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MiR-139-5p Inhibits the Development of Gastric Cancer through Targeting TPD52

MiR-139-5p Inhibits the Development of Gastric Cancer through Targeting TPD52 Hindawi Journal of Healthcare Engineering Volume 2022, Article ID 4033373, 10 pages https://doi.org/10.1155/2022/4033373 Research Article MiR-139-5p Inhibits the Development of Gastric Cancer through Targeting TPD52 1 2 1 1 1 Yuanbo Li , Yan Sun , Zhenlu Li , Shikuan Li , and Changliang Wu e Department of Emergency Surgery, e Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China Neuroelectro Physiological Laboratory, People’s Hospital of Linzi District, Zibo, Shandong Province, China Correspondence should be addressed to Changliang Wu; qdfywucl@qdu.edu.cn Received 29 November 2021; Revised 30 December 2021; Accepted 17 January 2022; Published 16 February 2022 Academic Editor: Enas Abdulhay Copyright © 2022 Yuanbo Li 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. Background. Many researchers have confirmed that miRNAs are involved in the pathogenesis of gastric cancer (GC). ,is study focused on investigating the specific functions of miR-139-5p in GC. Methods. MiR-139-5p and TPD52 expressions were observed by qRT-PCR or western blot in GC. ,e functional mechanism of miR-139-5p was explored by the luciferase reporter assay, transwell assay, and MTT assay. Results. MiR-139-5p downregulation and TPD52 upregulation were detected in GC. Adverse clinical features and prognosis in GC patients were related to low miR-139-5p expression. MiR-139-5p overexpression restrained GC cell proliferation and metastasis. Furthermore, miR-139-5p directly targeted TPD52. TPD52 silencing blocked GC pro- gression. And TPD52 upregulation weakened the antitumor effect of miR-139-5p in GC. Conclusion. MiR-139-5p inhibits GC cell proliferation and metastasis through downregulating TPD52. [12]. Moreover, abnormal miR-139-5p expression often 1. Introduction occurs in various cancers and diseases, including neuro- degeneration [13], breast cancer [14], glioblastoma [15], and Gastric cancer (GC) ranks third in human malignancies [1]. Moreover, it ranks first in gastrointestinal malig- thyroid carcinoma [16]. Recently, it has been reported that nancies, accounting for 95% of gastric malignancies [2]. obesity was associated with miR-139-5p [17]. Collectively, Now, surgery is still the most important treatment of early miR-139-5p is an important biomarker for human diseases, GC, and it is also the main method for the treatment of GC including cancers. [3]. Due to the late detection of GC, the effect of surgery is It has been proposed that tumor protein D52 (TPD52), not good. And 5-year survival rate is maintained at about which belongs to the TPD52-like protein family, functions as 30% [4]. Additionally, patients with early GC have a better an oncogene in prostate cancer [18]. Upregulation of TPD52 prognosis after treatment. ,e postoperative effect is was first found in human breast cancer [19]. TPD52 over- better for patients over 60 years old, while patients under expression was also detected in various human malignant 30 years old tend to have poor prognosis [5]. ,erefore, it tumors [20]. In addition, TPD52 expression was associated is necessary to strengthen the attention to the symptoms with the systemic progression of prostate cancer [21]. High of early GC and the monitoring of high-risk groups in TPD52 expression had an association with bad prognosis in order to increase the detection rate of patients with early breast cancer [22] and ovarian carcinoma [23] patients. GC. More importantly, oncogenic TPD52 regulated cell metas- MicroRNAs (miRNAs) regulate tumorigenesis by af- tasis in prostate cancer [24]. Nonetheless, the mechanism of fecting their target genes [6–8]. Recently, more and more TPD52 in GC is still unclear. Here, the functional mecha- miRNAs are found to express abnormally in GC, such as nism of TPD52 and miR-139-5p was investigated in GC. ,e miR-216a [9], miR-937 [10], and miR-1271 [11]. In par- relationship between miR-139-5p and prognosis in GC ticular, miR-139 participated in human GC development patients was also analyzed. 2 Journal of Healthcare Engineering was detected through the dual-luciferase assay system 2. Materials and Methods (Promega, USA). 2.1. Clinical Tissues. Sample tissues were obtained from sixty-seven GC patients in ,e Affiliated Hospital of Qingdao University. All participators provided informed 2.8. Statistical Analysis. Data were calculated by SPSS 19.0 consent. All GC patients only received surgery. ,eses tis- and GraphPad Prism 6. Differences were calculated by the sues were stored in a −80 C refrigerator. Our research was chi-squared test. Survival analysis was performed by the approved by the Institutional Ethics Committee of ,e Kaplan–Meier method with the log-rank test. All experi- Affiliated Hospital of Qingdao University. ments were performed in 3 replicates. Significant difference indicated P< 0.05. 2.2. Cell Culture and Transfection. HGC-27, SGC-7901, 3. Results MKN-45, and AGS GC cell lines and GES-1 cells (ATCC, USA) were inoculated in the RPMI-1640 medium with FBS 3.1. MiR-139-5p Downregulation and TPD52 Upregulation and incubated under suitable conditions (37 C, 5% CO ). Were Detected in GC. MiR-139-5p expression was evaluated MiR-139-5p mimics and inhibitor, TPD52 vector, and in GC. MiR-139-5p in GC tissues was downregulated siRNA (GeneCopoeia, Guangzhou, China) were transfected compared to the control (Figure 1(a), P< 0.01). Consis- in GC cells by using Lipofectamine 2000 (Invitrogen, tently, miR-139-5p was downregulated in HGC-27, SGC- Carlsbad, USA). 7901, MKN-45, and AGS cells compared to GES-1 cells (Figure 1(b), P< 0.05 or 0.01). Meanwhile, the expression of 2.3. Transwell Assay. Transwell chamber (8 μm pore size; TPD52 in GC was also detected. TPD52 upregulation was found in GC tissues (Figure 1(c), P< 0.01) and cells Millipore) is used to evaluate the migratory and invasive (Figure 1(d), P< 0.05 or 0.01). Additionally, lymph metas- ability of GC cells. ,e upper chamber was put with 4 ×10 tasis and TNM stage in GC patients were related to low miR- GC cells with the uncoated membrane. 10% FBS was added 139-5p expression (P< 0.05, Table 2). And low miR-139-5p in the lower chamber. ,e coated membrane was used for expression was related to worse prognosis (Figure 2) in GC the invasion assay. ,ese cells were cultured for 24 h. Finally, patients. moved cells were observed by using a microscope. 2.4. MTT Assay. Transfected cells (3 ×10 cells/well) were 3.2. MiR-139-5p Impeded GC Progression. ,e specific incubated in 96-well plates at 0, 24, 48, 72, and 96 h. Cell function of miR-139-5p in GC was investigated in HGC-27 viability was evaluated by the MTT assay. ,e absorbance cells containing miR-139-5p mimics or inhibitor. MiR-139- (OD � 490 nm) was examined with a spectrophotometer 5p expression was enhanced by its mimics (Figure 3(a), (,ermo Scientific). P< 0.01) and lowered by its inhibitor (Figure 3(b), P< 0.01) in HGC-27 cells. Functionally, miR-139-5p overexpression restrained HGC-27 cell proliferation (Figure 3(c), P< 0.01). 2.5. Quantitative RT-PCR. TRIzol reagent (Invitrogen, MiR-139-5p downregulation facilitated GC cell proliferation Carlsbad, USA) was applied to extract total RNA containing (Figure 3(d), P< 0.01). Meanwhile, miR-139-5p over- miRNA. Quantitative RT-PCR was performed with SYBR expression weakened the migratory and invasive abilities Green PCR Master Mix and primers. GAPDH or U6 was (Figures 4(a) and 4(b), P< 0.01) in HGC-27 cells, but miR- used as an internal reference. MiR-139-5p and TPD52 ex- 139-5p downregulation showed an opposite effect −△△ct pressions were assessed by the 2 method. ,e primers (Figures 4(a) and 4(b), P< 0.01). Collectively, miR-139-5p are shown in Table 1. impeded GC progression. 2.6. Western Blot Analysis. RIPA lysis buffer was applied to 3.3. TPD52 Was a Downstream Target of MiR-139-5p in GC. extract protein samples. Protein was then separated by 10% Furthermore, TargetScan (http://www.targetscan.org/ SDS-PAGE and transferred into the PVDF membrane. vert_71/) shows that TPD52 can bind to miR-139-5p Protein was incubated with anti-TPD52 (Abcam, Cam- (Figure 5(a)). Luciferase reporter gene assay showed that bridge, USA) and anti-GAPDH antibodies (Epitomics, miR-139-5p mimics blocked wild-type TPD52 luciferase Burlingame, USA) at 4 C overnight. Next, the membrane activity (Figure 5(b), P< 0.01), which had little effect on that was incubated with the corresponding secondary antibody of mutant TPD52. Additionally, miR-139-5p had a negative (Abcam, Cambridge, USA). Finally, protein bands were association with TPD52 expression in GC tissues observed by ECL (ECL, Pierce). (R � 0.7531, P< 0.001; Figure 5(c)). Consistently, miR-139- 5p overexpression reduced TPD52 expression in HGC-27 2.7. Dual-Luciferase Assay. pGL3 vectors (Promega, Madi- cells when miR-139-5p downregulation accelerated TPD52 son, USA) with the 3′-UTR of wild-type or mutant TPD52 expression (Figures 5(d) and 5(e), P< 0.01). ,erefore, miR- were built. GC cells with miR-139-5p mimics and the above 139-5p directly targets TPD52 and negatively regulates its vector were incubated for 48 h. Finally, luciferase activity expression in GC. Journal of Healthcare Engineering 3 Table 1: Primer sequence. Primers MiR-139-5p F: 5′-GCTCTACAGTGCACGTGTC-3′ R: 5′-GTGCAGGGTCCGAGGT-3′ TPD52 F: 5′-GGAAGAGGAGCAGGAAGAGC-3′ R: 5′-GATGACTGAGCCAACAGAG-3′ U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGGT-3′ GAPDH F: 5′-GCTGGCGCTGAGTATGGAGT-3′ R: 5′-CACAGTCTCTTGGTGATGG-3′ 0.8 1.5 ** 0.6 1.0 0.4 0.5 ** 0.2 ** 0.0 0.0 Normal tissues GC (a) (b) ** 2.5 2.5 ** ** 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Normal tissues GC (c) (d) Figure 1: Downregulation of miR-139-5p and upregulation of TPD52 were detected in GC. (a) MiR-139-5p expression in GC tissues (n � 67). (b) MiR-139-5p expression in HGC-27, SGC-7901, MKN-45, AGS, and GES-1 cell lines. (c) TPD52 expression in GC tissues ∗ ∗∗ (n � 67). (d) TPD52 expression in HGC-27, SGC-7901, MKN-45, AGS, and GES-1 cell lines. p< 0.05 and p< 0.01. 3.4. TPD52 Silence Blocked GC Progression. ,en, TPD52 recovered the reduced TPD52 expression mediated by miR- 139-5p mimics in HGC-27 cells (Figure 7(a), P< 0.01). ,en, function was investigated in HGC-27 cells with its siRNA. TPD52 expression was reduced by its siRNA (Figure 6(a), TPD52 vector lessened the suppressive effect of miR-139-5p on P< 0.01). Functionally, si-TPD52 restrained HGC-27 cell HGC-27 cell proliferation and metastasis (Figures 7(b)–7(d), proliferation (Figure 6(b), P< 0.01). Meanwhile, HGC-27 P< 0.01). In brief, upregulation of TPD52 partially impaired cell migration was suppressed by si-TPD52 (Figure 6(c), the antitumor effect of miR-139-5p in GC. P< 0.01). Similarly, downregulation of TPD52 also inhibited cell invasion in GC cells (Figure 6(d), P< 0.01). In brief, 4. Discussion TPD52 silencing can inhibit cell proliferation and metastasis ,e high tumor recurrence rate and mortality of GC are in GC. mainly caused by systemic metastasis. Many researchers have proposed that abnormally expressed miRNAs can affect 3.5. Upregulation of TPD52 Partially Impaired the Antitumor the occurrence and progression of GC [25, 26]. MiR-139-5p Effect of MiR-139-5p in GC. Finally, HGC-27 cells with miR- can affect the diagnosis, prognosis, and treatment of ma- 139-5p mimics and TPD52 vector were employed to further lignancy [27]. For instance, miR-139-5p affected cell me- explore their relationship. As we expected, TPD52 vector tastasis in colorectal cancer [28]. Especially, miR-139 was Relative expression of TPD52 mRNA Relative expression of miR-139-5p Relative expression of miR-139-5p Relative expression of TPD52 mRNA GES1 GES1 HGC-27 HGC-27 SGC-7901 SGC-7901 MKN45 MKN45 AGS AGS 4 Journal of Healthcare Engineering Table 2: Relationship between miR-139-5p expression and clinicopathological characteristics of GC patients. MiR-139-5p Characteristics Cases P value High Low Age (years) 0.586 ≥60 30 11 19 <60 37 17 20 Gender 0.421 Male 40 18 22 Female 27 10 17 Tumor size (cm) 0.052 <5 42 18 24 ≥5 25 10 15 TNM stage 0.038 I + II 47 20 27 III + IV 20 8 12 Differentiation 0.199 Low 35 15 20 Moderate or well 32 13 19 Lymph metastasis 0.007 Present 50 20 30 Absent 17 8 9 Statistical analyses were performed by the χ test. p< 0.05 was considered significant. 100 100 80 80 60 60 40 40 20 20 P=0.032* P=0.049* 0 0 0 12 24 36 48 60 0 12 24 36 48 60 OS time (Months) DFS time (Months) high miR-139-5p high miR-139-5p low miR-139-5p low miR-139-5p (a) (b) Figure 2: MiR-139-5p could predict the prognosis of GC patients. (a) High-miR-139-5p-expression patients showed longer OS. (b) High- miR-139-5p-expression patients showed longer DFS. associated with lymph node metastasis of human metastatic TPD52 was upregulated in GC, and TPD52 silencing can gastric tumors [29]. Moreover, miR-139-5p regulated aer- block GC progression. obic glycolysis via inhibiting PRKAA1 in GC [30]. ,us, the Previous studies have shown that TPD52 is a target of role of miR-139-5p was explored in GC. many miRNAs. Kumamoto et al. reported that TPD52 Here, miR-139-5p downregulation was detected in GC. knockdown can restrain the metastasis of lung squamous Adverse clinical features and prognosis in GC patients were cell carcinoma [32]. ,e same effect of TPD52 on GC was associated with its downregulation. ,e results are similar to also identified in this study. Moreover, miR-34a and previous studies [29]. Moreover, miR-139-5p over- miR-449 repressed breast cancer cell metastasis via expression restrained GC cell proliferation, migration, and targeting the oncogenic TPD52 [33, 34]. MiR-218 also invasion. Furthermore, miR-139-5p directly targets TPD52. inhibited tumor growth through targeting TPD52 in More importantly, miR-139-5p hindered GC progression by prostate cancer [35]. ,ese previous research studies are downregulating TPD52. Similarly, miR-139-5p also re- consistent with our conclusions in GC. In a word, the pressed cell proliferation in uterine leiomyoma through antitumor effect of miR-139-5p is affected by TPD52 in mediating TPD52 [31]. In addition, we also found that GC. Overall survival (%) Disease free survival (%) Journal of Healthcare Engineering 5 4 1.5 ** 1.0 0.5 0 0.0 NC miR-139 mimic NC miR-139 inhibitor HGC-27 HGC-27 (a) (b) 1.5 1.5 1.0 1.0 0.5 0.5 ** 0.0 0.0 24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h HGC-27 HGC-27 NC NC miR-139 mimic miR-139 inhibitor (c) (d) Figure 3: MiR-139-5p repressed cell proliferation in GC. ((a), (b)) MiR-139-5p expressions in HGC-27 cells with miR-139-5p mimics or ∗∗ inhibitor. ((c), (d)) Cell proliferation in HGC-27 cells with miR-139-5p mimics or inhibitor. p< 0.01. Migration 250 ** miR-139 mimics NC miR-139 inhibitor 100 um 100 um 100 um ** NC miR-139 miR-139 mimic inhibitor HGC-27 (a) Invasion 200 ** NC miR-139 mimcs miR-139inhibitor 100 um 100 um 100 um ** NC miR-139 miR-139 mimic inhibitor HGC-27 (b) Figure 4: MiR-139-5p overexpression repressed cell migration and invasion in GC. ((a), (b) Cell migration and invasion in HGC-27 cells ∗∗ with miR-139-5p mimics or inhibitor. p< 0.01. OD value (490 nm) Relative expression of miR-139-5p Relative expression of miR-139-5p OD value (490 nm) The number of invasive cells The number of migrated cells 6 Journal of Healthcare Engineering Predicted consequential pairing of target region (top) and miRNA (bottom) Position 1085-1092 of TPD52 3’ UTR 5’ ...AAACUGUAAAAUGAUACUGUAGA... hsa-miR-139-5p 3’ UGACCUCUGUGCACGUGACAUCU (a) 1.5 1.0 0.5 ** 0.0 NC TPD52-Wt TPD52-Mut HGC-27 (b) 2.5 P< 0.001 R =0.7531 2.0 1.5 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 miR-139-5p expression level in GC tissues (c) 4 miR-139 miR-139 inhibitor NC mimics ** TPD52 GAPDH ** NC miR-139 miR-139 mimic inhibitor HGC-27 (d) (e) Figure 5: MiR-139-5p directly targets TPD52. (a) ,e binding site between miR-139-5p and TPD52. (b) Luciferase reporter assay. (c) ,e correlation between miR-139-5p and TPD52 expression. ((d), (e)) ,e mRNA and protein expression of TPD52 were analyzed in HGC-27 ∗∗ cells containing miR-139-5p mimics or inhibitor. p< 0.01. Relative expression of TPD52 TPD52 expression level Relative luciferase activity in GC tissues Journal of Healthcare Engineering 7 1.5 1.5 1.0 1.0 0.5 0.5 ** ** 0.0 0.0 NC si-TPD52 24 h 48 h 72 h 96 h HGC-27 HGC-27 NC si-TPD52 (a) (b) Migration NC si-TPD52 ** 100 um 100 um NC si-TPD52 HGC-27 (c) Invasion NC si-TPD52 ** 100 um 100 um NC si-TPD52 HGC-27 (d) Figure 6: TPD52 silence blocked GC progression. (a) TPD52 expression in HGC-27 cells with TPD52 siRNA. ((b), (c), (d)) si-TPD52 ∗∗ regulated HGC-27 cell proliferation, migration, and invasion. p< 0.01. Relative expression of TPD52 mRNA OD value (490 nm) The number of invasive cells The number of migrated cells 8 Journal of Healthcare Engineering 1.5 1.5 ** 1.0 1.0 0.5 0.5 ** 0.0 0.0 NC miR-139 miR-139+ 24 h 48 h 72 h 96 h mimic TPD52 HGC-27 HGC-27 NC miR-139 mimic miR-139+TPD52 (a) (b) Migration 150 ** NC miR-139 miR-139+TPD52 100 um 100 um 100 um NC miR-139 miR-139+ mimic TPD52 HGC-27 (c) Invasion ** NC miR-139 miR-139+TPD52 100 um 100 um 100 um NC miR-139 miR-139+ mimic TPD52 HGC-27 (d) Figure 7: TPD52 upregulation impaired the inhibitory action of miR-139-5p in GC. (a) TPD52 expression in HGC-27 cells containing TPD52 vector and miR-139-5p mimics. ((b), (c), (d)) Cell proliferation, migration, and invasion in HGC-27 cells with the TPD52 vector and ∗∗ miR-139-5p mimics. p< 0.01. Relative expression of TPD52 OD value (490 nm) The number of invasive cells The number of migrated cells Journal of Healthcare Engineering 9 mediated EMT process,” Oncotarget, vol. 8, no. 51, 5. 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MiR-139-5p Inhibits the Development of Gastric Cancer through Targeting TPD52

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Hindawi Publishing Corporation
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Copyright © 2022 Yuanbo Li 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.
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2040-2295
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2040-2309
DOI
10.1155/2022/4033373
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Hindawi Journal of Healthcare Engineering Volume 2022, Article ID 4033373, 10 pages https://doi.org/10.1155/2022/4033373 Research Article MiR-139-5p Inhibits the Development of Gastric Cancer through Targeting TPD52 1 2 1 1 1 Yuanbo Li , Yan Sun , Zhenlu Li , Shikuan Li , and Changliang Wu e Department of Emergency Surgery, e Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China Neuroelectro Physiological Laboratory, People’s Hospital of Linzi District, Zibo, Shandong Province, China Correspondence should be addressed to Changliang Wu; qdfywucl@qdu.edu.cn Received 29 November 2021; Revised 30 December 2021; Accepted 17 January 2022; Published 16 February 2022 Academic Editor: Enas Abdulhay Copyright © 2022 Yuanbo Li 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. Background. Many researchers have confirmed that miRNAs are involved in the pathogenesis of gastric cancer (GC). ,is study focused on investigating the specific functions of miR-139-5p in GC. Methods. MiR-139-5p and TPD52 expressions were observed by qRT-PCR or western blot in GC. ,e functional mechanism of miR-139-5p was explored by the luciferase reporter assay, transwell assay, and MTT assay. Results. MiR-139-5p downregulation and TPD52 upregulation were detected in GC. Adverse clinical features and prognosis in GC patients were related to low miR-139-5p expression. MiR-139-5p overexpression restrained GC cell proliferation and metastasis. Furthermore, miR-139-5p directly targeted TPD52. TPD52 silencing blocked GC pro- gression. And TPD52 upregulation weakened the antitumor effect of miR-139-5p in GC. Conclusion. MiR-139-5p inhibits GC cell proliferation and metastasis through downregulating TPD52. [12]. Moreover, abnormal miR-139-5p expression often 1. Introduction occurs in various cancers and diseases, including neuro- degeneration [13], breast cancer [14], glioblastoma [15], and Gastric cancer (GC) ranks third in human malignancies [1]. Moreover, it ranks first in gastrointestinal malig- thyroid carcinoma [16]. Recently, it has been reported that nancies, accounting for 95% of gastric malignancies [2]. obesity was associated with miR-139-5p [17]. Collectively, Now, surgery is still the most important treatment of early miR-139-5p is an important biomarker for human diseases, GC, and it is also the main method for the treatment of GC including cancers. [3]. Due to the late detection of GC, the effect of surgery is It has been proposed that tumor protein D52 (TPD52), not good. And 5-year survival rate is maintained at about which belongs to the TPD52-like protein family, functions as 30% [4]. Additionally, patients with early GC have a better an oncogene in prostate cancer [18]. Upregulation of TPD52 prognosis after treatment. ,e postoperative effect is was first found in human breast cancer [19]. TPD52 over- better for patients over 60 years old, while patients under expression was also detected in various human malignant 30 years old tend to have poor prognosis [5]. ,erefore, it tumors [20]. In addition, TPD52 expression was associated is necessary to strengthen the attention to the symptoms with the systemic progression of prostate cancer [21]. High of early GC and the monitoring of high-risk groups in TPD52 expression had an association with bad prognosis in order to increase the detection rate of patients with early breast cancer [22] and ovarian carcinoma [23] patients. GC. More importantly, oncogenic TPD52 regulated cell metas- MicroRNAs (miRNAs) regulate tumorigenesis by af- tasis in prostate cancer [24]. Nonetheless, the mechanism of fecting their target genes [6–8]. Recently, more and more TPD52 in GC is still unclear. Here, the functional mecha- miRNAs are found to express abnormally in GC, such as nism of TPD52 and miR-139-5p was investigated in GC. ,e miR-216a [9], miR-937 [10], and miR-1271 [11]. In par- relationship between miR-139-5p and prognosis in GC ticular, miR-139 participated in human GC development patients was also analyzed. 2 Journal of Healthcare Engineering was detected through the dual-luciferase assay system 2. Materials and Methods (Promega, USA). 2.1. Clinical Tissues. Sample tissues were obtained from sixty-seven GC patients in ,e Affiliated Hospital of Qingdao University. All participators provided informed 2.8. Statistical Analysis. Data were calculated by SPSS 19.0 consent. All GC patients only received surgery. ,eses tis- and GraphPad Prism 6. Differences were calculated by the sues were stored in a −80 C refrigerator. Our research was chi-squared test. Survival analysis was performed by the approved by the Institutional Ethics Committee of ,e Kaplan–Meier method with the log-rank test. All experi- Affiliated Hospital of Qingdao University. ments were performed in 3 replicates. Significant difference indicated P< 0.05. 2.2. Cell Culture and Transfection. HGC-27, SGC-7901, 3. Results MKN-45, and AGS GC cell lines and GES-1 cells (ATCC, USA) were inoculated in the RPMI-1640 medium with FBS 3.1. MiR-139-5p Downregulation and TPD52 Upregulation and incubated under suitable conditions (37 C, 5% CO ). Were Detected in GC. MiR-139-5p expression was evaluated MiR-139-5p mimics and inhibitor, TPD52 vector, and in GC. MiR-139-5p in GC tissues was downregulated siRNA (GeneCopoeia, Guangzhou, China) were transfected compared to the control (Figure 1(a), P< 0.01). Consis- in GC cells by using Lipofectamine 2000 (Invitrogen, tently, miR-139-5p was downregulated in HGC-27, SGC- Carlsbad, USA). 7901, MKN-45, and AGS cells compared to GES-1 cells (Figure 1(b), P< 0.05 or 0.01). Meanwhile, the expression of 2.3. Transwell Assay. Transwell chamber (8 μm pore size; TPD52 in GC was also detected. TPD52 upregulation was found in GC tissues (Figure 1(c), P< 0.01) and cells Millipore) is used to evaluate the migratory and invasive (Figure 1(d), P< 0.05 or 0.01). Additionally, lymph metas- ability of GC cells. ,e upper chamber was put with 4 ×10 tasis and TNM stage in GC patients were related to low miR- GC cells with the uncoated membrane. 10% FBS was added 139-5p expression (P< 0.05, Table 2). And low miR-139-5p in the lower chamber. ,e coated membrane was used for expression was related to worse prognosis (Figure 2) in GC the invasion assay. ,ese cells were cultured for 24 h. Finally, patients. moved cells were observed by using a microscope. 2.4. MTT Assay. Transfected cells (3 ×10 cells/well) were 3.2. MiR-139-5p Impeded GC Progression. ,e specific incubated in 96-well plates at 0, 24, 48, 72, and 96 h. Cell function of miR-139-5p in GC was investigated in HGC-27 viability was evaluated by the MTT assay. ,e absorbance cells containing miR-139-5p mimics or inhibitor. MiR-139- (OD � 490 nm) was examined with a spectrophotometer 5p expression was enhanced by its mimics (Figure 3(a), (,ermo Scientific). P< 0.01) and lowered by its inhibitor (Figure 3(b), P< 0.01) in HGC-27 cells. Functionally, miR-139-5p overexpression restrained HGC-27 cell proliferation (Figure 3(c), P< 0.01). 2.5. Quantitative RT-PCR. TRIzol reagent (Invitrogen, MiR-139-5p downregulation facilitated GC cell proliferation Carlsbad, USA) was applied to extract total RNA containing (Figure 3(d), P< 0.01). Meanwhile, miR-139-5p over- miRNA. Quantitative RT-PCR was performed with SYBR expression weakened the migratory and invasive abilities Green PCR Master Mix and primers. GAPDH or U6 was (Figures 4(a) and 4(b), P< 0.01) in HGC-27 cells, but miR- used as an internal reference. MiR-139-5p and TPD52 ex- 139-5p downregulation showed an opposite effect −△△ct pressions were assessed by the 2 method. ,e primers (Figures 4(a) and 4(b), P< 0.01). Collectively, miR-139-5p are shown in Table 1. impeded GC progression. 2.6. Western Blot Analysis. RIPA lysis buffer was applied to 3.3. TPD52 Was a Downstream Target of MiR-139-5p in GC. extract protein samples. Protein was then separated by 10% Furthermore, TargetScan (http://www.targetscan.org/ SDS-PAGE and transferred into the PVDF membrane. vert_71/) shows that TPD52 can bind to miR-139-5p Protein was incubated with anti-TPD52 (Abcam, Cam- (Figure 5(a)). Luciferase reporter gene assay showed that bridge, USA) and anti-GAPDH antibodies (Epitomics, miR-139-5p mimics blocked wild-type TPD52 luciferase Burlingame, USA) at 4 C overnight. Next, the membrane activity (Figure 5(b), P< 0.01), which had little effect on that was incubated with the corresponding secondary antibody of mutant TPD52. Additionally, miR-139-5p had a negative (Abcam, Cambridge, USA). Finally, protein bands were association with TPD52 expression in GC tissues observed by ECL (ECL, Pierce). (R � 0.7531, P< 0.001; Figure 5(c)). Consistently, miR-139- 5p overexpression reduced TPD52 expression in HGC-27 2.7. Dual-Luciferase Assay. pGL3 vectors (Promega, Madi- cells when miR-139-5p downregulation accelerated TPD52 son, USA) with the 3′-UTR of wild-type or mutant TPD52 expression (Figures 5(d) and 5(e), P< 0.01). ,erefore, miR- were built. GC cells with miR-139-5p mimics and the above 139-5p directly targets TPD52 and negatively regulates its vector were incubated for 48 h. Finally, luciferase activity expression in GC. Journal of Healthcare Engineering 3 Table 1: Primer sequence. Primers MiR-139-5p F: 5′-GCTCTACAGTGCACGTGTC-3′ R: 5′-GTGCAGGGTCCGAGGT-3′ TPD52 F: 5′-GGAAGAGGAGCAGGAAGAGC-3′ R: 5′-GATGACTGAGCCAACAGAG-3′ U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGGT-3′ GAPDH F: 5′-GCTGGCGCTGAGTATGGAGT-3′ R: 5′-CACAGTCTCTTGGTGATGG-3′ 0.8 1.5 ** 0.6 1.0 0.4 0.5 ** 0.2 ** 0.0 0.0 Normal tissues GC (a) (b) ** 2.5 2.5 ** ** 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Normal tissues GC (c) (d) Figure 1: Downregulation of miR-139-5p and upregulation of TPD52 were detected in GC. (a) MiR-139-5p expression in GC tissues (n � 67). (b) MiR-139-5p expression in HGC-27, SGC-7901, MKN-45, AGS, and GES-1 cell lines. (c) TPD52 expression in GC tissues ∗ ∗∗ (n � 67). (d) TPD52 expression in HGC-27, SGC-7901, MKN-45, AGS, and GES-1 cell lines. p< 0.05 and p< 0.01. 3.4. TPD52 Silence Blocked GC Progression. ,en, TPD52 recovered the reduced TPD52 expression mediated by miR- 139-5p mimics in HGC-27 cells (Figure 7(a), P< 0.01). ,en, function was investigated in HGC-27 cells with its siRNA. TPD52 expression was reduced by its siRNA (Figure 6(a), TPD52 vector lessened the suppressive effect of miR-139-5p on P< 0.01). Functionally, si-TPD52 restrained HGC-27 cell HGC-27 cell proliferation and metastasis (Figures 7(b)–7(d), proliferation (Figure 6(b), P< 0.01). Meanwhile, HGC-27 P< 0.01). In brief, upregulation of TPD52 partially impaired cell migration was suppressed by si-TPD52 (Figure 6(c), the antitumor effect of miR-139-5p in GC. P< 0.01). Similarly, downregulation of TPD52 also inhibited cell invasion in GC cells (Figure 6(d), P< 0.01). In brief, 4. Discussion TPD52 silencing can inhibit cell proliferation and metastasis ,e high tumor recurrence rate and mortality of GC are in GC. mainly caused by systemic metastasis. Many researchers have proposed that abnormally expressed miRNAs can affect 3.5. Upregulation of TPD52 Partially Impaired the Antitumor the occurrence and progression of GC [25, 26]. MiR-139-5p Effect of MiR-139-5p in GC. Finally, HGC-27 cells with miR- can affect the diagnosis, prognosis, and treatment of ma- 139-5p mimics and TPD52 vector were employed to further lignancy [27]. For instance, miR-139-5p affected cell me- explore their relationship. As we expected, TPD52 vector tastasis in colorectal cancer [28]. Especially, miR-139 was Relative expression of TPD52 mRNA Relative expression of miR-139-5p Relative expression of miR-139-5p Relative expression of TPD52 mRNA GES1 GES1 HGC-27 HGC-27 SGC-7901 SGC-7901 MKN45 MKN45 AGS AGS 4 Journal of Healthcare Engineering Table 2: Relationship between miR-139-5p expression and clinicopathological characteristics of GC patients. MiR-139-5p Characteristics Cases P value High Low Age (years) 0.586 ≥60 30 11 19 <60 37 17 20 Gender 0.421 Male 40 18 22 Female 27 10 17 Tumor size (cm) 0.052 <5 42 18 24 ≥5 25 10 15 TNM stage 0.038 I + II 47 20 27 III + IV 20 8 12 Differentiation 0.199 Low 35 15 20 Moderate or well 32 13 19 Lymph metastasis 0.007 Present 50 20 30 Absent 17 8 9 Statistical analyses were performed by the χ test. p< 0.05 was considered significant. 100 100 80 80 60 60 40 40 20 20 P=0.032* P=0.049* 0 0 0 12 24 36 48 60 0 12 24 36 48 60 OS time (Months) DFS time (Months) high miR-139-5p high miR-139-5p low miR-139-5p low miR-139-5p (a) (b) Figure 2: MiR-139-5p could predict the prognosis of GC patients. (a) High-miR-139-5p-expression patients showed longer OS. (b) High- miR-139-5p-expression patients showed longer DFS. associated with lymph node metastasis of human metastatic TPD52 was upregulated in GC, and TPD52 silencing can gastric tumors [29]. Moreover, miR-139-5p regulated aer- block GC progression. obic glycolysis via inhibiting PRKAA1 in GC [30]. ,us, the Previous studies have shown that TPD52 is a target of role of miR-139-5p was explored in GC. many miRNAs. Kumamoto et al. reported that TPD52 Here, miR-139-5p downregulation was detected in GC. knockdown can restrain the metastasis of lung squamous Adverse clinical features and prognosis in GC patients were cell carcinoma [32]. ,e same effect of TPD52 on GC was associated with its downregulation. ,e results are similar to also identified in this study. Moreover, miR-34a and previous studies [29]. Moreover, miR-139-5p over- miR-449 repressed breast cancer cell metastasis via expression restrained GC cell proliferation, migration, and targeting the oncogenic TPD52 [33, 34]. MiR-218 also invasion. Furthermore, miR-139-5p directly targets TPD52. inhibited tumor growth through targeting TPD52 in More importantly, miR-139-5p hindered GC progression by prostate cancer [35]. ,ese previous research studies are downregulating TPD52. Similarly, miR-139-5p also re- consistent with our conclusions in GC. In a word, the pressed cell proliferation in uterine leiomyoma through antitumor effect of miR-139-5p is affected by TPD52 in mediating TPD52 [31]. In addition, we also found that GC. Overall survival (%) Disease free survival (%) Journal of Healthcare Engineering 5 4 1.5 ** 1.0 0.5 0 0.0 NC miR-139 mimic NC miR-139 inhibitor HGC-27 HGC-27 (a) (b) 1.5 1.5 1.0 1.0 0.5 0.5 ** 0.0 0.0 24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h HGC-27 HGC-27 NC NC miR-139 mimic miR-139 inhibitor (c) (d) Figure 3: MiR-139-5p repressed cell proliferation in GC. ((a), (b)) MiR-139-5p expressions in HGC-27 cells with miR-139-5p mimics or ∗∗ inhibitor. ((c), (d)) Cell proliferation in HGC-27 cells with miR-139-5p mimics or inhibitor. p< 0.01. Migration 250 ** miR-139 mimics NC miR-139 inhibitor 100 um 100 um 100 um ** NC miR-139 miR-139 mimic inhibitor HGC-27 (a) Invasion 200 ** NC miR-139 mimcs miR-139inhibitor 100 um 100 um 100 um ** NC miR-139 miR-139 mimic inhibitor HGC-27 (b) Figure 4: MiR-139-5p overexpression repressed cell migration and invasion in GC. ((a), (b) Cell migration and invasion in HGC-27 cells ∗∗ with miR-139-5p mimics or inhibitor. p< 0.01. OD value (490 nm) Relative expression of miR-139-5p Relative expression of miR-139-5p OD value (490 nm) The number of invasive cells The number of migrated cells 6 Journal of Healthcare Engineering Predicted consequential pairing of target region (top) and miRNA (bottom) Position 1085-1092 of TPD52 3’ UTR 5’ ...AAACUGUAAAAUGAUACUGUAGA... hsa-miR-139-5p 3’ UGACCUCUGUGCACGUGACAUCU (a) 1.5 1.0 0.5 ** 0.0 NC TPD52-Wt TPD52-Mut HGC-27 (b) 2.5 P< 0.001 R =0.7531 2.0 1.5 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 miR-139-5p expression level in GC tissues (c) 4 miR-139 miR-139 inhibitor NC mimics ** TPD52 GAPDH ** NC miR-139 miR-139 mimic inhibitor HGC-27 (d) (e) Figure 5: MiR-139-5p directly targets TPD52. (a) ,e binding site between miR-139-5p and TPD52. (b) Luciferase reporter assay. (c) ,e correlation between miR-139-5p and TPD52 expression. ((d), (e)) ,e mRNA and protein expression of TPD52 were analyzed in HGC-27 ∗∗ cells containing miR-139-5p mimics or inhibitor. p< 0.01. Relative expression of TPD52 TPD52 expression level Relative luciferase activity in GC tissues Journal of Healthcare Engineering 7 1.5 1.5 1.0 1.0 0.5 0.5 ** ** 0.0 0.0 NC si-TPD52 24 h 48 h 72 h 96 h HGC-27 HGC-27 NC si-TPD52 (a) (b) Migration NC si-TPD52 ** 100 um 100 um NC si-TPD52 HGC-27 (c) Invasion NC si-TPD52 ** 100 um 100 um NC si-TPD52 HGC-27 (d) Figure 6: TPD52 silence blocked GC progression. (a) TPD52 expression in HGC-27 cells with TPD52 siRNA. ((b), (c), (d)) si-TPD52 ∗∗ regulated HGC-27 cell proliferation, migration, and invasion. p< 0.01. Relative expression of TPD52 mRNA OD value (490 nm) The number of invasive cells The number of migrated cells 8 Journal of Healthcare Engineering 1.5 1.5 ** 1.0 1.0 0.5 0.5 ** 0.0 0.0 NC miR-139 miR-139+ 24 h 48 h 72 h 96 h mimic TPD52 HGC-27 HGC-27 NC miR-139 mimic miR-139+TPD52 (a) (b) Migration 150 ** NC miR-139 miR-139+TPD52 100 um 100 um 100 um NC miR-139 miR-139+ mimic TPD52 HGC-27 (c) Invasion ** NC miR-139 miR-139+TPD52 100 um 100 um 100 um NC miR-139 miR-139+ mimic TPD52 HGC-27 (d) Figure 7: TPD52 upregulation impaired the inhibitory action of miR-139-5p in GC. (a) TPD52 expression in HGC-27 cells containing TPD52 vector and miR-139-5p mimics. ((b), (c), (d)) Cell proliferation, migration, and invasion in HGC-27 cells with the TPD52 vector and ∗∗ miR-139-5p mimics. p< 0.01. Relative expression of TPD52 OD value (490 nm) The number of invasive cells The number of migrated cells Journal of Healthcare Engineering 9 mediated EMT process,” Oncotarget, vol. 8, no. 51, 5. 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Published: Feb 16, 2022

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