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LncRNA MSC-AS1 Promotes Colorectal Cancer Progression by Regulating miR-325/TRIM14 Axis

LncRNA MSC-AS1 Promotes Colorectal Cancer Progression by Regulating miR-325/TRIM14 Axis Hindawi Journal of Oncology Volume 2021, Article ID 9954214, 10 pages https://doi.org/10.1155/2021/9954214 Research Article LncRNA MSC-AS1 Promotes Colorectal Cancer Progression by Regulating miR-325/TRIM14 Axis 1 2 3 4 Changhong He , Xia Wang , Meichun Du , and Yanjun Dong Department of Clinical Laboratory, Yantaishan Hospital, Yantai 264000, China Blood Purification Centre, East Hospital, Qingdao Municipal Hospital, Qingdao 266071, China PICC Clinic, Qingdao Central Hospital Affiliated to Qingdao University, Qingdao 266042, China Department of Clinical Laboratory, People’s Hospital of Rizhao, Rizhao 276800, China Correspondence should be addressed to Yanjun Dong; dongyanjun@sdrzph.cn Received 29 March 2021; Accepted 29 April 2021; Published 11 May 2021 Academic Editor: Alamgeer Yuchi Copyright © 2021 Changhong He 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. LncRNA MSC-AS1 has been reported to be a tumor promoter in hepatocellular carcinoma. However, the function of MSC-AS1 in colorectal cancer (CRC) has not been elucidated. It is designed to study the expression level of MSC-AS1 and investigate its biological effect on the progression of CRC. Methods. 'e expression patterns of MSC-AS1, miR-325, and TRIM14 were explored by RT-qPCR in CRC tissues and cells. 'e protein expression of TRIM14 was tested by Western blot assay. 'e association between MSC-AS1 expression and clinicopathological data was analyzed by chi-squared test. CCK-8 assay, colony formation, and Transwell assay were used to investigate the effect of MSC-AS1 on cell growth, invasion, and migration in CRC cells. 'e correlations among MSC-AS1, miR-325, and TRIM14 were analyzed by Pearson’s correlation coefficient analysis. Results. We found that MSC-AS1 and TRIM14 were upregulated in CRC tissues, while miR-325 was downregulated in CRC tissues. Functional experiments demonstrated that MSC-AS1 knockdown inhibited cell proliferation, migration, and invasion abilities in CRC cells. Additionally, miR-325 was proved to be a target miRNA of MSC-AS1, and TRIM14 might be a downstream gene of miR-325. Besides that, MSC-AS1 counteracted the inhibitory effect of miR-325 on the cell progression and TRIM14 expression. Conclusion. Our results indicated that MSC-AS1 facilitated CRC progression by sponging miR-325 to upregulate TRIM14 expression. We suggested that MSC-AS1 might be a potential lncRNA-target for CRC therapy. ranks first among malignancies. In recent years, molecular 1. Introduction targeted therapy has become a hot spot in tumor therapy. Colorectal cancer (CRC) is one of the most common gas- 'ese non-protein-coding RNAs are called noncoding trointestinal malignancies, which seriously threatens human RNAs, including long noncoding RNAs (lncRNAs) with a health. 'e incidence of CRC has been on the rise world- length of more than 200 nucleotides. Although the mode and wide. In 2020, about 1.9 million patients were diagnosed regulatory mechanism of lncRNAs in tumors have not been with CRC worldwide, and more than 935,000 patients died thoroughly studied, lncRNAs are verified to play a vital role directly or indirectly from CRC [1]. A large number of in the occurrence and development of human cancers [5]. At studies have shown that dietary habits (alcohol consump- present, it has been found that related lncRNAs are involved tion, red meat, processed meat, and refined grains, etc.), in the occurrence, metastasis and invasion, early diagnosis, lifestyle (low physical labor, smoking, etc.), obesity, diabetes, prognosis evaluation, and radiotherapy and chemotherapy and genetic factors are the most important causes of CRC efficacy of CRC [6, 7]. LncNEAT1 was reported to promote [2, 3]. 'e treatment of CRC is generally based on surgery, CRC progression by suppressing miR-486-5p expression followed by radiotherapy, chemotherapy, or other treat- and regulating NR4A1/Wnt/β-catenin pathway [8]. Lnc- ments [4]. Although the survival rate of CRC has been HSD17B11-1:1 acted as a tumor enhancing factor by reg- improved in recent years, the economic burden of CRC ulating miR-338-3p and MACC1 in CRC [9]. In addition, 2 Journal of Oncology lncGNAT1-1 acted as a tumor inhibitor in CRC by mod- from CRC tissues and cells. 'en, the concentration of RNA ulating RKIP-NF-kappaB-Snail pathway [10]. LncRNA MSC was determined by ultraviolet spectrophotometer. Accord- antisense RNA 1 (MSC-AS1) has been reported to accelerate ing to the instructions of PrimeScript RT reagent kit, the osteogenic differentiation by regulating miR-140e-5p and cDNA was synthesized by reverse transcription. RT-qPCR BMP2 [11]. Moreover, MSC-AS1 promoted tumor pro- was performed according to the instructions of SYBR TM gression in kidney renal clear cell carcinoma [12]. Never- Premix Ex Taq . 'e ABI software was used to analyze and -△△Ct theless, the role of MSC-AS1 in CRC remains unclear. process the data to obtain the Ct value. Finally, 2 Plentiful reports have confirmed that lncRNAs act as method was used to calculate the relative expression of target miRNA sponges to upregulate downstream genes to affect gene. tumor progression. For example, lncRNA UCA1 was found to promote CRC cell proliferation and 5-FU resistance by 2.4. Dual-Luciferase Reporter Assay. StarBase (http:// suppressing miR-204-5p [13]. KCNQ1OT1 was reported to starbase.sysu.edu.cn/) was performed to seek the potential facilitate CRC cell progression by inhibiting miR216b-5p to binding miRNAs of MSC-AS1. TargetScan (http://www. increase ZNF146 expression [14]. Zhang et al. found that targetscan.org/vert_72/) was used to explore the potential LINC00152 was downregulated in CRC and induced cell downstream genes of miR-325. 'e mutant and wild se- apoptosis in CRC cells by regulating miR-376c-3p [15]. miR- quence fragments of MSC-AS1 and TRIM14 were cloned 325 was found to inhibit cell growth and metastasis by and combined with the Promega vector. MSC-AS1-Mut and targeting MT3 in bladder cancer [16]. In the current work, MSC-AS1-Wt and TRIM14-Mut and TRIM14-Wt were bioinformatics analysis confirmed that miR-325 might be a transfected into cells with miR-325 mimic or NC mimic, target miRNA of MSC-AS1. respectively. After transfection for 48 h, the relative lucif- In this article, we explored the expression of MSC-AS1 in erase activity of target gene was detected by dual-luciferase CRC tissues. In the meantime, the function of MSC-AS1 reporter assay kit. knockdown on cell proliferation, migration, and invasion was also detected. Most importantly, we demonstrated that MSC-AS1 influenced the tumor progression by regulating 2.5. MTT Assay. 'e cells in logarithmic phase were miR-325/TRIM14 axis in CRC. resuspended. 100 μl cell suspension (1 × 10 cells/ml) was inoculated in 96-well plate at 37 C and 5% CO . After in- cubation for 24, 48, 72, and 96 h, the plates were added with 2. Materials and Methods 20 μl MTT solution (5 mg/ml, Sigma) and incubated for 2.1. Clinical Specimens. 46 tissues from patients with CRC another 4 h. 'en, 200 μl DMSO was added and shook on the were obtained from Yantaishan Hospital. 'e CRC tissues shaker for 10 min. 'e absorbance value was measured by and paracancerous tissues were surgically collected and using a microplate analyzer at 490 nm. According to the stored in liquid nitrogen. All patients did not receive any experimental data, the growth curve was plotted. preoperative treatment (radiotherapy or chemotherapy). Before surgery, the purpose and significance of this study 2.6. Transwell Assay. For cell migration, after transfection were introduced to all patients or their family members; and for 48 h, cells were digested with trypsin and resuspended we have obtained the informed consents signed by all pa- with 400 μl serum-free medium. Cell suspension (1 × 10 tients or their family members. 'is study was approved by cells/ml) was seeded in a 24-well plate. Cells in each group the Ethics Committee of Yantaishan Hospital. were added to the upper chamber of Transwell chamber, and 600 μL DMEM with 10% FBS was added to the lower 2.2. Cell Culture and Cell Transfection. CRC cell lines HT29, chamber of Transwell chamber. Cells were cultured in a SW620, HCT116, and SW480 and normal human epithelial constant temperature incubator containing 5% CO at 37 C cells NCM460 were obtained from TongPai (Shanghai) for 24 h. 'en, the chamber was fixed with 4% formaldehyde Biotechnology Co., LTD (Shanghai, China). 'e frozen CRC for 15 min and stained with crystal violet for 20 min. 'e cells were placed in a 37 C water bath until they were migratory cells were observed and photographed under a completely dissolved. Cells were resuspended with DMEM light microscope. 5 high-power visual fields were randomly containing 10% PBS. 'en, CRC cells were cultured in an selected to count the number of migratory cells. incubator at 37 C and 5% CO . Cell passage was carried out For cell invasion, 50 μl Matrigel blue was diluted and when the cell confluence reached 70–80%. spread in the upper chamber of Transwell chamber. 'e CRC cells (2×10 cells/well) in logarithmic growth phase remaining steps were the same as those in the cell migration were cultured into 6-well plate. MSC-AS1 siRNA, negative experiment. 'e invaded cells were observed under a light control (si-NC), pcDNA3.1-MSC-AS1 (MSC-AS1 vector), microscope and photographed. 5 high-power fields were miR-325 mimic, and NC mimic were transfected into cells randomly selected to count the number of invaded cells. by Lipofectamine 2000. After culturing at 37 C and 5% CO for 48 h, follow-up experiments were carried out. 2.7. Western Blot Assay. Total protein was extracted from CRC cells by TRIzol. 'e protein samples (30 μg) were 2.3.QuantitativeReal-TimePolymeraseChainReaction(qRT- boiled at 100 C for 5 min. After SDS-PAGE gel electro- PCR). TRIzol reagent was performed to extract total RNA phoresis, the protein samples were transferred to the PVDF Journal of Oncology 3 membranes. After blocking with 5% skim milk for 2 hours, (Figure 3(b)). Next, the expression level of miR-325 in CRC the membranes were incubated with antibodies overnight at tissues and cells was detected. Results indicated that miR- 325 was significantly downregulated in CRC tissues and cells 4 C. After incubation with secondary antibodies for another 1 h, the protein signal of CLCA4 was detected by ECL compared with normal groups (Figures 3(c) and 3(d)). reagent. Additionally, the expression of miR-325 was increased in HCT116 cells with MSC-AS1 knockdown (Figure 3(e)). Nevertheless, RT-qPCR results indicated that the expression 2.8. Statistical Analysis. All data were expressed as of MSC-AS1 was reduced in CRC cells with miR-325 mimic mean± SD and analyzed by using SPSS 22.0 and GraphPad (Figure 3(f)). Besides, Pearson’s correlation analysis was Prism 6.0. 'e differences between the two groups were used to detect the relationship between MSC-AS and miR- detected by Student’s t-test. 'e differences among multiple 325 in CRC tissues. 'e results showed that MSC-AS1 ex- groups were detected by one-way ANOVA. 'e correlations pression was inversely related with miR-325 expression in between MSC-AS, miR-325, and TRIM14 were detected by CRC (Figure 3(g)). In sum, our findings suggested that Pearson’s correlation analysis. p< 0.05 was considered MSC-AS1 might be a sponge of miR-325. statistically significant. 3.4. TRIM14 Might Be a Target Gene of miR-325. 3. Results Subsequently, we seek out downstream target gene of miR- 3.1. MSC-AS1 Overexpression Was Discovered in CRC Tissues 325. 'e StarBase software showed that TRIM14 might be a and Cells. First, GEPIA database showed that MSC-AS1 was potential target gene of miR-325 (Figure 4(a)). Dual-lucif- upregulated in colon adenocarcinoma (COAD) erase reporter results displayed that miR-325 mimic led to (Figure 1(a)). Next, RT-qPCR assay was used to detect the decrease in TRIM14-Wt but not TRIM14-Mut (Figure 4(b)). expression pattern of MSC-AS1 in 46 CRC tissues from 'en, the mRNA expression of TRIM14 in CRC tissues and Yantaishan Hospital. It is noted that there was an upward cells was detected. We found that TRIM14 was obviously trend of MSC-AS in CRC tissues compared with control upregulated in CRC tissues compared with nontumor tissues tissues (Figure 1(b)). Next, we measured the expression level (Figure 4(c)). Furthermore, the expression of TRIM14 was reduced in HT29 and HCT116 cells (Figure 4(d)). Fur- of MSC-AS1 in CRC cells (HT29, SW620, HCT116, and SW480) and normal human epithelial cells NCM460. As thermore, there was a negative correlation between miR-325 expected, the expression of MSC-AS1 was obviously higher and TRIM14 in CRC tissues (Figure 4(e)). Our results in CRC cells than in NCM460 cells (Figure 1(c)). Further- confirmed that TRIM14 might be a target gene of miR-325. more, MSC-AS1 was closely associated with lymph node metastasis (p � 0.014) and TNM stage (p � 0.040) (Table 1). 3.5. MSC-AS1 Regulated CRC Progression by Inhibiting miR- Our data indicated that MSC-AS1 was involved in the 325 Expression. To investigate the mechanism of MSC-AS1/ progression of CRC and might be a potential diagnosis target miR-325, MSC-AS1 vector was transfected into CRC cells in patient with CRC. with miR-325 mimic. RT-qPCR results displayed that the expression of miR-325 was significantly increased in cells 3.2. MSC-AS1 Might Be a Carcinogen in CRC Cells. In order when they are transfected with miR-325 mimic, while it to investigate the function of MSC-AS1 in CRC, MSC-AS1 declined when mimic cells were transfected with MSC-AS1 siRNA was transfected into HCT116 cells (Figure 2(a)). vector (Figure 5(a)). Next, CCK-8 assay and Transwell assay 'en, CCK-8 assay and Transwell assay were performed. were used to measure the function of MSC-AS1/miR-325 in CRC cells. We found that cell proliferative ability was CCK-8 results displayed that MSC-AS1 downregulation weakened cell proliferative ability in HCT116 cells dramatically declined in cells with miR-325 mimic. How- ever, MSC-AS1 overexpression weakened the inhibitory (Figure 2(b)). Moreover, Transwell assay displayed that cell migration was obviously inhibited by MSC-AS1 knockdown effect of miR-325 mimic on cell proliferation (Figure 5(b)). in HCT116 cells (Figure 2(c)). Similarly, cell invasion ability Furthermore, Transwell results indicated that miR-325 of CRC cells was blocked by MSC-AS1 knockout mimic reduced cell migration capability in HCT116 and (Figure 2(d)). Altogether, our results indicated that MSC- SW480 cells, while MSC-AS1 transfection reversed the effect AS1 silencing blocked cell progression in CRC cells. of miR-325 (Figure 5(c)). Likewise, MSC-AS1 upregulation destroyed the inhibitory effect of miR-325 mimic on cell invasion ability (Figure 5(d)). 'erefore, our data suggested 3.3. MSC-AS1 Acted as a Sponge of miR-325 in CRC. In our that MSC-AS1 regulated CRC progression by restraining study, StarBase database was performed to seek the potential miR-325 expression. miRNAs of MSC-AS1. As shown in Figure 3(a), there were special binding sites between MSC-AS1 and miR-325. Dual-luciferase reporter assay and RT-qPCR assay were 3.6. MSC-AS1 Accelerated TRIM14 Expression by Sponging miR-325 in CRC Cells. Next, we explored how MSC-AS1 performed. We found that the luciferase activity of MSC- AS1-WT was obviously reduced when cells were transfected regulated the TRIM14 expression by targeting miR-325. As shown in Figure 6(a), the mRNA expression of TRIM14 was with miR-325 mimic. However, the luciferase activity of MSC-AS1-MUT was not significantly changed in mimic cells significantly reduced by miR-325 mimic, while the 4 Journal of Oncology ∗∗ COAD Nontumor tissues CRC tissues (num(T) = 275; num(N) = 349) (a) (b) ∗∗ 3 ∗∗ ∗∗ ∗∗ NCM460 HT29 SW620 HCT116 SW480 (c) Figure 1: MSC-AS1 expression in CRC tissues and cells. (a) 'e expression level of MSC-AS1 in GEPIA database. (b) 'e expression of MSC-AS1 in 46 CRC tissues from Yantaishan Hospital. (c) 'e expression of MSC-AS1 in CRC cells (HT29, SW620, HCT116, and SW480). ∗∗ p< 0.01. Table 1: Correlation between the expression level of MSC-AS1 and clinical characteristics of CRC patients (n � 46). MSC-AS1 expression Clinical characteristics Number of cases n � 46 p value Low (n � 22) High (n � 24) Age (years) 0.619 ≤60 12 5 7 >60 34 17 17 Gender 0.351 Male 26 14 12 Female 20 8 12 Tumor size 0.382 ≤5 cm 24 10 14 >5 cm 22 12 10 Location 0.229 Proximal 11 7 4 Distal 35 15 20 TNM stage 0.040∗ I-II 18 12 6 III-IV 28 10 18 Lymph node metastasis 0.014∗ Absent 27 17 10 Present 19 5 14 p< 0.05: the difference is significant. Relative expression of MSC-AS1 Relative expression of MSC-AS1 Journal of Oncology 5 2.0 1.5 1.0 ∗∗ ∗∗ 0.5 0.0 HCT116 si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (a) HCT116 2.0 1.5 ∗∗ 1.0 ∗∗ 0.5 0.0 0 24 48 72 96 Time (h) si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (b) HCT116 HCT116 si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 ∗∗ ∗∗ si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (c) Figure 2: Continued. Cell viability OD 490nm Relative expression of MSC-AS1 The number of migration cells 6 Journal of Oncology HCT116 HCT116 si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 ∗∗ ∗∗ si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (d) Figure 2: MSC-AS1 might be a carcinogen in CRC cells. (a) 'e expression of MSC-AS1 was reduced by MSC-AS1 siRNAs. (b) MSC-AS1 knockdown obviously suppressed cell proliferation in HCT116 cells. ((c) and (d)) MSC-AS1 knockdown inhibited cell migration and ∗∗ invasion abilities in HCT116 cells. p< 0.01. miRNA Gene name Target site Alignment Target: 5′ au AGUUAUUGGUAAUACUACUAGu 3′ hsa-miR-325 MSC-AS1 chr8: 72966522 – 72966545[+] miRNA: 3′ ug UGAAUGACCUGUG – GAUGAUc C 5′ (a) HCT116 2.0 ∗∗ 1.5 1.5 1.0 1.0 ∗∗ 0.5 0.5 0.0 0.0 Nontumor tissues CRC tissues MSC-AS1-Wt MSC-AS1-Mut miR-NC miR-325 mimic (b) (c) 2.0 2.5 ∗∗ ∗∗ 2.0 1.5 1.5 1.0 ∗∗ 1.0 ∗∗ ∗∗ 0.5 ∗∗ 0.5 0.0 0.0 si-NC si-MSC- si-MSC- NCM460 HT29 SW620 HCT116 SW480 AS1 #1 AS1 #2 (d) (e) 1.0 2.0 R = 0.5772 0.8 p < 0.001 1.5 0.6 1.0 0.4 ∗∗ 0.5 0.2 0.0 0.0 mic-NC miR-325 micic 0 1 2 3 4 5 Relative expression of MSC-AS1 (f) (g) Figure 3: MSC-AS1 acted as a sponge of miR-325 in CRC cells. (a) StarBase showed that there were special binding sites between MSC-AS1 and miR-325. (b) 'e relative luciferase activity of MSC-AS1-Wt and MSC-AS1-Mut. (c) 'e expression of miR-325 in CRC tissues. (d) 'e expression of miR-325 in CRC cells. (e) 'e expression of miR-325 was increased by MSC-AS1 knockdown. (f) 'e expression of MSC-AS1 ∗∗ was reduced by miR-325 mimic. (g) Correlation analysis between MSC-AS1 and miR-325 expression in 46 CRC tissues. p< 0.01. Relative expression of Relative expression of miR-325 Relative luciferase activity MSC-AS1 Relative expression of miR-325 Relative expression of miR-325 Relative expression of miR-325 The number of invasion cells Journal of Oncology 7 TRIM14-Wt 5′- UGCCUGUAAUCCCAGCUACUAGG -3′ miR-325 3′- UGUGAAUGACCUGUGGAUGAUCC -5′ TRIM14-Mut 5′- UGCCUGUAAUCCCAGGAUGAUCG -3′ (a) HCT116 1.5 ∗∗ 1.0 0.5 0.0 0 TRIM14-Wt TRIM14-Mut Nontumor tissues CRC tissues miR-NC miR-325 mimic (b) (c) 1.0 0.8 ∗∗ 2 = 0.4746 0.6 p < 0.001 ∗∗ 0.4 0.2 0.0 0 1 2 3 NCM460 HT29 SW620 HCT116 SW480 Relative expression of TRIM14 (d) (e) Figure 4: TRIM14 might be a target gene of miR-325. (a) 'ere were special binding sites between miR-325 and TRIM14. (b) 'e luciferase activity of TRIM14-Wt and TRIM14-Mut. (c) 'e expression of TRIM14 in CRC tissues. (d) 'e expression of TRIM14 in CRC cells. (e) ∗∗ Correlation analysis between TRIM14 and miR-325 expression in 46 CRC tissues. p< 0.01. suppression effect of miR-325 mimic on TRIM14 expression recent years, which is first found to be overexpressed in was counteracted by MSC-AS1 vector. In addition, MSC- hepatocellular carcinoma tissues [19]. Cao et al. confirmed AS1 vector impaired the inhibitory effect of miR-325 on the that MSC-AS1 played a carcinogenic lncRNA role in he- protein expression of TRIM14 (Figure 6(b)). 'erefore, our patocellular carcinoma by increasing PGK1 expression [20]. data suggested that MSC-AS1 regulated TRIM14 expression As we have mentioned, MSC-AS1 expression was increased by restraining miR-325 expression. in CRC; and we discovered that depletion of MSC-AS1 suppressed tumor progression in CRC. 'erefore, our re- sults indicated that MSC-AS1 might be carcinogenic 4. Discussion lncRNA in CRC. Accumulating studies have manifested that lncRNAs can Recently, the mortality and incidence of CRC have been on the rise worldwide, posing a serious threat to human health. act as ceRNAs to sponge miRNAs and thus upregulate target Due to the lack of effective CRC prevention and low rate of genes. MSC-AS1 was found to regulate the tumor pro- early diagnosis, most patients are already in the mid and late gression by inhibiting miR-124 expression to increase CDK6 stages when they are diagnosed. 'e IARC estimates that, by expression in osteosarcoma [21]. Moreover, MSC-AS1 acted 2025, there will be more than 20 million new cases of CRC as a role of carcinogenic factor in glioma by sponging miR- worldwide. In order to find out the effective treatment, it is 373-3p and upregulating CPEB4 [22]. Li et al. reported that urgent to study the mechanism of CRC by using molecular the expression of miR-325 was reduced in CRC [23]. biology technology. Similarly, the low expression of miR-325 in CRC was dis- covered in our study. Additionally, we confirmed that MSC- At present, the role of lncRNAs in tumor progression has become a research hotspot. Studies have found abnormal AS1 might act as a sponge of miR-325 in CRC. Functionally, MSC-AS1 promoted cell proliferation, invasion, and mi- expression of lncRNAs in various human cancers, which play a key role in tumor development, metastasis, early gration in CRC cells by sponging miR-325. diagnosis, prognosis evaluation, radiotherapy and chemo- Tripartite motif containing 14 (TRIM14) is a member of therapy efficacy, etc. Alaiyan et al. reported that CCAT1 was the TRIM family. Studies have found that TRIM14 was gradually upregulated during the progression of CRC and involved in a variety of biological functions, including cell was associated with tumor metastasis [17]. HAGLR was proliferation, cell apoptosis, inflammatory response, cell proved to promote cell growth, migration, and invasion metastasis, and immune response [24–26]. TRIM14 was viabilities and inhibit cell apoptosis in CRC [18]. LncRNA confirmed to facilitate cell proliferation, migration, and MSC-AS1 is a new lncRNA that has been discovered in invasion and block cell apoptosis in CRC cells [27, 28]. Relative luciferase activity Relative expression of TRIM14 Relative expression of miR-325 Relative expression of TRIM14 8 Journal of Oncology 3.0 2.5 ∗∗ 2.0 ∗∗ 1.5 1.0 0.5 0.0 HCT116 miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (a) 2.0 1.5 ∗∗ 1.0 ∗∗ 0.5 0.0 0 24 48 72 96 Time (h) miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (b) HCT116 MSC-AS1 vector 150 miR-NC miR-325 mimic ∗∗ + miR-325 mimic ∗∗ HCT116 miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (c) Figure 5: Continued. Cell viability OD 490nm Relative expression of miR-325 The number of migration cells Journal of Oncology 9 HCT116 MSC-AS1 vector miR-NC miR-325 mimic +miR-325 mimic ∗∗ ∗∗ HCT116 miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (d) Figure 5: MSC-AS1 regulated CRC progression by inhibiting miR-325 expression. (a) 'e expression of miR-325 in HCT116 cells transfected with miR-325 mimic or MSC-AS1 vector. (b) miR-325 mimic suppressed cell proliferation, while MMSC-AS1 impaired the inhibitory effect of miR-325. (c, d) miR-325 mimic suppressed cell migration and invasion, while MMSC-AS1 impaired the inhibitory effect ∗∗ of miR-325. p< 0.01. 2.0 miR-149-5p MSC-AS1 vector miR-NC mimic + miR-325 mimic 1.5 TRIM14 1.0 ∗∗ ∗∗ GAPDH 0.5 0.0 NC miR-325 MSC-AS1 vector mimic + miR-325 mimic (a) (b) Figure 6: MSC-AS1 accelerated TRIM14 expression by sponging miR-325 in CRC cells. (a) miR-325 mimic reduced the mRNA expression of TRIM14, while MMSC-AS1 impaired the inhibitory effect of miR-325. (b) miR-325 mimic reduced the protein expression of TRIM14, ∗∗ while MMSC-AS1 impaired the inhibitory effect of miR-325. p< 0.01. 'erefore, TRIM14 was confirmed to play a role as an References oncogene in CRC. In the current work, we confirmed that [1] H. Sung, J. Ferlay, R. L Siegel et al., “Global cancer statistics TRIM14 might be a downstream target gene of miR-325. 2020: GLOBOCAN estimates of incidence and mortality Most importantly, our findings demonstrated that MSC-AS1 worldwide for 36 cancers in 185 countries,” CA: A Cancer regulated TRIM14 expression by restraining miR-325 Journal for Clinicians, vol. 71, no. 3, pp. 209–249, 2021. expression. [2] G. Breau and U. Ellis, “Risk factors associated with young- Above all, we demonstrated that MSC-AS1 promoted onset colorectal adenomas and cancer: a systematic review tumor progression by sponging miR-325 to increase and meta-analysis of observational research,” Cancer Control TRIM14 expression in CRC. 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LncRNA MSC-AS1 Promotes Colorectal Cancer Progression by Regulating miR-325/TRIM14 Axis

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Hindawi Publishing Corporation
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Copyright © 2021 Changhong He 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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1687-8450
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1687-8469
DOI
10.1155/2021/9954214
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Abstract

Hindawi Journal of Oncology Volume 2021, Article ID 9954214, 10 pages https://doi.org/10.1155/2021/9954214 Research Article LncRNA MSC-AS1 Promotes Colorectal Cancer Progression by Regulating miR-325/TRIM14 Axis 1 2 3 4 Changhong He , Xia Wang , Meichun Du , and Yanjun Dong Department of Clinical Laboratory, Yantaishan Hospital, Yantai 264000, China Blood Purification Centre, East Hospital, Qingdao Municipal Hospital, Qingdao 266071, China PICC Clinic, Qingdao Central Hospital Affiliated to Qingdao University, Qingdao 266042, China Department of Clinical Laboratory, People’s Hospital of Rizhao, Rizhao 276800, China Correspondence should be addressed to Yanjun Dong; dongyanjun@sdrzph.cn Received 29 March 2021; Accepted 29 April 2021; Published 11 May 2021 Academic Editor: Alamgeer Yuchi Copyright © 2021 Changhong He 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. LncRNA MSC-AS1 has been reported to be a tumor promoter in hepatocellular carcinoma. However, the function of MSC-AS1 in colorectal cancer (CRC) has not been elucidated. It is designed to study the expression level of MSC-AS1 and investigate its biological effect on the progression of CRC. Methods. 'e expression patterns of MSC-AS1, miR-325, and TRIM14 were explored by RT-qPCR in CRC tissues and cells. 'e protein expression of TRIM14 was tested by Western blot assay. 'e association between MSC-AS1 expression and clinicopathological data was analyzed by chi-squared test. CCK-8 assay, colony formation, and Transwell assay were used to investigate the effect of MSC-AS1 on cell growth, invasion, and migration in CRC cells. 'e correlations among MSC-AS1, miR-325, and TRIM14 were analyzed by Pearson’s correlation coefficient analysis. Results. We found that MSC-AS1 and TRIM14 were upregulated in CRC tissues, while miR-325 was downregulated in CRC tissues. Functional experiments demonstrated that MSC-AS1 knockdown inhibited cell proliferation, migration, and invasion abilities in CRC cells. Additionally, miR-325 was proved to be a target miRNA of MSC-AS1, and TRIM14 might be a downstream gene of miR-325. Besides that, MSC-AS1 counteracted the inhibitory effect of miR-325 on the cell progression and TRIM14 expression. Conclusion. Our results indicated that MSC-AS1 facilitated CRC progression by sponging miR-325 to upregulate TRIM14 expression. We suggested that MSC-AS1 might be a potential lncRNA-target for CRC therapy. ranks first among malignancies. In recent years, molecular 1. Introduction targeted therapy has become a hot spot in tumor therapy. Colorectal cancer (CRC) is one of the most common gas- 'ese non-protein-coding RNAs are called noncoding trointestinal malignancies, which seriously threatens human RNAs, including long noncoding RNAs (lncRNAs) with a health. 'e incidence of CRC has been on the rise world- length of more than 200 nucleotides. Although the mode and wide. In 2020, about 1.9 million patients were diagnosed regulatory mechanism of lncRNAs in tumors have not been with CRC worldwide, and more than 935,000 patients died thoroughly studied, lncRNAs are verified to play a vital role directly or indirectly from CRC [1]. A large number of in the occurrence and development of human cancers [5]. At studies have shown that dietary habits (alcohol consump- present, it has been found that related lncRNAs are involved tion, red meat, processed meat, and refined grains, etc.), in the occurrence, metastasis and invasion, early diagnosis, lifestyle (low physical labor, smoking, etc.), obesity, diabetes, prognosis evaluation, and radiotherapy and chemotherapy and genetic factors are the most important causes of CRC efficacy of CRC [6, 7]. LncNEAT1 was reported to promote [2, 3]. 'e treatment of CRC is generally based on surgery, CRC progression by suppressing miR-486-5p expression followed by radiotherapy, chemotherapy, or other treat- and regulating NR4A1/Wnt/β-catenin pathway [8]. Lnc- ments [4]. Although the survival rate of CRC has been HSD17B11-1:1 acted as a tumor enhancing factor by reg- improved in recent years, the economic burden of CRC ulating miR-338-3p and MACC1 in CRC [9]. In addition, 2 Journal of Oncology lncGNAT1-1 acted as a tumor inhibitor in CRC by mod- from CRC tissues and cells. 'en, the concentration of RNA ulating RKIP-NF-kappaB-Snail pathway [10]. LncRNA MSC was determined by ultraviolet spectrophotometer. Accord- antisense RNA 1 (MSC-AS1) has been reported to accelerate ing to the instructions of PrimeScript RT reagent kit, the osteogenic differentiation by regulating miR-140e-5p and cDNA was synthesized by reverse transcription. RT-qPCR BMP2 [11]. Moreover, MSC-AS1 promoted tumor pro- was performed according to the instructions of SYBR TM gression in kidney renal clear cell carcinoma [12]. Never- Premix Ex Taq . 'e ABI software was used to analyze and -△△Ct theless, the role of MSC-AS1 in CRC remains unclear. process the data to obtain the Ct value. Finally, 2 Plentiful reports have confirmed that lncRNAs act as method was used to calculate the relative expression of target miRNA sponges to upregulate downstream genes to affect gene. tumor progression. For example, lncRNA UCA1 was found to promote CRC cell proliferation and 5-FU resistance by 2.4. Dual-Luciferase Reporter Assay. StarBase (http:// suppressing miR-204-5p [13]. KCNQ1OT1 was reported to starbase.sysu.edu.cn/) was performed to seek the potential facilitate CRC cell progression by inhibiting miR216b-5p to binding miRNAs of MSC-AS1. TargetScan (http://www. increase ZNF146 expression [14]. Zhang et al. found that targetscan.org/vert_72/) was used to explore the potential LINC00152 was downregulated in CRC and induced cell downstream genes of miR-325. 'e mutant and wild se- apoptosis in CRC cells by regulating miR-376c-3p [15]. miR- quence fragments of MSC-AS1 and TRIM14 were cloned 325 was found to inhibit cell growth and metastasis by and combined with the Promega vector. MSC-AS1-Mut and targeting MT3 in bladder cancer [16]. In the current work, MSC-AS1-Wt and TRIM14-Mut and TRIM14-Wt were bioinformatics analysis confirmed that miR-325 might be a transfected into cells with miR-325 mimic or NC mimic, target miRNA of MSC-AS1. respectively. After transfection for 48 h, the relative lucif- In this article, we explored the expression of MSC-AS1 in erase activity of target gene was detected by dual-luciferase CRC tissues. In the meantime, the function of MSC-AS1 reporter assay kit. knockdown on cell proliferation, migration, and invasion was also detected. Most importantly, we demonstrated that MSC-AS1 influenced the tumor progression by regulating 2.5. MTT Assay. 'e cells in logarithmic phase were miR-325/TRIM14 axis in CRC. resuspended. 100 μl cell suspension (1 × 10 cells/ml) was inoculated in 96-well plate at 37 C and 5% CO . After in- cubation for 24, 48, 72, and 96 h, the plates were added with 2. Materials and Methods 20 μl MTT solution (5 mg/ml, Sigma) and incubated for 2.1. Clinical Specimens. 46 tissues from patients with CRC another 4 h. 'en, 200 μl DMSO was added and shook on the were obtained from Yantaishan Hospital. 'e CRC tissues shaker for 10 min. 'e absorbance value was measured by and paracancerous tissues were surgically collected and using a microplate analyzer at 490 nm. According to the stored in liquid nitrogen. All patients did not receive any experimental data, the growth curve was plotted. preoperative treatment (radiotherapy or chemotherapy). Before surgery, the purpose and significance of this study 2.6. Transwell Assay. For cell migration, after transfection were introduced to all patients or their family members; and for 48 h, cells were digested with trypsin and resuspended we have obtained the informed consents signed by all pa- with 400 μl serum-free medium. Cell suspension (1 × 10 tients or their family members. 'is study was approved by cells/ml) was seeded in a 24-well plate. Cells in each group the Ethics Committee of Yantaishan Hospital. were added to the upper chamber of Transwell chamber, and 600 μL DMEM with 10% FBS was added to the lower 2.2. Cell Culture and Cell Transfection. CRC cell lines HT29, chamber of Transwell chamber. Cells were cultured in a SW620, HCT116, and SW480 and normal human epithelial constant temperature incubator containing 5% CO at 37 C cells NCM460 were obtained from TongPai (Shanghai) for 24 h. 'en, the chamber was fixed with 4% formaldehyde Biotechnology Co., LTD (Shanghai, China). 'e frozen CRC for 15 min and stained with crystal violet for 20 min. 'e cells were placed in a 37 C water bath until they were migratory cells were observed and photographed under a completely dissolved. Cells were resuspended with DMEM light microscope. 5 high-power visual fields were randomly containing 10% PBS. 'en, CRC cells were cultured in an selected to count the number of migratory cells. incubator at 37 C and 5% CO . Cell passage was carried out For cell invasion, 50 μl Matrigel blue was diluted and when the cell confluence reached 70–80%. spread in the upper chamber of Transwell chamber. 'e CRC cells (2×10 cells/well) in logarithmic growth phase remaining steps were the same as those in the cell migration were cultured into 6-well plate. MSC-AS1 siRNA, negative experiment. 'e invaded cells were observed under a light control (si-NC), pcDNA3.1-MSC-AS1 (MSC-AS1 vector), microscope and photographed. 5 high-power fields were miR-325 mimic, and NC mimic were transfected into cells randomly selected to count the number of invaded cells. by Lipofectamine 2000. After culturing at 37 C and 5% CO for 48 h, follow-up experiments were carried out. 2.7. Western Blot Assay. Total protein was extracted from CRC cells by TRIzol. 'e protein samples (30 μg) were 2.3.QuantitativeReal-TimePolymeraseChainReaction(qRT- boiled at 100 C for 5 min. After SDS-PAGE gel electro- PCR). TRIzol reagent was performed to extract total RNA phoresis, the protein samples were transferred to the PVDF Journal of Oncology 3 membranes. After blocking with 5% skim milk for 2 hours, (Figure 3(b)). Next, the expression level of miR-325 in CRC the membranes were incubated with antibodies overnight at tissues and cells was detected. Results indicated that miR- 325 was significantly downregulated in CRC tissues and cells 4 C. After incubation with secondary antibodies for another 1 h, the protein signal of CLCA4 was detected by ECL compared with normal groups (Figures 3(c) and 3(d)). reagent. Additionally, the expression of miR-325 was increased in HCT116 cells with MSC-AS1 knockdown (Figure 3(e)). Nevertheless, RT-qPCR results indicated that the expression 2.8. Statistical Analysis. All data were expressed as of MSC-AS1 was reduced in CRC cells with miR-325 mimic mean± SD and analyzed by using SPSS 22.0 and GraphPad (Figure 3(f)). Besides, Pearson’s correlation analysis was Prism 6.0. 'e differences between the two groups were used to detect the relationship between MSC-AS and miR- detected by Student’s t-test. 'e differences among multiple 325 in CRC tissues. 'e results showed that MSC-AS1 ex- groups were detected by one-way ANOVA. 'e correlations pression was inversely related with miR-325 expression in between MSC-AS, miR-325, and TRIM14 were detected by CRC (Figure 3(g)). In sum, our findings suggested that Pearson’s correlation analysis. p< 0.05 was considered MSC-AS1 might be a sponge of miR-325. statistically significant. 3.4. TRIM14 Might Be a Target Gene of miR-325. 3. Results Subsequently, we seek out downstream target gene of miR- 3.1. MSC-AS1 Overexpression Was Discovered in CRC Tissues 325. 'e StarBase software showed that TRIM14 might be a and Cells. First, GEPIA database showed that MSC-AS1 was potential target gene of miR-325 (Figure 4(a)). Dual-lucif- upregulated in colon adenocarcinoma (COAD) erase reporter results displayed that miR-325 mimic led to (Figure 1(a)). Next, RT-qPCR assay was used to detect the decrease in TRIM14-Wt but not TRIM14-Mut (Figure 4(b)). expression pattern of MSC-AS1 in 46 CRC tissues from 'en, the mRNA expression of TRIM14 in CRC tissues and Yantaishan Hospital. It is noted that there was an upward cells was detected. We found that TRIM14 was obviously trend of MSC-AS in CRC tissues compared with control upregulated in CRC tissues compared with nontumor tissues tissues (Figure 1(b)). Next, we measured the expression level (Figure 4(c)). Furthermore, the expression of TRIM14 was reduced in HT29 and HCT116 cells (Figure 4(d)). Fur- of MSC-AS1 in CRC cells (HT29, SW620, HCT116, and SW480) and normal human epithelial cells NCM460. As thermore, there was a negative correlation between miR-325 expected, the expression of MSC-AS1 was obviously higher and TRIM14 in CRC tissues (Figure 4(e)). Our results in CRC cells than in NCM460 cells (Figure 1(c)). Further- confirmed that TRIM14 might be a target gene of miR-325. more, MSC-AS1 was closely associated with lymph node metastasis (p � 0.014) and TNM stage (p � 0.040) (Table 1). 3.5. MSC-AS1 Regulated CRC Progression by Inhibiting miR- Our data indicated that MSC-AS1 was involved in the 325 Expression. To investigate the mechanism of MSC-AS1/ progression of CRC and might be a potential diagnosis target miR-325, MSC-AS1 vector was transfected into CRC cells in patient with CRC. with miR-325 mimic. RT-qPCR results displayed that the expression of miR-325 was significantly increased in cells 3.2. MSC-AS1 Might Be a Carcinogen in CRC Cells. In order when they are transfected with miR-325 mimic, while it to investigate the function of MSC-AS1 in CRC, MSC-AS1 declined when mimic cells were transfected with MSC-AS1 siRNA was transfected into HCT116 cells (Figure 2(a)). vector (Figure 5(a)). Next, CCK-8 assay and Transwell assay 'en, CCK-8 assay and Transwell assay were performed. were used to measure the function of MSC-AS1/miR-325 in CRC cells. We found that cell proliferative ability was CCK-8 results displayed that MSC-AS1 downregulation weakened cell proliferative ability in HCT116 cells dramatically declined in cells with miR-325 mimic. How- ever, MSC-AS1 overexpression weakened the inhibitory (Figure 2(b)). Moreover, Transwell assay displayed that cell migration was obviously inhibited by MSC-AS1 knockdown effect of miR-325 mimic on cell proliferation (Figure 5(b)). in HCT116 cells (Figure 2(c)). Similarly, cell invasion ability Furthermore, Transwell results indicated that miR-325 of CRC cells was blocked by MSC-AS1 knockout mimic reduced cell migration capability in HCT116 and (Figure 2(d)). Altogether, our results indicated that MSC- SW480 cells, while MSC-AS1 transfection reversed the effect AS1 silencing blocked cell progression in CRC cells. of miR-325 (Figure 5(c)). Likewise, MSC-AS1 upregulation destroyed the inhibitory effect of miR-325 mimic on cell invasion ability (Figure 5(d)). 'erefore, our data suggested 3.3. MSC-AS1 Acted as a Sponge of miR-325 in CRC. In our that MSC-AS1 regulated CRC progression by restraining study, StarBase database was performed to seek the potential miR-325 expression. miRNAs of MSC-AS1. As shown in Figure 3(a), there were special binding sites between MSC-AS1 and miR-325. Dual-luciferase reporter assay and RT-qPCR assay were 3.6. MSC-AS1 Accelerated TRIM14 Expression by Sponging miR-325 in CRC Cells. Next, we explored how MSC-AS1 performed. We found that the luciferase activity of MSC- AS1-WT was obviously reduced when cells were transfected regulated the TRIM14 expression by targeting miR-325. As shown in Figure 6(a), the mRNA expression of TRIM14 was with miR-325 mimic. However, the luciferase activity of MSC-AS1-MUT was not significantly changed in mimic cells significantly reduced by miR-325 mimic, while the 4 Journal of Oncology ∗∗ COAD Nontumor tissues CRC tissues (num(T) = 275; num(N) = 349) (a) (b) ∗∗ 3 ∗∗ ∗∗ ∗∗ NCM460 HT29 SW620 HCT116 SW480 (c) Figure 1: MSC-AS1 expression in CRC tissues and cells. (a) 'e expression level of MSC-AS1 in GEPIA database. (b) 'e expression of MSC-AS1 in 46 CRC tissues from Yantaishan Hospital. (c) 'e expression of MSC-AS1 in CRC cells (HT29, SW620, HCT116, and SW480). ∗∗ p< 0.01. Table 1: Correlation between the expression level of MSC-AS1 and clinical characteristics of CRC patients (n � 46). MSC-AS1 expression Clinical characteristics Number of cases n � 46 p value Low (n � 22) High (n � 24) Age (years) 0.619 ≤60 12 5 7 >60 34 17 17 Gender 0.351 Male 26 14 12 Female 20 8 12 Tumor size 0.382 ≤5 cm 24 10 14 >5 cm 22 12 10 Location 0.229 Proximal 11 7 4 Distal 35 15 20 TNM stage 0.040∗ I-II 18 12 6 III-IV 28 10 18 Lymph node metastasis 0.014∗ Absent 27 17 10 Present 19 5 14 p< 0.05: the difference is significant. Relative expression of MSC-AS1 Relative expression of MSC-AS1 Journal of Oncology 5 2.0 1.5 1.0 ∗∗ ∗∗ 0.5 0.0 HCT116 si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (a) HCT116 2.0 1.5 ∗∗ 1.0 ∗∗ 0.5 0.0 0 24 48 72 96 Time (h) si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (b) HCT116 HCT116 si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 ∗∗ ∗∗ si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (c) Figure 2: Continued. Cell viability OD 490nm Relative expression of MSC-AS1 The number of migration cells 6 Journal of Oncology HCT116 HCT116 si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 ∗∗ ∗∗ si-NC si-MSC-AS1 #1 si-MSC-AS1 #2 (d) Figure 2: MSC-AS1 might be a carcinogen in CRC cells. (a) 'e expression of MSC-AS1 was reduced by MSC-AS1 siRNAs. (b) MSC-AS1 knockdown obviously suppressed cell proliferation in HCT116 cells. ((c) and (d)) MSC-AS1 knockdown inhibited cell migration and ∗∗ invasion abilities in HCT116 cells. p< 0.01. miRNA Gene name Target site Alignment Target: 5′ au AGUUAUUGGUAAUACUACUAGu 3′ hsa-miR-325 MSC-AS1 chr8: 72966522 – 72966545[+] miRNA: 3′ ug UGAAUGACCUGUG – GAUGAUc C 5′ (a) HCT116 2.0 ∗∗ 1.5 1.5 1.0 1.0 ∗∗ 0.5 0.5 0.0 0.0 Nontumor tissues CRC tissues MSC-AS1-Wt MSC-AS1-Mut miR-NC miR-325 mimic (b) (c) 2.0 2.5 ∗∗ ∗∗ 2.0 1.5 1.5 1.0 ∗∗ 1.0 ∗∗ ∗∗ 0.5 ∗∗ 0.5 0.0 0.0 si-NC si-MSC- si-MSC- NCM460 HT29 SW620 HCT116 SW480 AS1 #1 AS1 #2 (d) (e) 1.0 2.0 R = 0.5772 0.8 p < 0.001 1.5 0.6 1.0 0.4 ∗∗ 0.5 0.2 0.0 0.0 mic-NC miR-325 micic 0 1 2 3 4 5 Relative expression of MSC-AS1 (f) (g) Figure 3: MSC-AS1 acted as a sponge of miR-325 in CRC cells. (a) StarBase showed that there were special binding sites between MSC-AS1 and miR-325. (b) 'e relative luciferase activity of MSC-AS1-Wt and MSC-AS1-Mut. (c) 'e expression of miR-325 in CRC tissues. (d) 'e expression of miR-325 in CRC cells. (e) 'e expression of miR-325 was increased by MSC-AS1 knockdown. (f) 'e expression of MSC-AS1 ∗∗ was reduced by miR-325 mimic. (g) Correlation analysis between MSC-AS1 and miR-325 expression in 46 CRC tissues. p< 0.01. Relative expression of Relative expression of miR-325 Relative luciferase activity MSC-AS1 Relative expression of miR-325 Relative expression of miR-325 Relative expression of miR-325 The number of invasion cells Journal of Oncology 7 TRIM14-Wt 5′- UGCCUGUAAUCCCAGCUACUAGG -3′ miR-325 3′- UGUGAAUGACCUGUGGAUGAUCC -5′ TRIM14-Mut 5′- UGCCUGUAAUCCCAGGAUGAUCG -3′ (a) HCT116 1.5 ∗∗ 1.0 0.5 0.0 0 TRIM14-Wt TRIM14-Mut Nontumor tissues CRC tissues miR-NC miR-325 mimic (b) (c) 1.0 0.8 ∗∗ 2 = 0.4746 0.6 p < 0.001 ∗∗ 0.4 0.2 0.0 0 1 2 3 NCM460 HT29 SW620 HCT116 SW480 Relative expression of TRIM14 (d) (e) Figure 4: TRIM14 might be a target gene of miR-325. (a) 'ere were special binding sites between miR-325 and TRIM14. (b) 'e luciferase activity of TRIM14-Wt and TRIM14-Mut. (c) 'e expression of TRIM14 in CRC tissues. (d) 'e expression of TRIM14 in CRC cells. (e) ∗∗ Correlation analysis between TRIM14 and miR-325 expression in 46 CRC tissues. p< 0.01. suppression effect of miR-325 mimic on TRIM14 expression recent years, which is first found to be overexpressed in was counteracted by MSC-AS1 vector. In addition, MSC- hepatocellular carcinoma tissues [19]. Cao et al. confirmed AS1 vector impaired the inhibitory effect of miR-325 on the that MSC-AS1 played a carcinogenic lncRNA role in he- protein expression of TRIM14 (Figure 6(b)). 'erefore, our patocellular carcinoma by increasing PGK1 expression [20]. data suggested that MSC-AS1 regulated TRIM14 expression As we have mentioned, MSC-AS1 expression was increased by restraining miR-325 expression. in CRC; and we discovered that depletion of MSC-AS1 suppressed tumor progression in CRC. 'erefore, our re- sults indicated that MSC-AS1 might be carcinogenic 4. Discussion lncRNA in CRC. Accumulating studies have manifested that lncRNAs can Recently, the mortality and incidence of CRC have been on the rise worldwide, posing a serious threat to human health. act as ceRNAs to sponge miRNAs and thus upregulate target Due to the lack of effective CRC prevention and low rate of genes. MSC-AS1 was found to regulate the tumor pro- early diagnosis, most patients are already in the mid and late gression by inhibiting miR-124 expression to increase CDK6 stages when they are diagnosed. 'e IARC estimates that, by expression in osteosarcoma [21]. Moreover, MSC-AS1 acted 2025, there will be more than 20 million new cases of CRC as a role of carcinogenic factor in glioma by sponging miR- worldwide. In order to find out the effective treatment, it is 373-3p and upregulating CPEB4 [22]. Li et al. reported that urgent to study the mechanism of CRC by using molecular the expression of miR-325 was reduced in CRC [23]. biology technology. Similarly, the low expression of miR-325 in CRC was dis- covered in our study. Additionally, we confirmed that MSC- At present, the role of lncRNAs in tumor progression has become a research hotspot. Studies have found abnormal AS1 might act as a sponge of miR-325 in CRC. Functionally, MSC-AS1 promoted cell proliferation, invasion, and mi- expression of lncRNAs in various human cancers, which play a key role in tumor development, metastasis, early gration in CRC cells by sponging miR-325. diagnosis, prognosis evaluation, radiotherapy and chemo- Tripartite motif containing 14 (TRIM14) is a member of therapy efficacy, etc. Alaiyan et al. reported that CCAT1 was the TRIM family. Studies have found that TRIM14 was gradually upregulated during the progression of CRC and involved in a variety of biological functions, including cell was associated with tumor metastasis [17]. HAGLR was proliferation, cell apoptosis, inflammatory response, cell proved to promote cell growth, migration, and invasion metastasis, and immune response [24–26]. TRIM14 was viabilities and inhibit cell apoptosis in CRC [18]. LncRNA confirmed to facilitate cell proliferation, migration, and MSC-AS1 is a new lncRNA that has been discovered in invasion and block cell apoptosis in CRC cells [27, 28]. Relative luciferase activity Relative expression of TRIM14 Relative expression of miR-325 Relative expression of TRIM14 8 Journal of Oncology 3.0 2.5 ∗∗ 2.0 ∗∗ 1.5 1.0 0.5 0.0 HCT116 miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (a) 2.0 1.5 ∗∗ 1.0 ∗∗ 0.5 0.0 0 24 48 72 96 Time (h) miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (b) HCT116 MSC-AS1 vector 150 miR-NC miR-325 mimic ∗∗ + miR-325 mimic ∗∗ HCT116 miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (c) Figure 5: Continued. Cell viability OD 490nm Relative expression of miR-325 The number of migration cells Journal of Oncology 9 HCT116 MSC-AS1 vector miR-NC miR-325 mimic +miR-325 mimic ∗∗ ∗∗ HCT116 miR-NC miR-325 mimic MSC-AS1 vector + miR-325 mimic (d) Figure 5: MSC-AS1 regulated CRC progression by inhibiting miR-325 expression. (a) 'e expression of miR-325 in HCT116 cells transfected with miR-325 mimic or MSC-AS1 vector. (b) miR-325 mimic suppressed cell proliferation, while MMSC-AS1 impaired the inhibitory effect of miR-325. (c, d) miR-325 mimic suppressed cell migration and invasion, while MMSC-AS1 impaired the inhibitory effect ∗∗ of miR-325. p< 0.01. 2.0 miR-149-5p MSC-AS1 vector miR-NC mimic + miR-325 mimic 1.5 TRIM14 1.0 ∗∗ ∗∗ GAPDH 0.5 0.0 NC miR-325 MSC-AS1 vector mimic + miR-325 mimic (a) (b) Figure 6: MSC-AS1 accelerated TRIM14 expression by sponging miR-325 in CRC cells. (a) miR-325 mimic reduced the mRNA expression of TRIM14, while MMSC-AS1 impaired the inhibitory effect of miR-325. (b) miR-325 mimic reduced the protein expression of TRIM14, ∗∗ while MMSC-AS1 impaired the inhibitory effect of miR-325. p< 0.01. 'erefore, TRIM14 was confirmed to play a role as an References oncogene in CRC. In the current work, we confirmed that [1] H. Sung, J. Ferlay, R. L Siegel et al., “Global cancer statistics TRIM14 might be a downstream target gene of miR-325. 2020: GLOBOCAN estimates of incidence and mortality Most importantly, our findings demonstrated that MSC-AS1 worldwide for 36 cancers in 185 countries,” CA: A Cancer regulated TRIM14 expression by restraining miR-325 Journal for Clinicians, vol. 71, no. 3, pp. 209–249, 2021. expression. [2] G. Breau and U. Ellis, “Risk factors associated with young- Above all, we demonstrated that MSC-AS1 promoted onset colorectal adenomas and cancer: a systematic review tumor progression by sponging miR-325 to increase and meta-analysis of observational research,” Cancer Control TRIM14 expression in CRC. 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Journal of OncologyHindawi Publishing Corporation

Published: May 11, 2021

References