. 1889–1899, 2013. and Commentaries in Antimicrobial and Anticancer Chemo- [55] S. Komatsu, D. Ichikawa, T. Kawaguchi et al., “Plasma therapy, vol. 16, no. 3–5, pp. 47–59, 2013. microRNA profiles: identification of miR-23a as a novel [71] P. Magee, L. Shi, and M. Garofalo, “Role of microRNAs in biomarker for chemoresistance in esophageal squamous cell chemoresistance,” Annals of Translational Medicine, vol. 3, carcinoma,” Oncotarget, vol. 7, no. 38, pp. 62034–62048, 2016. no. 21, p. 332, 2015. [56] A. Kuehbacher, C. Urbich, A. M. Zeiher, and S. Dimmeler, [72] L. Zhao, D. Zou, X. Wei et al., “MiRNA-221-3p desensitizes “Role of dicer and drosha for endothelial microrna expression pancreatic cancer cells to 5-fluorouracil by targeting RB ,” and angiogenesis,” Circulation Research, vol. 101, no. 1, Tumour Biology, vol. 37, no. 12, 2016. pp. 59–68, 2007. [73] R. L. Zheng, Y.-J. Jiang, and X. Wang, “Role of micrornas on [57] O. C. Maes, H. Sarojini, and E. Wang, “Stepwise up-regulation therapy resistance in Non-Hodgkin’s lymphoma,” In- of MicroRNA expression levels from replicating to reversible ternational Journal of Clinical and Experimental Medicine, and irreversible growth arrest states in WI-38 human fibro- vol. 7, no. 11, pp. 3818–3832, 2014. [74] H. Wang, X. Zhang, Y. Liu et al., “Downregulated miR-31 level blasts,” Journal of Cellular Physiology, vol. 221, no. 1, associates with poor prognosis of gastric cancer and its res- pp. 109–119, 2009. toration suppresses tumor cell malignant phenotypes by [58] H. Wu, J. R. Neilson, P. Kumar et al., “miRNA profiling of inhibiting E2F2,” Oncotarget, vol. 7, no. 24, pp. 36577–36589, naive, effector and memory CD8 T cells,” PLoS One, vol. 2, no. 10, Article ID e1020, 2007. [75] A. Korourian, R. Roudi, A. Shariftabrizi, and Z. Madjd, [59] S. S. Chang, W. W. Jiang, I. Smith et al., “MicroRNA “MicroRNA-31 inhibits RhoA-mediated tumor invasion and alterations in head and neck squamous cell carcinoma,” chemotherapy resistance in MKN-45 gastric adenocarcinoma International Journal of Cancer, vol. 123, no. 12, cells,” Experimental Biology and Medicine, vol. 242, no. 18, pp. 2791–2797, 2008. pp. 1842–1847, 2017. [60] J. Y. Lee, H. J. Kim, N. A. Yoon et al., “Tumor suppressor p53 [76] J. Lv, K. Xia, P. Xu et al., “miRNA expression patterns in plays a key role in induction of both tristetraprolin and let-7 in chemoresistant breast cancer tissues,” Biomedicine & Phar- human cancer cells,” Nucleic Acids Research, vol. 41, no. 11, macotherapy, vol. 68, no. 8, pp. 935–942, 2014. pp. 5614–5625, 2013. [77] M. Li, W. Chen, H. Zhang et al., “MiR-31 regulates the cis- [61] S. Liang, L. He, X. Zhao et al., “MicroRNA let-7f inhibits platin resistance by targeting Src in gallbladder cancer,” tumor invasion and metastasis by targeting myh9 in human Oncotarget, vol. 7, no. 50, pp. 83060–83070, 2016. gastric cancer,” PLoS One, vol. 6, no. 4, Article ID e18409, [78] T. Mitamura, H. Watari, L. Wang et al., “Downregulation of miRNA-31 induces taxane resistance in ovarian cancer cells [62] A. I. Damanakis, S. Eckhardt, A. Wunderlich et al., through increase of receptor tyrosine kinase MET,” Onco- “MicroRNAs let7 expression in thyroid cancer: correlation genesis, vol. 2, no. 3, p. e40, 2013. with their deputed targets HMGA and SLC A ,” Journal of 2 5 5 [79] R. Hummel, C. Sie, D. I. Watson et al., “MicroRNA signatures Cancer Research and Clinical Oncology, vol. 142, no. 6, in chemotherapy resistant esophageal cancer cell lines,” World pp. 1213–1220, 2016. Journal of Gastroenterology, vol. 20, no. 40, pp. 14904–14912, [63] L. X. Yan, X.-F. Huang, Q. Shao et al., “MicroRNA miR-21 overexpression in human breast cancer is associated with [80] E. Elyakim, E. Sitbon, A. Faerman et al., “hsa-miR-191 is a advanced clinical stage, lymph node metastasis and patient candidate oncogene target for hepatocellular carcinoma poor prognosis,” RNA, vol. 14, no. 11, pp. 2348–2360, 2008. therapy,” Cancer Research, vol. 70, no. 20, pp. 8077–8087, [64] H. Zheng, L. Zhang, Y. Zhao et al., “Plasma mirnas as di- agnostic and prognostic biomarkers for ovarian cancer,” PLoS [81] J. Zhao, C.-R. Qiao, Z. Ding et al., “A novel pathway in NSCLC One, vol. 8, no. 11, Article ID e77853, 2013. cells: miR191, targeting NFIA, is induced by chronic hypoxia, Journal of Oncology 11 and promotes cell proliferation and migration,” Molecular Medicine Reports, vol. 15, no. 3, pp. 1319–1325, 2017. [82] S. K. Patnaik, E. Kannisto, and S. Yendamuri, “Over- expression of microrna miR-30a or miR-191 in A549 lung cancer or BEAS-2B normal lung cell lines does not alter phenotype,” PLoS One, vol. 5, no. 2, Article ID e9219, 2010. [83] B. Guo, Q. Hui, Y. Zhang, P. Chang, and K. Tao, “miR-194 is a negative regulator of GEF-H1 pathway in melanoma,” On- cology Reports, vol. 36, no. 4, pp. 2412–2420, 2016. [84] Q. Kong and X.-S. Chen, T. Tian, X.-Y. Xia and P. Xu, Microrna-194 suppresses prostate cancer migration and in- vasion by downregulating human nuclear distribution pro- tein,” Oncology Reports, vol. 37, no. 2, pp. 803–812, 2017. [85] R. Nofech-Mozes, H. W. Z. Khella, A. Scorilas et al., “MicroRNA-194 is a marker for good prognosis in clear cell renal cell carcinoma,” Cancer Medicine, vol. 5, no. 4, pp. 656–664, 2016. [86] X. Zhu, D. Li., F. Yu et al., “miR-194 inhibits the proliferation, invasion, migration, and enhances the chemosensitivity of non-small cell lung cancer cells by targeting forkhead box a1 protein,” Oncotarget, vol. 7, no. 11, pp. 13139–13152, 2016. [87] P. Li, H. Liu, A.-K. Yang et al., “MiR-194 functions as a tumor suppressor in laryngeal squamous cell carcinoma by targeting Wee1,” Journal of Hematology & Oncology, vol. 10, no. 1, p. 32, [88] J. Bao, J. H. Zou, C. Y. Li, and G. Q. Zheng, “miR-194 inhibits gastric cancer cell proliferation and tumorigenesis by tar- geting KDM5B,” European Review for Medical and Phar- macological Sciences, vol. 20, no. 21, pp. 4487–4493, 2016. [89] G. Cui, D. Liu, W. Li et al., “Original research: miR-194 inhibits proliferation and invasion and promotes apoptosis by targeting KDM5B in esophageal squamous cell carcinoma cells,” Experimental Biology and Medicine, vol. 242, no. 1, pp. 45–52, 2017. MEDIATORS of INFLAMMATION The Scientific Gastroenterology Journal of World Journal Research and Practice Diabetes Research Disease Markers Hindawi Hindawi Publishing Corporation Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 http://www www.hindawi.com .hindawi.com V Volume 2018 olume 2013 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 International Journal of Journal of Immunology Research Endocrinology Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 Submit your manuscripts at www.hindawi.com BioMed PPAR Research Research International Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 Journal of Obesity Evidence-Based Journal of Journal of Stem Cells Complementary and Ophthalmology International Alternative Medicine Oncology Hindawi Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2013 Parkinson’s Disease Computational and Behavioural Mathematical Methods AIDS Oxidative Medicine and in Medicine Neurology Research and Treatment Cellular Longevity Hindawi Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Oncology Hindawi Publishing Corporation

MicroRNAs as Potential Biomarkers for Chemoresistance in Adenocarcinomas of the Esophagogastric Junction

Download PDF
12 pages

Loading next page...
 
/lp/hindawi-publishing-corporation/micrornas-as-potential-biomarkers-for-chemoresistance-in-nCMmLFkbdw

References (89)

Publisher
Hindawi Publishing Corporation
Copyright
Copyright © 2019 Christina Just et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN
1687-8450
eISSN
1687-8469
DOI
10.1155/2019/4903152
Publisher site
See Article on Publisher Site

Abstract

Hindawi Journal of Oncology Volume 2019, Article ID 4903152, 11 pages https://doi.org/10.1155/2019/4903152 Research Article MicroRNAs as Potential Biomarkers for Chemoresistance in Adenocarcinomas of the Esophagogastric Junction 1 2 1 3 Christina Just, Juliana Knief , Pamela Lazar-Karsten, Ekaterina Petrova, 3 4 3 2 Richard Hummel, Christoph Ro¨cken , Ulrich Wellner, and Christoph Thorns Department of Pathology, Section of Hematopathology and Endocrine Pathology, University Hospital of Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany Department of Pathology, Marienkrankenhaus Hamburg, Alfredstrasse 9, 22087 Hamburg, Germany Department of Surgery, University Hospital of Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany Department of Pathology, University Hospital of Schleswig-Holstein, Campus Kiel, Arnold-Heller-Strasse 3, 24105 Kiel, Germany Correspondence should be addressed to Juliana Knief; knief.patho@marienkrankenhaus.org Received 14 February 2019; Accepted 15 July 2019; Published 29 July 2019 Guest Editor: Kenneth J. Vega Copyright © 2019 Christina Just et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Concerning adenocarcinomas of the esophagogastric junction, neoadjuvant chemotherapy is regularly implemented, but patients’ response varies greatly, with some cases showing no therapeutic effect, being deemed as chemoresistant. Small, noncoding RNAs (miRNAs) have evolved as key players in biological processes, including malignant diseases, often promoting tumor growth and expansion. In addition, specific miRNAs have been implicated in the development of chemoresistance through evasion of apoptosis, cell cycle alterations, and drug target modification. We performed a retrospective study of 33 patients receiving neoadjuvant chemotherapy by measuring their miRNA expression profiles. Histologic tumor regression was evaluated using resection specimens, while miRNA profiles were prepared using preoperative biopsies without prior therapy. A preselected panel of 96 miRNAs, known to be of importance in various malignancies, was used to test for significant differences between responsive (chemosensitive) and nonresponsive (chemoresistant) cases. *e cohort consisted of 12 nonresponsive and 21 responsive cases with the following 4 miRNAs differentially expressed between both the groups: hsa-let-7f-5p, hsa-miRNA- 221-3p, hsa-miRNA-31-5p, and hsa-miRNA-191-5p. *e former 3 showed upregulation in chemoresistant cases, while the latter showed upregulation in chemosensitive cases. In addition, significant correlation between high expression of hsa- miRNA-194-5p and prolonged survival could be demonstrated (p value <0.0001). In conclusion, we identified a panel of 3 miRNAs predicting chemoresistance and a single miRNA contributing to chemosensitivity. *ese miRNAs might function as prognostic biomarkers and enable clinicians to better predict the effect of one or more reliably select patients benefitting from (neoadjuvant) chemotherapy. increasingly been used not only as diagnostic but also as 1. Introduction prognostic biomarkers, and several studies have suggested Since the discovery of microRNAs (miRNAs), which are the existence of tissue-specific miRNA signatures which small, noncoding RNAs with a length of 19–22 nucleotides might be used to classify different cancer types [3–5]. [1], multiple studies have focused on the importance of their Regarding human cancer in general, miRNAs have been function and participation in human diseases ranging from found to act not only as oncogenes, promoting tumor inflammatory disorders and autoimmune diseases to ma- growth and dissemination, but also as tumor suppressors, lignant tumors including melanoma, various epithelial inhibiting tumor cell proliferation and migration and in- cancers, and hematological malignancies [2]. miRNAs have ducing apoptosis [6]. Sometimes, their function varies in a 2 Journal of Oncology single organ, showing divergent behavior in specific histo- study. All patients received neoadjuvant chemotherapy. logic tumor types (for example, adenocarcinoma vs. squa- Only tumor-containing samples of preoperative biopsies mous cell carcinoma of the esophagus) [7]. (without prior therapy) were used for miRNA analysis, while Concerning carcinomas of the upper gastrointestinal whole-resection specimens (postchemotherapeutic) were tract, especially gastric cancer and adenocarcinoma of the taken to determine the degree of histological regression and esophagogastric junction, a multitude of miRNAs have been thereby treatment response. All cases were collected as part identified as useful biomarkers: for example, upregulation of of routine clinical care at the University Hospital of miRNA-17-5p, miRNA-20a, miRNA-106b, miRNA-150, Schleswig-Holstein, Campus Luebeck, during 1997–2013. and miRNA-93 has been reported to inhibit apoptosis and to All analyses performed were in accordance with the Dec- promote cell cycle progression, whereas downregulation of laration of Helsinki and had been approved by the local miRNA-29 and miRNA-375 has been shown to increase cell Ethics Committee beforehand (reference number 14-242A). growth and migration [4, 8]. Other commonly dysregulated miRNAs include miRNA-21 and miRNA-19a/b which both 2.2. Histologic Examination. Samples were carefully exam- promote lymph node and distant metastases as well as in- ined by two researchers (CJ and JK) with a light microscope vasion of blood vessels when overexpressed [4]. In addition, (Axioskop, Zeiss, Jena, Germany), and histologic tumor a seven-miRNA panel consisting of miRNA-10b, miRNA- types were determined using the current WHO standard 21, miRNA-223, miRNA-338, let-7a, miRNA-30a-5p, and [28]. Regression after chemotherapy was determined using miRNA-126 has been found to reliably predict survival in haematoxylin- and eosin-(H&E-) stained slides and rated gastric cancer patients and is related not only to overall but according to the system devised by Becker et al. [29]. Re- also relapse free survival [9]. Besides, various miRNAs have gression grades 1a and 1b were considered as having been found to be associated with poor survival in both responded to therapy (responder group), while regression gastric carcinomas and adenocarcinomas of the esoph- grade 3 was considered nonresponsive (nonresponder agogastric junction, including miRNA-16, miRNA-21, group). Cases with regression grade 2 were not included in miRNA-29, miRNA-125b, miRNA-130a, miRNA-141, the study as an assignment to either group could not reliably miRNA-203a, miRNA-222, miRNA-302c, and miRNA-451 be undertaken (partial response). [8, 10–15]. Complementing their diagnostic and prognostic sig- nificance, miRNAs have also been found to contribute to 2.3. RNA Isolation and miRNA Profiling. RNA for profiling chemoresistance and/or chemosensitivity via regulation of of miRNA was isolated from FFPE tissue using the apoptosis, DNA damage, and repair mechanisms, and ep- RecoverAll total nucleic acid isolation kit (Applied Bio- ithelial-mesenchymal transition and modulation of drug systems, Carlsbad, California, USA). RNA concentrations targets, drug-metabolizing enzymes, and drug efflux were quantified using the NanoDrop Spectrophotometer transporters [16, 17]. Especially in gastric cancer, the fol- (Nanodrop Technologies, Montchanin, New Castle, Dela- lowing miRNAs (amongst others) have been found to ware, USA). Afterwards, reverse transcription (RT) using substantially contribute to chemoresistance: let-7b, miRNA- amounts of 20 ng of total RNA by applying the miRCURY 106a, miRNA-142, miRNA-143, miRNA-21, miRNA-338, LNA Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, miRNA-340, miRNA-497, miRNA-503, and miRNA- Denmark), containing synthetic RNA Spike Ins, was per- 582—their target genes including PTEN (phosphatase and formed. 5 μl of the RT products was combined with the tension homolog), BCL (B-cell lymphoma 2), and IGF1R PCR master mix and nuclease-free water from the miR- (insulin-like growth factor 1 receptor) [18–25]. For esoph- CURY LNA ExiLENT SYBR Green master mix (Exiqon, ageal (adeno-) carcinoma, miRNA-141, miRNA-148a, Vedbaek, Denmark). After that, 10 μl of the PCR Master miRNA-200c, miRNA-221, miRNA-27a, and miRNA-296 mix-cDNA mix was added to each 384-well plate of the are said to contribute to chemoresistance [26]. *is miR- miRCURY LNA Universal RT miR Ready-to-Use PCR, mediated chemoresistance is mainly directed against com- Cancer focus panel, V4 (Exiqon, Vedbaek, Denmark). Fi- monly used therapeutic agents such as cisplatin, 5-fluoro- nally, qPCR was performed by using the LightCycler 480 uracil, and vincristine [27]. instrument (Roche molecular systems Inc., Mannheim, Following these observations and taking into account Germany). All reactions were carried out according to the that data concerning chemoresistance in carcinomas of the manufacturer’s instructions. Initial data analysis was exe- esophagogastric junction are still scarce and many studies cuted by using the LightCycler 480 Software (Roche mo- have focused on cell lines rather than tissue specimen, our lecular systems Inc., Mannheim, Germany) to obtain raw Ct study aimed to further contribute to the knowledge for this values. Ct values were used to determine the amount of entity by comparing miRNA profiles in chemoresistant and miRNA in a sample (both parameters showing an inverse chemosensitive cancer tissue. correlation). 2. Materials and Methods 2.4. Preprocessing of Data. In order to compensate for 2.1. Selection of Cases. Samples from formalin-fixed, par- variations in quality of extracted RNA, extraction yield, and affin-embedded (FFPE) tissue containing adenocarcinomas efficiency of reverse transcription, normalization of data was of the esophagogastric junction were included in the present carried out using GenEx Software Version 6.1 (Trial Version; Journal of Oncology 3 MultiD Analyses AB, Munich). *e first normalization cases as mucinous adenocarcinoma, and 4 cases as un- method used was the Normfinder algorithm which has been differentiated/unclassifiable according to the current WHO described in detail earlier [30]. In our analysis, a panel of 38 standard [36]. After thorough histologic examination of miRs from the data set and a single miR (hsa-miRNA-103a- whole-resection specimens, the cohort consisted of 12 3p) were selected for preprocessing as hsa-miRNA-103a-3p nonresponders (regression grade 3) and 21 responders was stably expressed throughout the cohort and has been (regression grades 1a and 1b). Tumor regression was de- reported as being highly reliable for normalization of data termined as mentioned above; representative examples of [31]. Secondly, external controls (so-called Spike regression grading are shown in Figure 1. Further charac- Ins—preformulated, commercially available RNAs with teristic features of the study cohort are summarized in defined lengths and binding capacities), which were in- Table 1. cluded in the beginning as potential references, were used to normalize the data [32]. *e last normalization method used 3.2. Correlation with Clinical Characteristics. Correlation was the global mean algorithm which—in three step- between both groups showed that undifferentiated carci- s—reduces nonspecific background noise, calculates the nomas and poorly differentiated carcinomas (differentiation arithmetic mean value for all samples, and then subtracts grade 3) were more common in the nonresponder group, this value from each individual value [33]. *e global mean while responders showed a higher proportion of tubular method is usually applied in cases where large numbers of adenocarcinomas (p values 0.034 and 0.043, respectively). miRNAs are tested and has been reported to be superior to No differences could be detected concerning gender, depth other normalization methods in this particular setting [34]. of infiltration, lymphovascular/perineural invasion, nodal status, or presence of distant metastases (p values between 2.5. Analysis of Subgroups. To test for significant differences 0.087 and 0.443, as shown in Table 1). in miRNA expression profiles between responder and nonresponder groups, the Mann–Whitney-U test for un- 3.3. ;erapy Regimen. Neoadjuvant chemotherapy was ad- paired samples was applied using SPSS Statistics Version 22 ministered in all cases, and 3 patients received radiation (IBM, Ehringen, Germany). Because exploratory data therapy in addition (both belonging to the responder group analysis was performed, adjusting for multiple testing was with irradiation doses of 45 Gy, 59 Gy, and 66 Gy, not required. Afterwards, overlaps of differentially expressed respectively). miRNAs between the applied normalization procedures Concerning the nonresponder group, neoadjuvant described above were compared. chemotherapy consisted in most cases of 5-fluorouracil combined with leucovorin, oxaliplatin, and docetaxel (so- 2.6. Correlation with Clinical Characteristics. To estimate called FLOT regimen; 8/12 patients). *e remaining differences in clinical features (age, gender, differentiation patients received either 5-fluorouracil in combination with cisplatin (2 cases) or epirubicin combined with oxaliplatin (2 grade, nodal status, depth of infiltration, perineural invasion, lymphovascular invasion, and presence of distant metasta- cases). ses) between both groups and between clinical features and In the responder group, a combination of cisplatin and miRNA expression levels, the χ test was applied and a p 5-fluorouracil was administered in most cases (12/21 pa- tients). Only 4 patients received treatment using the FLOT value <0.05 was considered statistically significant. regimen, and 2 were treated with 5-fluorouracil in combi- nation with leucovorin and etoposide. *e remaining three 2.7. Correlation with Overall Survival. To assess the prog- patients received chemotherapy without 5-fluorouracil nostic value of miRNA expression, the median for each containing oxaliplatin, etoposide, and irinotecan. analyzed miRNA was calculated. Cases were then di- chotomized, either showing an expression level above or below the median as described previously [35]. Overall 3.4. miRNA Analysis. Overall, the following 4 miRNAs were survival curves were visualized via Kaplan–Meier estimates differentially expressed between responder and non- using SPSS Statistics Version 22 (IBM, Ehringen, Germany). responder groups: hsa-let-7f-5p, hsa-miRNA-191-5p, hsa- In addition, Cox regression analysis was used to test for miRNA-221-3p, and hsa-miRNA-31-5p. Concerning the different normalisation methods, only independence, taking into consideration gender, depth of infiltration, differentiation grade, nodal status, and presence minor variations were detected: applying the Normfinder algorithm, hsa-let-7f-5p, hsa-miRNA-191-5p, and hsa- or absence of distant metastases. Data were adjusted for multiple testing using the Bon- miRNA-31-5p were differentially expressed (p values 0.03, 0.005, and 0.047). For normalisation with Spike Ins, dif- ferroni procedure; after that, a p value <0.00052 was con- sidered statistically significant for this test. ferences in expression of hsa-miRNA-221-3p and hsa- miRNA-31-5p could be observed (p values 0.022 and 0.02). For normalisation with global mean, hsa-let-7f-5p, hsa- 3. Results miRNA-191-5p, hsa-miRNA-221-3p, and hsa-miRNA-31- 5p were differentially expressed (p values 0.025, 0.014, 0.04, 3.1. Histology. Overall, 24 cases were classified as tubular adenocarcinoma, 3 cases as poorly cohesive carcinoma, 2 and 0.033, respectively). Finally, for normalisation using 4 Journal of Oncology (a) (b) (c) Figure 1: Pictures of histologic tumor regression: (a) regression grade 1a (no vital tumor); (b) regression grade 1b (<10% vital tumor cells); (c) regression grade 3 (>50% vital tumor cells). H&E staining, magnification 200x. hsa-miRNA-103a-3p, hsa-let-7f-5p and hsa-miRNA-31-5p testing, significant differences in survival according to high showed differences in expression (p values 0.038 and 0.036). or low expression of miRNAs were detected only for hsa- *roughout all normalization methods, there was higher miRNA-194-5p with a p value <0.0001. Survival times in expression of hsa-let-7f-5p, hsa-miRNA-221-3p, and hsa- patients with higher expression were nearly three times miRNA-31-5p in the nonresponder group, while hsa- longer than in those with low expression (97.67 months vs. miRNA-191-5p showed higher expression in the responder 32.69 months). Cox regression analysis, however, could not group. *e respective miRNA expression patterns (Ct show independence for prediction of patients’ survival after values) are depicted in Figure 2. taking into consideration gender, depth of infiltration, differentiation grade, nodal status, and presence or absence of distant metastases (p � 0.462; hazard ratio 1.469; 95% 3.5. miRNAs and Tumor Differentiation Grade. Correlation confidence interval 0.527–4.096). In addition, there was no between miRNA expression profiles and tumor differenti- correlation between expression levels of hsa-miRNA-194-5p ation grade found that poorer differentiation (G3) was and drug response (p � 0.456). significantly associated with decreased levels of hsa-miRNA- Appropriate survival curves and mean survival times 200a-3p and elevated levels of hsa-miRNA-21-5p, hsa- including 95% confidence interval are shown in Figure 3 and miRNA-222-3p, hsa-miRNA-25-3p, and hsa-let-7d-5p (p Table 2. values 0.03–0.031). 4. Discussion 3.6. miRNAs and Patients’ Prognosis. Complete survival data *e mechanisms underlying chemotherapy and multidrug were available for 29 patients with a mean follow-up period resistance in human cancer are polymorphic [37]. In this of 44.52 months (range 1–100 months). During follow-up, context, miRNAs have been found to regulate apoptosis, 12 patients (41.38%) died. After adjusting for multiple DNA repair, and epithelial-mesenchymal transition and Journal of Oncology 5 Table 1: Characteristics of adenocarcinoma of the esophagogastric cell cycle regulation has been reported as a key mechanism junction according to the responder or nonresponder status. in tumor progression; in addition, in cervical cancer, increased expression levels are associated with epithelial- Characteristics Responders Nonresponders p value mesenchymal transition, migration, and invasion by Total n 21 12 targeting TWIST2 [41, 42]. For human glioblastomas, Gender cervical and colon carcinoma cells downregulation of Male 18 8 0.198 PTEN and activation of Akt and STAT3—mediated by Female 3 4 increased levels of hsa-miRNA-221-3p—have been shown WHO classification Tubular 17 7 as key players in tumor cell survival and radio- and Poorly cohesive 2 1 chemoresistance [43–46]. In addition, in hepatocellular Mucinous 2 0 0.034 carcinoma, upregulation of hsa-miRNA-221-3p decreases Undifferentiated 0 4 the expression of HDAC6, a tumor suppressor, and Others promotes tumorigenesis [47]. One study focusing solely Differentiation grade on esophageal adenocarcinomas showed that the che- Well (G1) 0 0 moresistance was conveyed through alteration of the Moderately (G2) 19 7 0.043 Wnt/β-catenin pathway and DKK2, CDH1, CD44, MYC, Poor (G3) 2 5 and ABCG2 expression [26]. Concerning hsa-miRNA-31- pT (low) 5p, only few studies have addressed how increased ex- pT0 2 0 pT1a 2 0 pression contributes to chemoresistance: in malignant 0.136 pT1b 5 0 pleural mesothelioma and hepatocellular carcinoma, it pT2 3 1 promotes chemoresistance by targeting OCT1 and ABCB9 pT (high) [48, 49]. In ovarian cancer, chemoresistance is increased pT3 8 10 by modulation of specific calcium-regulated potassium pT4a 1 1 channels [50]. Conversely, an opposite effect has also been pT4b 0 0 reported: overexpression of hsa-miRNA-31-5p decreases pN levels of stathmin 1, a microtubule-depolymerizing pN0 12 6 molecule that leads to reduced chemosensitivity in pN1 3 4 0.203 ovarian cancer [51]. In addition, in osteosarcoma, upre- pN2 6 1 gulation of hsa-miRNA-31-5p inhibits tumor cell mi- pN3 0 1 LVSI gration and invasion by targeting PI3K3C2A [52]. Present 6 5 Regarding hsa-miRNA-191-5p, the few studies conducted 0.443 Absent 15 7 so far show that overexpression promotes chemo- Perineural invasion resistance by modulating p53 and TET1 in chol- Present 1 3 angiocarcinomas [53]. Furthermore, an association with 0.087 Absent 20 9 various estrogen-dependent genes such as ANXA1, Distant metastases PIWIL2, CASP4, ESR1/ESR2, PLAC1, and SOCS2, has Present 4 1 0.409 been shown in breast cancer—however, up to date, no Absent 17 11 such data concerning adenocarcinomas of the esoph- LVSI: lymphovascular space invasion; bold lettering in p values indicates a agogastric junction have been published [54]. statistically significant difference. *e miRNA signature we discovered seems to differ from previously published data; other studies found that to modulate drug targets, drug-metabolizing enzymes, or especially in esophageal carcinoma—in addition to miRNA- drug efflux transporters [16, 17]. In our study, analyzing 221—miRNA-141, miRNA-200c, miRNA-148a, miRNA- adenocarcinomas of the esophagogastric junction, we 296, miRNA-23, miRNA-223, and miRNA-27a are sub- stantially contributing to chemoresistance [26, 55]. Never- identified a panel of four miRNAs which were differen- tially expressed between patients responding or not theless, as some studies focus on plasma-circulating miRNAs or cell lines and not exclusively on expression in tumor responding to neoadjuvant chemotherapy. We found higher expression of hsa-let-7f-5p, hsa-miRNA-221-3p, tissue, results are comparable only to a limited degree [13]. Ours is—to our knowledge—the first study focusing on and hsa-miRNA-31-5p in the nonresponder group, while hsa-miRNA-191-5p showed higher expression in the re- miRNA expression profiles in adenocarcinomas of the esophagogastric junction and their meaning for therapeutic sponder group. *e molecular mechanisms contributing to chemoresistance or chemosensitivity concerning these response based on tissue specimens. four miRNAs are—up to date—not fully understood. Hsa-let-7f-5p is located on the long arm of chromosome 9 Nevertheless, the function and effects of these miRNAs as (9q22.3) and shows involvement in immune cell differenti- stated in the literature might give some clues as to what ation, angiogenesis, and cellular growth arrest [56–58]. It is commonly affected in multiple human cancers, including the underlying mechanisms might be: for hsa-let-7f-5p, a proangiogenic effect has been reported; thus, over- melanoma, lung, and head/neck cancer, with downregulation in the majority of cases [59–61]. Nevertheless, upregulation is expression might contribute to tumor progression via tumor neoangiogenesis [38–40]. For hsa-miRNA-221-3p, also encountered, for instance, in papillary, follicular, and 6 Journal of Oncology 4 2 –1 –1 Non responder Responder Non responder Responder (a) (b) –1 –2 –2 Non responder Responder Non responder Responder (c) (d) Figure 2: Boxplots and Ct values according to differentially expressed miRNAs in responder and nonresponder groups: (a) Ct values for hsa-let-7f-5p; (b) Ct values for hsa-miRNA-191-5p; (c) Ct values for hsa-miRNA-221-3p; (d) Ct values for hsa-miRNA-31-5p. Lower Ct values indicate higher miR expression, while higher Ct values indicate lower miR levels. chemo- and/or radio sensitivity and induces tumor cell anaplastic thyroid cancer as well as breast cancer and ovarian cancer [62–64]. It has been associated not only with che- apoptosis [26, 68, 69]. In addition, in tissue experiments, hsa-miRNA-221-3p has been shown to promote resistance mosensitivity and treatment response in gastric cancer [65] but also with chemotherapy resistance in breast cancer [37]. to a variety of regularly used therapeutic agents including 5- In our study, increased expression of hsa-let-7f-5p showed a fluorouracil, tyrosine kinase inhibitors, and antiandrogens distinctive association with chemoresistance which seems to [37, 70–73]. be in contrast to previously published data concerning car- *ese findings are difficult to compare with our study cinomas of the upper gastrointestinal tract [65]. Still, as data population as both responders and nonresponders had been concerning the association of miR expression levels and treated using a combination chemotherapy containing 5- chemotherapy response are still often controversial and fluorouracil in most cases (28/33 cases). sometimes different functions (either as a tumor suppressor Downregulation of hsa-miRNA-31-5p has been de- scribed in a variety of human cancers, for instance, triple- or as an oncogene) have been reported for different histologic tumor types (adenocarcinoma vs. squamous cell carcinoma) negative breast carcinoma [67] and indicates shortened in a single organ, our data might only mirror a specific effect overall survival in gastric cancer [74] as well as the presence for a defined subset of patients [7]. of locally advanced tumor stages, implicating the function as Hsa-miRNA-221-3p is well characterized, and its a miRNA with tumor suppressor properties. function and involvement in human cancer has been ex- Data concerning its association with therapy response tensively described. It is commonly known as an onco- are more controversial: while some studies report that miRNA, promoting tumor proliferation, invasion, dissem- overexpression promotes chemoresistance in gastric and ination, and metastasis [66, 67]. Multiple analyses have ovarian cancer [50, 75] and breast carcinomas [76], others focused on cell lines where overexpression is commonly could demonstrate that upregulation in gallbladder car- associated with chemoresistance and knockdown restores cinomas leads to increased chemosensitivity [77]. In hsa-miR-221-3p hsa-let-7f-5p hsa-miR-31-5p hsa-miR-191-5p Journal of Oncology 7 Concerning its association with chemotherapy outcome, 1.0 single studies have found hsa-miRNA-191-5p to promote chemoresistance [54], while others could show no influence on chemosensitivity or chemoresistance at all [82]. Overall, 0.8 literature addressing this particular issue is still very sparse. In contrast to the data described above, in our study, high levels of hsa-miRNA-191-5p showed a clear-cut association 0.6 with chemosensitive cases responding to neoadjuvant chemotherapy. It remains to be seen whether these results can be 0.4 reproduced in larger studies or if a similar effect can be shown in other cancer entities. It may be conceivable that our findings reflect—as it is possibly the case with both hsa- 0.2 let-7f-5p and hsa-miRNA-31-5p—an effect which is only discernible in a defined subset of tumors or specific tumor p < 0.0001 entities. 0.0 Due to the small case number in our study and the often limited availability of both pretherapeutic biopsies and re- 0 20.0 40.0 60.0 80.0 100.0 Follow-up (months) section specimen in a single institution, we additionally con- sulted a web database (GEO database) to supplement our data. Expression levels of hsa-miR-194 Here, we could find only one additional study analyzing High expression High expression- chemotherapy response in a very small cohort (8 cases) of censored upper gastrointestinal carcinomas, namely, stomach cancer, Low expression Low expression- proposing a miRNA signature used for predicting chemo- censored therapy outcome [65]. *is signature, however, differed from Figure 3: Kaplan–Meier curve showing survival differences our findings, showing an association of chemoresistance and according to high or low expression of hsa-miRNA-194-5p with a p high expression of let-7g, miR-342, miR-16, miR-181, miR-1, value <0.0001. Survival times are given in months. and miR-34. Whether this might be due to small case numbers in both studies or whether this reflects differences between two Table 2: Average survival times according to high or low ex- cancer entities with a fundamentally different pathophysiology pression of hsa-miRNA-194-5p. remains to be seen. Another study analyzed esophageal ade- nocarcinomas (14 cases) and reported that miRNA-221 con- Average survival (months) SD 95% CI p value tributes to chemoresistance via the Wnt/β-catenin pathway, hsa-miRNA-194-5p supporting our findings [26]. High 97.67 1.91 93.93–101.4 <0.0001 In addition, we correlated the miRNA expression with Low 32.69 7.89 17.22–48.16 patients’ overall survival and could demonstrate that overall survival was significantly correlated with expression levels of hsa-miRNA-194-5p (p value <0.0001) although in- addition, hsa-miRNA-31-5p expression levels seem to influence the therapy response not only in general but also dependence could not be demonstrated applying Cox re- gression analysis (p � 0.462; hazard ratio 1.469; 95% according to different chemotherapeutic agents: in cell line experiments, it has been demonstrated that down- confidence interval 0.527–4.096). In accordance with our regulation can promote resistance to platinum-based results, where higher levels of hsa-miRNA-194-5p corre- therapies and paclitaxel [78], while upregulation corre- sponded to prolonged survival (97.67 months vs. lates with resistance against 5-fluorouracil [79]. Our study 32.69 months), overexpression has been reported to inhibit cell proliferation and to act as a tumor suppressor in la- adds to the current understanding as we could demon- strate increased chemotherapy resistance in cases with ryngeal SCC, prostate cancer, melanoma, bladder cancer, NSCLC, and clear cell renal carcinoma [83–87]. In addition, higher hsa-miRNA-31-5p expression levels. It seems as if the therapeutic response might not only depend on the overexpression has been found to inhibit growth and pro- liferation in gastric cancer cell lines and esophageal squa- tumor subtype analyzed but also on the therapy applied and that hsa-miRNA-31-5p might exhibit both oncogenic mous cell carcinoma [88, 89]. Other miRNAs which are and tumor suppressive functions according to a specific commonly linked to prolonged or shortened overall survival context. in gastric or esophageal carcinomas—for instance, miRNA- *roughout the literature, hsa-miRNA-191-5p is de- 16, miRNA-21, miRNA-29, miRNA-125b, miRNA-130a, scribed as having oncogenic properties, leading to increased miRNA-141, miRNA-203a, miRNA-222, miRNA-302c, and tumor cell proliferation, invasion, and inhibition of apo- miRNA-451—could not be reproduced in our study [4, 8, 9, 11, 14]. *is might well be due to a smaller number of ptosis [80]. *is holds true for various epithelial cancer types such as breast, pancreatic, and hepatocellular carcinomas patients included in our study which limits the statistical reliability as well as the fact that, after adjusting for multiple [81] or cholangiocarcinoma. Here, overexpression is addi- tionally associated with decreased overall survival [53]. testing, only a p value<0.00052 was considered statistically Cumulative survival 8 Journal of Oncology [3] G. Juodzbalys, D. Kasradze, M. Cicciu` et al., “Modern mo- significant which excluded a few otherwise statistically lecular biomarkers of head and neck cancer. Part I. Epigenetic significant miRNAs. diagnostics and prognostics: systematic review,” Cancer Biomarkers: Section A of Disease Markers, vol. 17, no. 4, 5. Conclusion pp. 487–502, 2016. [4] H.-S. Liu, “MicroRNAs as potential biomarkers for gastric Our results imply that miRNAs might play an important role cancer,” World Journal of Gastroenterology, vol. 20, no. 34, in the evolution of chemoresistance and/or chemosensitivity pp. 12007–12017, 2014. in adenocarcinomas of the esophagogastric junction. Nev- [5] M. Zhou, H. Hara, Y. Dai et al., “Circulating organ-specific ertheless, as—due to the restricted availability of both pre- MicroRNAs serve as biomarkers in organ-specific diseases: and posttherapeutic tissue samples in a single institu- implications for organ allo-and xeno-transplantation,” tion—the number of patients in our study was limited, International Journal of Molecular Sciences, vol. 17, no. 8, statistical results should be interpreted with caution. In p. 1232, 2016. addition, only tumor tissue was compared without estab- [6] A. M. Strubberg and B. B. Madison, “MicroRNAs in the lishing baseline levels of miRNA expression in nonneo- etiology of colorectal cancer: pathways and clinical implica- plastic mucosa. tions,” Disease Models & Mechanisms, vol. 10, no. 3, Regardless of the abovementioned limitations, our re- pp. 197–214, 2017. sults contribute to other studies postulating that miRNAs [7] R. Hezova, A. Kovarikova, J. Srovnal et al., “MiR-205 func- might be a pretherapeutic means to predict therapy response tions as a tumor suppressor in adenocarcinoma and an on- cogene in squamous cell carcinoma of esophagus,” Tumor in the future and better stratify patients who benefit from Biology, vol. 37, no. 6, pp. 8007–8018, 2016. neoadjuvant therapy or who might not benefit at all and [8] D. Wang, Z. Fan, F. Liu, and J. Zuo, “Hsa-mir-21 and Hsa-mir- therefore could be spared of adverse effects of noneffective 29 in tissue as potential diagnostic and prognostic biomarkers treatment strategies. for gastric cancer,” Cellular Physiology and Biochemistry, It remains to be seen if the miRNA signature we vol. 37, no. 4, pp. 1454–1462, 2015. established for adenocarcinomas of the esophagogastric [9] X. Li, Y. Zhang, Y. Zhang, J. Ding, K. Wu, and D. Fan, junction can be reproduced in future studies or for different “Survival prediction of gastric cancer by a seven-microRNA tumor entities. signature,” Gut, vol. 59, no. 5, pp. 579–585, 2010. [10] H. Jiang, W.-W. Yu, L.-L. Wang, and Y. Peng, “miR-130a acts Abbreviations as a potential diagnostic biomarker and promotes gastric cancer migration, invasion and proliferation by targeting Ct: Cycle threshold RUNX3,” Oncology Reports, vol. 34, no. 3, pp. 1153–1161, hsa: Homo sapiens miRNA: MicroRNA. [11] W. Liu, Z. Dong, J. Liang et al., “Downregulation of potential tumor suppressor miR-203a by promoter methylation con- Data Availability tributes to the invasiveness of gastric cardia adenocarcinoma,” Cancer Investigation, vol. 34, no. 10, pp. 506–516, 2016. *e data used to support the findings of this study are [12] Y. B. Lu, J. J. Hu, W. J. Sun, X. H. Duan, and X. Chen, available from the corresponding author upon request. “Prognostic value of miR-141 downregulation in gastric cancer,” Genetics and Molecular Research, vol. 14, no. 4, pp. 17305–17311, 2015. Disclosure [13] M. Odenthal, J. Hee, and I. Gockel, “Serum microRNA Part of this study has been presented as a scientific poster at profiles as prognostic/predictive markers in the multimodality the ASCP Annual Meeting 2017 (Chicago, Illinois). therapy of locally advanced adenocarcinomas of the gastro- esophageal junction,” International Journal of Cancer, vol. 137, no. 1, pp. 230–237, 2015. Conflicts of Interest [14] C. Ren, H. Chen, C. Han, D. Fu, D. Wang, and M. Shen, “High expression of miR-16 and miR-451 predicating better prog- *e authors declare that they have no conflicts of interest. nosis in patients with gastric cancer,” Journal of Cancer Re- search and Clinical Oncology, vol. 142, no. 12, pp. 2489–2496, Authors’ Contributions [15] J. G. Wu, J.-J. Wang, X. Jiang et al., “MiR-125b promotes cell Christina Just and Juliana Knief contributed equally to this migration and invasion by targeting PPP1CA-RB signal work. pathways in gastric cancer, resulting in a poor prognosis,” Gastric Cancer: Official Journal of the International Gastric References Cancer Association and the Japanese Gastric Cancer Associ- ation, vol. 18, no. 4, pp. 729–739, 2015. [1] R. C. Lee, R. L. Feinbaum, and V. Ambros, “*e C. elegans [16] G. Xiong, M. Feng, G. Yang et al., “*e underlying mecha- heterochronic gene lin-4 encodes small RNAs with antisense nisms of non-coding RNAs in the chemoresistance of pan- complementarity to lin-14,” Cell, vol. 75, no. 5, pp. 843–854, creatic cancer,” Cancer Letters, vol. 397, pp. 94–102, 2017. [2] J. Song, Y. Ouyang, J. Che et al., “Potential value of miR-221/ [17] T. Zheng, J. Wang, X. Chen, and L. Liu, “Role of microrna in anticancer drug resistance. International journal of cancer,” 222 as diagnostic, prognostic, and therapeutic biomarkers for diseases,” Frontiers in Immunology, vol. 8, p. 56, 2017. International Journal of Cancer, vol. 126, no. 1, pp. 2–10, 2010. Journal of Oncology 9 [18] Y. Fang, H. Shen, H. Li et al., “miR-106a confers cisplatin mean normalization,” in Methods in Molecular Biology, resistance by regulating PTEN/Akt pathway in gastric cancer Springer, New York, NY, USA, 2012. cells,” Acta biochimica et biophysica Sinica, vol. 45, no. 11, [35] D. Calatayud, C. Dehlendorff, M. K. Boisen et al., “Tissue pp. 963–972, 2013. MicroRNA profiles as diagnostic and prognostic biomarkers [19] X. Han, J.-J. Zhang, Z.-Q. Han, H.-B. Zhang, and Z.-A. Wang, in patients with resectable pancreatic ductal adenocarcinoma “Let-7b attenuates cisplatin resistance and tumor growth in and periampullary cancers,” Biomarker Research, vol. 5, no. 1, p. 8, 2017. gastric cancer by targeting AURKB,” Cancer Gene ;erapy, vol. 25, no. 11-12, pp. 300–308, 2018. [36] F. T. Bosman, F. Carneiro, R. H. Hruban, and N. D. *eise, Who Classification of Tumours of the Digestive System, In- [20] X. Liu, H. Cai, W. Sheng, H. Huang, Z. Long, and Y. Wang, “microRNAs expression profile related with response to ternational Agency for Research on Cancer, Lyon, France, 4th preoperative radiochemotherapy in patients with locally ad- edition, 2010. vanced gastric cancer,” BMC Cancer, vol. 18, no. 1, p. 1048, [37] K. R. Kutanzi, O. V. Yurchenko, F. A. Beland, V. F. Checkhun, 2018. and I. P. Pogribny, “MicroRNA-mediated drug resistance in [21] T. Wang, G. Ge, Y. Ding et al., “MiR-503 regulates cisplatin breast cancer,” Clinical Epigenetics, vol. 2, no. 2, pp. 171–185, resistance of human gastric cancer cell lines by targeting 2011. [38] A. Eirin, S. M. Riester, X.-Y. Zhu et al., “MicroRNA and IGF1R and BCL ,” Chinese Medical Journal, vol. 127, no. 12, pp. 2357–2362, 2014. mRNA cargo of extracellular vesicles from porcine adipose tissue-derived mesenchymal stem cells,” Gene, vol. 551, no. 1, [22] S. M. Yang, C. Huang, X.-F. Li, M.-Z. Yu, Y. He, and J. Li, “miR-21 confers cisplatin resistance in gastric cancer cells by pp. 55–64, 2014. regulating PTEN,” Toxicology, vol. 306, pp. 162–168, 2013. [39] X. Qin, F. Chang, Z. Wang, and W. Jiang, “Correlation of [23] Y. Zhang, Q. Lu, and X. Cai, “MicroRNA-106a induces circulating pro-angiogenic mirnas with cardiotoxicity in- multidrug resistance in gastric cancer by targeting RUNX3,” duced by epirubicin/cyclophosphamide followed by docetaxel FEBS Letters, vol. 587, no. 18, pp. 3069–3075, 2013. in patients with breast cancer,” Cancer Biomarkers, vol. 23, [24] W. Zhu, D. Zhu, S. Lu et al., “miR-497 modulates multidrug no. 4, pp. 473–484, 2018. [40] C. Urbich, A. Kuehbacher, and S. Dimmeler, “Role of resistance of human cancer cell lines by targeting BCL ,” Medical Oncology, vol. 29, no. 1, pp. 384–391, 2012. microRNAs in vascular diseases, inflammation, and angio- genesis,” Cardiovasc Research, vol. 79, no. 4, pp. 581–588, [25] M. Zhuang, Q. Shi, X. Zhang et al., “Involvement of miR-143 in cisplatin resistance of gastric cancer cells via targeting 2008. IGF R and BCL ,” Tumour Biology, vol. 36, no. 4, [41] X. Y. He, Z.-L. Tan, Q. Mou et al., “microRNA-221 enhances 1 2 pp. 2737–2745, 2015. MYCN via targeting nemo-like kinase and functions as an [26] Y. Wang, Y. Zhao, A. Herbst et al., “miR-221 mediates oncogene related to poor prognosis in neuroblastoma,” chemoresistance of esophageal adenocarcinoma by direct Clinical Cancer Research, vol. 23, no. 11, pp. 2905–2918, targeting of DKK2 expression,” Annals of Surgery, vol. 264, 2017. [42] W. F. Wei, C.-F. Zhou, X.-G. Wu et al., “MicroRNA-221-3p, a no. 5, pp. 804–814, 2016b. [27] I. Riquelme, P. Letelier, A. Riffo-Campos, P. Brebi, and J. Roa, TWIST2 target, promotes cervical cancer metastasis by di- rectly targeting THBS2,” Cell Death & Disease, vol. 8, no. 12, “Emerging role of miRNAs in the drug resistance of gastric cancer,” International Journal of Molecular Sciences, vol. 17, p. 3220, 2017. no. 3, p. 424, 2016. [43] J. Du, W. LiNa, C. Li et al., “MicroRNA-221 targets PTEN to [28] J.-F. Flejou, “who classification of digestive tumors: the fourth reduce the sensitivity of cervical cancer cells to gefitinib edition,” Annales de Pathologie, vol. 31, no. 5, pp. S27–S31, through the PI K/Akt signaling pathway,” Tumour Biology, 2011. vol. 37, no. 3, pp. 3939–3947, 2016. [29] K. Becker, J. D. Mueller, C. Schulmacher et al., “Histo- [44] W. Li, F. Guo, P. Wang, S. Hong, and C. Zhang, “miR-221/222 morphology and grading of regression in gastric carcinoma confers radioresistance in glioblastoma cells through acti- treated with neoadjuvant chemotherapy,” Cancer, vol. 98, vating Akt independent of PTEN status,” Current Molecular no. 7, pp. 1521–1530, 2003. Medicine, vol. 14, no. 1, pp. 185–195, 2014. [30] C. L. Andersen, J. L. Jensen, and T. F. Ørntoft, “Normalization [45] Y. Ren, M. Yang, R. Ma et al., “Microcystin-LR promotes of real-time quantitative reverse transcription-PCR data: a migration via the cooperation between microRNA-221/PTEN model-based variance estimation approach to identify genes and STAT3 signal pathway in colon cancer cell line DLD-1,” suited for normalization, applied to bladder and colon cancer Ecotoxicology and Environmental Safety, vol. 167, pp. 107–113, data sets,” Cancer Research, vol. 64, no. 15, pp. 5245–5250, 2019. 2004. [46] Q. Xie, Y. Yan, Z. Huang, X. Zhong, and L. Huang, [31] H. J. Peltier and G. J. Latham, “Normalization of microRNA “MicroRNA-221 targeting PI3-K/Akt signaling axis induces expression levels in quantitative RT-PCR assays: identification cell proliferation and BCNU resistance in human glioblas- of suitable reference RNA targets in normal and cancerous toma,” Neuropathology, vol. 34, no. 5, pp. 455–464, 2014. human solid tissues,” RNA, vol. 14, no. 5, pp. 844–852, 2008. [47] H. J. Bae, K. H. Jung, J. W. Eun et al., “MicroRNA-221 governs [32] P. Fardin, S. Moretti, B. Biasotti, A. Ricciardi, S. Bonassi, and tumor suppressor HDAC6 to potentiate malignant progres- L. Varesio, “Normalization of low-density microarray using sion of liver cancer,” Journal of Hepatology, vol. 63, no. 2, external spike-in controls: analysis of macrophage cell lines pp. 408–419, 2015. expression profile,” BMC Genomics, vol. 8, no. 1, p. 17, 2007. [48] H. L. Moody, M. J. Lind, and S. G. Maher, “MicroRNA-31 [33] P. Mestdagh, P. V. Vlierberghe, A. D. Weer et al., “A novel and regulates chemosensitivity in malignant pleural mesotheli- universal method for microRNA RT-qPCR data normaliza- oma,” Molecular ;erapy—Nucleic Acids, vol. 8, pp. 317–329, tion,” Genome Biology, vol. 10, no. 6, p. R64, 2009. 2017. [34] B. D’Haene, P. Mestdagh, J. Hellemans, and J. Vandesompele, [49] K. T. Que, Y. Zhou, Y. You et al., “MicroRNA-31-5p regulates “miRNA expression profiling: from reference genes to global chemosensitivity by preventing the nuclear location of PARP1 10 Journal of Oncology in hepatocellular carcinoma,” Journal of Experimental & [65] C. H. Kim, H. K. Kim, R. L. Rettig et al., “miRNA signature Clinical Cancer Research, vol. 37, no. 1, p. 268, 2018. associated with outcome of gastric cancer patients following chemotherapy,” BMC Medical Genomics, vol. 4, no. 1, p. 79, 2011. [50] P. Samuel, R. C. Pink, D. P. Caley et al., “Over-expression of [66] W. Liu, N. Song, H. Yao, L. Zhao, H. Liu, and G. Li, “miR-221 miR-31 or loss of KCNMA1 leads to increased cisplatin re- and miR-222 simultaneously target reck and regulate growth sistance in ovarian cancer cells,” Tumour Biology, vol. 37, and invasion of gastric cancer cells,” Medical Science Monitor: no. 2, pp. 2565–2573, 2016. International Medical Journal of Experimental and Clinical [51] M. K. Hassan, H. Watari, T. Mitamura et al., “P18/Stathmin1 Research, vol. 21, pp. 2718–2725, 2015. is regulated by miR-31 in ovarian cancer in response to [67] M. Ouyang, Y. Li, S. Ye et al., “MicroRNA profiling implies taxane,” Oncoscience, vol. 2, pp. 294–308, 2015. new markers of chemoresistance of triple-negative breast [52] L. M. Chao, W. Sun, H. Chen, B. Y. Liu, P. F. Li, and cancer,” PLoS One, vol. 9, no. 5, Article ID e96228, 2014. D. W Zhao, “MicroRNA-31 inhibits osteosarcoma cell pro- [68] Z. Chun-Zhi, H. Lei, Z. An-ling et al., “MicroRNA-221 and liferation, migration and invasion by targeting PIK3C2A,” microRNA-222 regulate gastric carcinoma cell proliferation European review for medical and pharmacological sciences, and radioresistance by targeting PTEN,” BMC Cancer, vol. 10, vol. 22, no. 21, pp. 7205–7213, 2018. no. 1, p. 367, 2010. [53] H. Li, Z.-Q. Zhou, Z.-R. Yang et al., “MicroRNA-191 acts as a [69] C. W. Dai, Q.-W. Bai, G.-S. Zhang et al., “MicroRNA let-7f is tumor promoter by modulating the TET1-p53 pathway in down-regulated in patients with refractory acute myeloid intrahepatic cholangiocarcinoma,” Hepatology, vol. 66, no. 1, leukemia and is involved in chemotherapy resistance of pp. 136–151, 2017a. adriamycin-resistant leukemic cells,” Leukemia & Lymphoma, [54] N. Nagpal, H. M. Ahmad, B. Molparia, and R. Kulshreshtha, vol. 55, no. 7, pp. 1645–1648, 2014. “MicroRNA-191, an estrogen-responsive microRNA, func- [70] M. Garofalo and C. M. Croce, “MicroRNAs as therapeutic tions as an oncogenic regulator in human breast cancer,” targets in chemoresistance,” Drug Resistance Updates: Reviews Carcinogenesis, vol. 34, no. 8, pp. 1889–1899, 2013. and Commentaries in Antimicrobial and Anticancer Chemo- [55] S. Komatsu, D. Ichikawa, T. Kawaguchi et al., “Plasma therapy, vol. 16, no. 3–5, pp. 47–59, 2013. microRNA profiles: identification of miR-23a as a novel [71] P. Magee, L. Shi, and M. Garofalo, “Role of microRNAs in biomarker for chemoresistance in esophageal squamous cell chemoresistance,” Annals of Translational Medicine, vol. 3, carcinoma,” Oncotarget, vol. 7, no. 38, pp. 62034–62048, 2016. no. 21, p. 332, 2015. [56] A. Kuehbacher, C. Urbich, A. M. Zeiher, and S. Dimmeler, [72] L. Zhao, D. Zou, X. Wei et al., “MiRNA-221-3p desensitizes “Role of dicer and drosha for endothelial microrna expression pancreatic cancer cells to 5-fluorouracil by targeting RB ,” and angiogenesis,” Circulation Research, vol. 101, no. 1, Tumour Biology, vol. 37, no. 12, 2016. pp. 59–68, 2007. [73] R. L. Zheng, Y.-J. Jiang, and X. Wang, “Role of micrornas on [57] O. C. Maes, H. Sarojini, and E. Wang, “Stepwise up-regulation therapy resistance in Non-Hodgkin’s lymphoma,” In- of MicroRNA expression levels from replicating to reversible ternational Journal of Clinical and Experimental Medicine, and irreversible growth arrest states in WI-38 human fibro- vol. 7, no. 11, pp. 3818–3832, 2014. [74] H. Wang, X. Zhang, Y. Liu et al., “Downregulated miR-31 level blasts,” Journal of Cellular Physiology, vol. 221, no. 1, associates with poor prognosis of gastric cancer and its res- pp. 109–119, 2009. toration suppresses tumor cell malignant phenotypes by [58] H. Wu, J. R. Neilson, P. Kumar et al., “miRNA profiling of inhibiting E2F2,” Oncotarget, vol. 7, no. 24, pp. 36577–36589, naive, effector and memory CD8 T cells,” PLoS One, vol. 2, no. 10, Article ID e1020, 2007. [75] A. Korourian, R. Roudi, A. Shariftabrizi, and Z. Madjd, [59] S. S. Chang, W. W. Jiang, I. Smith et al., “MicroRNA “MicroRNA-31 inhibits RhoA-mediated tumor invasion and alterations in head and neck squamous cell carcinoma,” chemotherapy resistance in MKN-45 gastric adenocarcinoma International Journal of Cancer, vol. 123, no. 12, cells,” Experimental Biology and Medicine, vol. 242, no. 18, pp. 2791–2797, 2008. pp. 1842–1847, 2017. [60] J. Y. Lee, H. J. Kim, N. A. Yoon et al., “Tumor suppressor p53 [76] J. Lv, K. Xia, P. Xu et al., “miRNA expression patterns in plays a key role in induction of both tristetraprolin and let-7 in chemoresistant breast cancer tissues,” Biomedicine & Phar- human cancer cells,” Nucleic Acids Research, vol. 41, no. 11, macotherapy, vol. 68, no. 8, pp. 935–942, 2014. pp. 5614–5625, 2013. [77] M. Li, W. Chen, H. Zhang et al., “MiR-31 regulates the cis- [61] S. Liang, L. He, X. Zhao et al., “MicroRNA let-7f inhibits platin resistance by targeting Src in gallbladder cancer,” tumor invasion and metastasis by targeting myh9 in human Oncotarget, vol. 7, no. 50, pp. 83060–83070, 2016. gastric cancer,” PLoS One, vol. 6, no. 4, Article ID e18409, [78] T. Mitamura, H. Watari, L. Wang et al., “Downregulation of miRNA-31 induces taxane resistance in ovarian cancer cells [62] A. I. Damanakis, S. Eckhardt, A. Wunderlich et al., through increase of receptor tyrosine kinase MET,” Onco- “MicroRNAs let7 expression in thyroid cancer: correlation genesis, vol. 2, no. 3, p. e40, 2013. with their deputed targets HMGA and SLC A ,” Journal of 2 5 5 [79] R. Hummel, C. Sie, D. I. Watson et al., “MicroRNA signatures Cancer Research and Clinical Oncology, vol. 142, no. 6, in chemotherapy resistant esophageal cancer cell lines,” World pp. 1213–1220, 2016. Journal of Gastroenterology, vol. 20, no. 40, pp. 14904–14912, [63] L. X. Yan, X.-F. Huang, Q. Shao et al., “MicroRNA miR-21 overexpression in human breast cancer is associated with [80] E. Elyakim, E. Sitbon, A. Faerman et al., “hsa-miR-191 is a advanced clinical stage, lymph node metastasis and patient candidate oncogene target for hepatocellular carcinoma poor prognosis,” RNA, vol. 14, no. 11, pp. 2348–2360, 2008. therapy,” Cancer Research, vol. 70, no. 20, pp. 8077–8087, [64] H. Zheng, L. Zhang, Y. Zhao et al., “Plasma mirnas as di- agnostic and prognostic biomarkers for ovarian cancer,” PLoS [81] J. Zhao, C.-R. Qiao, Z. Ding et al., “A novel pathway in NSCLC One, vol. 8, no. 11, Article ID e77853, 2013. cells: miR191, targeting NFIA, is induced by chronic hypoxia, Journal of Oncology 11 and promotes cell proliferation and migration,” Molecular Medicine Reports, vol. 15, no. 3, pp. 1319–1325, 2017. [82] S. K. Patnaik, E. Kannisto, and S. Yendamuri, “Over- expression of microrna miR-30a or miR-191 in A549 lung cancer or BEAS-2B normal lung cell lines does not alter phenotype,” PLoS One, vol. 5, no. 2, Article ID e9219, 2010. [83] B. Guo, Q. Hui, Y. Zhang, P. Chang, and K. Tao, “miR-194 is a negative regulator of GEF-H1 pathway in melanoma,” On- cology Reports, vol. 36, no. 4, pp. 2412–2420, 2016. [84] Q. Kong and X.-S. Chen, T. Tian, X.-Y. Xia and P. Xu, Microrna-194 suppresses prostate cancer migration and in- vasion by downregulating human nuclear distribution pro- tein,” Oncology Reports, vol. 37, no. 2, pp. 803–812, 2017. [85] R. Nofech-Mozes, H. W. Z. Khella, A. Scorilas et al., “MicroRNA-194 is a marker for good prognosis in clear cell renal cell carcinoma,” Cancer Medicine, vol. 5, no. 4, pp. 656–664, 2016. [86] X. Zhu, D. Li., F. Yu et al., “miR-194 inhibits the proliferation, invasion, migration, and enhances the chemosensitivity of non-small cell lung cancer cells by targeting forkhead box a1 protein,” Oncotarget, vol. 7, no. 11, pp. 13139–13152, 2016. [87] P. Li, H. Liu, A.-K. Yang et al., “MiR-194 functions as a tumor suppressor in laryngeal squamous cell carcinoma by targeting Wee1,” Journal of Hematology & Oncology, vol. 10, no. 1, p. 32, [88] J. Bao, J. H. Zou, C. Y. Li, and G. Q. Zheng, “miR-194 inhibits gastric cancer cell proliferation and tumorigenesis by tar- geting KDM5B,” European Review for Medical and Phar- macological Sciences, vol. 20, no. 21, pp. 4487–4493, 2016. [89] G. Cui, D. Liu, W. Li et al., “Original research: miR-194 inhibits proliferation and invasion and promotes apoptosis by targeting KDM5B in esophageal squamous cell carcinoma cells,” Experimental Biology and Medicine, vol. 242, no. 1, pp. 45–52, 2017. MEDIATORS of INFLAMMATION The Scientific Gastroenterology Journal of World Journal Research and Practice Diabetes Research Disease Markers Hindawi Hindawi Publishing Corporation Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 http://www www.hindawi.com .hindawi.com V Volume 2018 olume 2013 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 International Journal of Journal of Immunology Research Endocrinology Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 Submit your manuscripts at www.hindawi.com BioMed PPAR Research Research International Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 Journal of Obesity Evidence-Based Journal of Journal of Stem Cells Complementary and Ophthalmology International Alternative Medicine Oncology Hindawi Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2013 Parkinson’s Disease Computational and Behavioural Mathematical Methods AIDS Oxidative Medicine and in Medicine Neurology Research and Treatment Cellular Longevity Hindawi Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018

Journal

Journal of Oncology – Hindawi Publishing Corporation

Published: Jul 29, 2019

There are no references for this article.