Mir-137 Identified By Gain-of-function Screening Inhibits Proliferation Of Gastric Cancer Cells

【Background】MiRNAs is a hot research field of molecular biology in recent years. Functional studies suggest that miRNAs take active parts in the regulation of almost every cellular process investigated up to now and that changes in their expression profiles are observed in human pathologies, including cancer. They have emerged as key post-transcriptional regulators of gene expression in mammals. There is a growing body of evidence to indicate that miRNAs play a crucial role in carcinogenesis, and act as oncogenes or tumor suppressors in function.Gastric cancer is the second most prevalent cause of malignant tumor related death in the world. The highest incidence is in northeast Asia, where Japan, Korea, and China located in. Gastric cancer is a terrible disease to Chinese. Up to date, efficient targets of anti-tumor therapy. MiRNAs, whose potential role in gastric cancer is unclear, provide us new hopes to cure gastric cancer. In this study, we shall investigate the function of miRNAs in gastric cancer proliferation, which is the principle malignant phynotype of gastric cancer.【Aims】To find miRNAs that inhibit proliferation of gastric cancer cell based upon high throughput gain-of-function screening; validate the effect of key miRNA in inhibiting gastric cancer cells; investigate the possible mechanism of the key miRNA inhibiting gastric cancer cell proliferation; evaluate the relationship between the expression of key miRNA in gastric cancer tissues and prognosis in patients; and provide a novel approach inhibiting gastric cancer cell proliferation on the post-transcriptional level.【Methods】1. High throughput and gain-of-function screening was performed based on the principle phenotype of gastric cancer cell line SGC 7901, using miR-Ribo? miRNAs mimics Library (Human) v14.0 version (volume: 904 human miRNAs mimics and aided by RT-CES system. We obtained twelve miRNAs that inhibited the proliferation of SGC 7901 cell and especially miR-137 that played the pivotal role in inhibition of cancer proliferation.2. miRNA-137 levels were assessed by real time RT-PCR, the objects consisting of human gastric cancer cell lines (SGC7901,AGS,MKN45), immortal human gastric mucous epithelial cell GES-1, eleven primary gastric adenocarcinoma tissues and their pair-matched nontumor tissues from patients undergoing gastric resection.3. In vitro experiments, including MTT assay,plat clone formation assay,and anchorage-independent growth assay in soft agar were performed to validate that miR-137 had an effect on proliferation of gastric cancer cells, accompanying with upregulation of miR-137 expression in SGC7901and AGS.4. We constructed lentivirus vector of overexpressing miR-137, and established stable cell line LV-SGC7901 transfected with the above lentivirus vector. Then LV-miR-137 SGC7901 cells or LV-Cont SGC7901 cells were inoculated subcutaneously into the left and right flanks of nude mice, respectively.5. We investigated miR-137 shed effect on cell cycle and apoptosis as well as DNA replication of SGC7901 and AGS cells via FACS and EdU staining, accompanying with miR-137 overexpression.6. Aided by informatics analysis, we predicted that the probable genes targeted by miR-137 and classified them based on functions. Then according to previous studies and pre-experimental results, we concluded that probably miR-137 targeted CDK6.7. We detected the expression of miR-137 and CDK6 in three identical primary gastric adenocarcinoma tissues as well as normal mucous tissues as parallels via real time RT-PCR and Western Blot, respectively.8. The CDK6 mRNA and protein levels were determined by real time RT-PCR and Western Blot in SGC7901 and AGS cells transfected with miR-137 mimics or that in GES-1 cells transfected with miR-137 inhibitor. We constructed dual luciferase reporter system containing wild type or mutate CDK6 3,UTR, then cotransfected with miR-137 mimics, ultimately tested luciferase activity changes. 9. We investigated miR-137 had an effect on proliferation and cell cycle of SGC7901 and AGS cells via MTT and FACS, accompanying with CDK6 downregulation by siCDK6. 10. We tested miR-137 levels in thirty eight primary gastric adenocarcinoma tissues embedded in paraffin, and made statistical analysis by linking its expression with pathological characteristics and prognosis.【Results】1. Twelve miRNAs molecules which significantly inhibited cell proliferation of gastric cancer were obtained using the high-throughput screening technique. Among those miRNAs, miR-137 may play the key inhibitory effect.High throughput and gain-of-function screening was performed based on the principle phenotype of gastric cancer cell line SGC 7901, using miR-Ribo? miRNA mimics Library (Human) v14.0 version (volume: 904 human miRNAs mimics and aided by RT-CES system. We obtained twelve miRNAs that inhibited the proliferation of SGC 7901 cells, including miR-30b,miR-137,miR-363,miR-124*,miR-15a,miR-149,miR-15b*,miR-376b,miR-571,miR-622,miR-339-3p,miR-1321. Among them, miR-137 especially played the pivotal role(Z Score=-3.84,Relative growth rate=0.51).2. The expression of miR-137 was significantly down-regulated in gastric cancer cell lines and primary gastric adenocarcinoma tissues.The relative expression of miR-137 was found to be significantly lower in the primary gastric adenocarcinoma tissues compared to their pair-matched nontumor tissues. To confirm the association between miR-137 expression and gastric cancer, we examined miR-137 expression in SGC7901 and AGS using real time RT-PCR. The results showed that miR-137 expression was substantially reduced in SGC7901 and AGS compared with GES-1.3. The upregulation of miR-137 significantly inhibited cell proliferation of gastric cancer in vitro and in vivo, leading to cell cycle G1/S arrest.To investigate the role of miR-137 in gastric carcinogenesis, we examined the effect of miR-137 overexpression on the proliferation of SGC7901 and AGS. The cells were transfected with miR-137 mimics or NC. Phase-contrast microscopy, MTT assays , plat clone formation assay,and anchorage-independent growth assay in soft agar showed that overexpression of miR-137 significantly inhibited the proliferation of both AGS and SGC7901. To test the potential effect of miR-137 on gastric cancer cell proliferation in vivo, we constructed lentivirus vector of overexpressing miR-137, and established stable cell line LV-miR-137 SGC7901 transfected with the above lentivirus vector. LV-miR-137 SGC7901 cells or LV-Cont SGC7901cells were injected subcutaneously into both flanks of athymic nude mice. A month later, the mean wet weight of tumors with LV-miR-137 SGC7901 was significantly lower than that of LV-Cont miR-137, indicating that miR-137 may inhibit xenograft tumor formation of gastric cancer cells in vivo. Thus, these data suggested that miR-137 may play a crucial role in the proliferation of gastric cancer cells in vitro. To further explore the role of miR-137 in apoptosis, overexpression of miR-137 in SGC7901 and AGS resulted in significantly G1/S arrest compared with NC.4. Bioinformatics data suggests that CDK6 might be the target gene of miR-137.Bioinformatics analyses revealed that CDK6, predicted by both TargetScan, PicTar, miRanda and NAhybrid, was a potential target of miR-137. Moreover, CDK6 is a cell-cycle regulator. Thus, CDK6 might be the target gene of miR-137.5. The expressions of miR-137 and CDK6 were negatively correlated in gastric cancer tissues and normal mucosa.Bioinformatics data suggests that CDK6 might be the target gene of miR-137. We detected the expression of miR-137 and CDK6 in three identical primary gastric adenocarcinoma tissues as well as normal mucous tissues as parallels. The results indicated that miR-137 expression was correlated reciprocally with CDK6.6. miR-137 negatively regulated the expression of CDK6.We further assessed the effect of miR-137 on CDK6 protein level and RNA level by performing western blot and real time RT-PCR analysis in gastric cancer cell line SGC7901 and AGS,which was known to low express miR-137. Overexpression of miR-137 mimics in SGC7901 and AGS resulted in significantly reduced CDK6 protein expression level and CDK6 mRNA expression level. In contrast, silencing of miR-137 using miR-137 inhibitor in GES-1 cells resulted in marked up regulation of CDK6 protein level and CKD6 mRNA level. These data indicated that miR-137 regulated CDK6 expression in gastric cancer both at the RNA and protein levels. Further more,we constructed dual luciferase reporter system containing wild type or mutant CDK6 3′UTR, psiCHECK2-CDK6 and psiCHECK2-CDK6-mut. Then cotransfected with miR-137 mimics into SGC7901 respectively,and ultimately tested luciferase activity changes. The luciferase activity of SGC7901 transfected with miR-137 mimics and psiCHECK2-CDK6/ psiCHECK2-CDK6-mut1(bases 7133-7139)/psiCHECK2-CDK6-mut2(bases 7114-7120) was significantly decreased compared with the lucCtrl and psiCHECK2-CDK6-mut3(bases 7133-7139 and 7114-7120). So, these data suggested that miR-137 might inhibit CDK6 through interaction between miR-137 and 3′UTR of CDK6 in bases 7114-7120 and/ or bases 7133-7139.7. miR-137 inhibited gastric cancer cell proliferation through negatively regulating the expression of CDK6.To investigate the role of CDK6 in gastric proliferation, we examined the effect of CDK6 on the proliferation of SGC7901 and AGS. The cells were transfected with siCDK6 or NC, MTT assays showed that downregulation of CDK6 significantly inhibited the proliferation of both AGS and SGC7901. Furthermore, downregulation of CDK6 in SGC7901 and AGS resulted in significantly G1/S arrest compared with NC. Importantly, downregulation of CDK6 in gastric cancer cells could inhibit proliferation and block G1/S transition, which was consistent with the effect of miR-137 overexpression in SGC7901 and AGS. Summarily, these findings indicate that miR-137 may mediate its antiproliferative or tumor suppressor-like activity at least in part by altering cell-cycle progression via inhibiting expression of CDK6.8. The expression level of miR-137 in primary gastric adenocarcinoma was significantly correlated to clinical pathological features and prognosis.To further assess the signature of miR-137 in prognosis of gastric cancer patients, we tested miR-137 levels in thirty eight primary gastric adenocarcinoma tissues embedded in paraffin, and made statistical analysis by linking its expression with pathological characteristics and prognosis. The data indicated that the expression level of miR-137 in primary gastric adenocarcinoma was significantly correlated to clinical pathological features and prognosis. The median survival of patients in miR-137 high expressers(n=19)was significant better than that in miR-137 low expressers (n=19)(Log Rank=6.981,P=0.008). 【Conclusion】Based upon high-throughput screening on phenotype of gastric cancer cell proliferation, we for the first time identified a miRNAs group that suppresse gastric cancer cell proliferation, in which miR-137 may play a key role. MiR-137 leads to G1/S arrest through negatively regulating the expression of CDK6, providing a novel approach that inhibits gastric cancer cell proliferation on the post-transcriptional level. Thus, miR-137 may be regarded as a new target of gastric cancer treatments. Since miR-137 is correlated with the prognosis of gastric cancer, it would also be able to act as a warning molecule for gastric cancer.