The Role Of MiRNA-375 In Gastric Cancer

miRNAs are a new class of naturally occurring small noncoding, single-stranded RNAs of 19-25 nucleotides that negatively regulate genes by triggering either mRNAs degradation or translational repression through either perfect or imperfect binding to the 3'-untranslated region (3'-UTR) of their target mRNAs. Plenty of studies have demonstrated that miRNAs could contribute to most, if not all, basic biological processes, such as development, cell proliferation, apoptosis, migration and invasion. An increasing number of studies make it clear that miRNAs are associated with various diseases, including cancers. Emerging evidence has showed the indeed association of aberrantly expressed miRNAs with solid tumor and blood tumor development and progression. To explore the potential mechanism of miRNAs dysregulation, scientists further study downstream targets of miRNAs and reveal common miRNA-driven pathways. It is clear that miRNAs may function as potential oncogenes or tumor suppressors, and alteration in miRNA expression may play a critical role in tumorigenesis and cancer progression. Identification of cancer specific miRNAs and their targets is critical for understanding their roles in carcinogenesis and may be important for defining novel therapeutic targets.Although some miRNAs that are associated with gastric cancer have been identified to date, the role of many of them in stomach tumorigenesis and the underlying mechanism remain to be determined. China appears to have both relatively high incidence rate and mortality rate of gastric cancer in the world. The estimated cases of gastric cancer is 755,000 pre year worldwide and about 35% of them is occurred in China. Otherwise, the mechanism of gastric cancer has not been completely understood. And the 5-year survival rate of gastric cancer is very low. To improve survival, it would be desirable to develop biomarkers that could facilitate the diagnosis of gastric cancer in early stage. In this study, we firstly screened differently expressed miRNAs between gastric cancer tissues and adjacent non-cancer tissues by microarray and found that miR-375 was one of the most frequently down-regulated miRNAs in gastric cancer tissues. Expression level of miR-375 in gastric cancer was further validated by quantitative Real-time PCR. We further found that miR-375 was able to significantly inhibit gastric cancer cell proliferation, migration and invasion. And the downstream target of miR-375 was predicted and validated to explore the potential mechanism of miR-375 in gastric cancer. Thus, our study may shed light on potential pathogenesis of gastric cancer and may help to develop gastric cancer targeted therapies.Materials and Methods1. Samples and cell culture Gastric epithelial cell lines were maintained at 37℃in an atmosphere of 5% CO2 in medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Pair-matched tumorous and adjacent non-tumorous gastric tissues from 48 patients undergoing resection for gastric cancer were obtained from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (Hangzhou, China). All the samples were divided into two parts. One part was immediately snapped frozen and stored in liquid nitrogen until RNA extraction. Another part was stored in formalin for pathology analysis. Gastric mucosa samples including 17 non-malignant mucosa and 17 malignant mucosa that were obtained from gastroscopy were also stored in liquid nitrogen until use.2. miRNA microarray and Quantitative Real-Time PCRMicroarray analysis was performed to obtain the miRNA expression profile in gastric cancer tissues and adjacent non-cancer tissues. miR-375 expression in gastric cell lines and gastric tissues from both gastric resection and gastroscopy was examined by quantitative Real-time PCR analysis.3. The effects of miR-375 on gastric cancer cells proliferation, migration and invasionCells were firstly transfected with miR-375 precursor or negative control using siPORTM Amine Transfection Agent following manufacturer's protocol. Cell counting, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) assay, Scratch-wound healing assay, Transwell migration assay and Matrigel invasion assay were performed post-transfection.4. The effects of miR-375 on Xenograft tumor growthMGC-803 cells transfected with either miR-375 or the empty vector were inoculated subcutaneously into the left and right flanks of nude mice, respectively. The volume of the implanted tumor was measured every two day with a vernier caliper, using the formula:V=LX W2/2; where V, volume (mm3); L, biggest diameter (mm); W, smallest diameter (mm).The mice were euthanized and the tumors were weighted three weeks after inoculation.5. Prediction and validation of miR-375 targetsBy using the algorithms TargetScan, PicTar, and miRanda, we considered Jak2 as a putative target of miR-375. Luciferase activity assay and Western blot analysis were generated to validate the relationship between miR-375 and Jak2. Jak2 effects on gastric cancer cell proliferation, migration and invasion were examined by cell counting, MTT assay, Scratch-wound healing assay, Transwell migration assay and Matrigel invasion assay separately.6. Prediction and validation of transcription factor that regulates miR-375 expressionBy using the online algorithm (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite), we considered transcription factor Snail, which has six binding sites in the sequence containing the promoter of the miR-375 gene, as a putative upstream regulator of miR-375 expression. Luciferase activity assay was generated to validate the relationship between Snail and miR-375 promoter activity.Results1. miR-375 is frequently downregulated in gastric cancer tissues and cell lines.2. Overexpression of miR-375 inhibits gastric cancer cell proliferation, migration, invasion and Xenograft tumor growth.3. miR-375 represses Jak2 protein expression though binding to 3'-UTR of Jak2 and miR-375 expression level and Jak2 protein level in gastric cancer were inversely correlated.4. Jak2 is involved in the regulation of cells proliferation, migration and invasion by miR-375.5. miR-375 is directly and negatively regulated by the transcription factor Snail.Conclusion1. miR-375 was underexpressed significantly in gastric cancer compared with their non-tumor counterparts from patients undergoing gastric resection, which was confirmed by the data from gastroscopy and gastric cancer cell lines. The downregulation of miR-375 may partially due to the negative regulation by Snail in gastric cancer. These findings indicate that miR-375 may function as tumor suppressor gene in gastric cancer.2. Overexpression of miR-375 or downregulation of Jak2 could inhibit gastric cancer cell proliferation, migration and invasion. And overexpression of Jak2 could partially or completely reverse the inhibition effect of miR-375 on cell proliferation, migration and invasion. These findings indicate that miR-375 may influence gastric caner cell proliferation, migration and invasion by downregulating Jak2 protein potentially.3. miR-375 expression was inversely correlated with Jak2 protein level in gastric cancer. Both of them might become candidate biomarkers of gastric cancer diagnosis or treatment.