| Background : Gastric cancer is one of the most common malignant tumors in the world.Early diagnosis and treatment can improve the prognosis,but the early symptoms are not obvious.Because of the lack of effective early diagnostic markers,most of the patients were in advanced stages.Therefore,the discovery of effective molecular markers of gastric cancer is an effective way to improve the condition.The development of gastric cancer is influenced by a variety of factors,including epigenetics.Epigenetic regulation refers to a mutation that does not alter the DNA sequence,but is a genetic phenotype,including DNA methylation,histone modification,non-coding RNA regulation,etc.Micro RNA(mi RNA)is a kind of small molecule RNA,which regulates the differentiation,proliferation and apoptosis of organisms.In the process of tumor formation and development,mi RNA can play a role in tumor suppressor gene or oncogene in different tissues and cells.In recent years,the methylation of the promoter region of mi RNA was found to affect its expression.Moreover,mi RNA plays a vital role in the development of tumor.Mi R-634 has been found to be abnormal in some malignancies,including ovarian cancer and cervical cancer,which are closely related to the development of tumor,blood vessel formation and invasion and metastasis.However,no relevant reports have been reported in gastric cancer.The purpose of this study was to explore the expression levels of mi R-634 in gastric cancer tissues and cell lines.The effects of expression and down-regulation of mi R-634 expression on gastric cancer cell line SGC-7901,MGC-803,AGS proliferation,migration and invasion were studied.The purpose of this study was to investigate the hypermethylation status of mi R-634 gene promoter region.And the effect of mi R-634 and its target gene JAG1 on gastric cancer metastasis in vitro and in vivo.Materials and Methods:83 cases of fresh gastric carcinoma tissue and paired tissue adjacent to carcinoma from January 2014 to January 2016 in the fourth hospital affiliated to medical university and China university of medical sciences tumor research institute.The study was approved by the medical ethics committee of the Fourth Affiliated Hospital of China Medical University,and all patients provided informed consent.The mi R-634 mimics and mi R-634 inhibitors were transfected by lentivirus into human gastric cancer SGC-7901 and MGC-803 cells,and the mi R-634 cells without transfection were used as the control group(NC group).Quanitative real-time Polymerase Chain Reaction PCR(q RT-PCR)was used to detect gastric cancer cell lines and normal gastric mucosa cell lines(GES-1)and 83 cases of gastric cancer and adjacent tissue.Methylation specific PCR(MSP)was used to detect the methylation status of mi RNA gene promoter region in gastric cancer cells and gastric cancer tissues,and drug intervention was carried out with methylated drug 5-aza-2-deoxycytidine(5-aza-dc).Cell viability was measured by the CCK8 assay.The migration and invasion ability of the cells were detected by scratch assays and Transwell? chamber assays,respectively.The cell proliferation was detected by plate cloning experiment.The luciferase assay verified the binding of mi R-634 to the target gene JAG1.q RT-PCR and western blotting assays were used to detect the expression of mi R-634 and JAG1,the EMT,and Notch signaling pathway-related proteins(JAG1,Notch1,Notch2,E-cadherin,and N-cadherin).In vivo experiment,we constructed AGS that expressed mi R-634 and stabilized the cell lines,and established the subcutaneous transplanted tumor and peritoneal metastasis model of nude mice.The effect of expression of mi R-634 on tumor growth and metastasis in mice was analyzed,and the tissue section was observed by hematoxylin(H&E).Results:1.The expression level of mi R-634 in gastric cancer cell lines was significantly lower than that in normal human gastric mucosa.The expression level of mi R-634 in 83 cases of gastric cancer was significantly lower than that in normal tissues.Low mi R-634 expression was significantly correlated with tumor size(P=0.029).In the OS analysis,patients with high mi R-634 expression in GC patients had longer survival time than those with low expression of mi R-634(P = 0.038).2.5-aza-d C can reverse the methylation of mi R-634 gene and restore the expression of mi R-634.The cell lines of gastric cancer were highly methylated without 5-Aza-d C treatment,and the gastric cancer cell lines were non-methylated after 5-Aza-d C treatment.The abnormal methylation of mi R-634 gene promoter region may be one of the important mechanisms for their expression in gastric cancer cells.In addition,the incidence of methylation of mi R-634 gene promoter region in gastric cancer tissues was significantly higher than that of the corresponding adjacent tissues.3.JAG1 is highly expressed in gastric cancer tissues and cells.Moreover,JAG1 is the direct target gene of mi R-634.There was no significant correlation between JAG1 expression and age(P=0.496),gender(P=0.794)or stage(P=1.000),but significantly correlated with tumor size(P=0.026).RFS analysis suggested that the high mi R-634 expression group had a lower recurrence rate than the mi R-634 group(P =0.012).4.Mi R-634 inhibited the proliferation,invasion and metastasis of gastric cancer cells.Mi R-634 expressed in AGS cells increased the expression level of E-cadherin and decreased the expression level of N-cadherin.Subcutaneous injection of AGS cells from the Lv-mi R-634-mimics and the non-lentivirus-mi R-634(NC group)was given to the nude mice.There were significant differences in tumor size between the two groups(p<0.05).Intraperitoneal injection of nude mice showed liver metastasis in the NC group,while no distant liver metastasis occurred in mice with mi R-634-mimics injection group.Conclusions:The expression level of mi R-634 in gastric cancer tissues and cell lines was significantly lower than that in normal adjacent tissues and control cells.The survival of cells was significantly decreased,and number of cells migrating and invading was decreased in the mi R-634 mimics group.However,in the mi R-634 inhibitor group,the opposite results were observed.Over-expression of mi R-634 inhibited the proliferation,migration,and invasion of gastric cancer cell lines,and the mi R-634 target gene was JAG1.Compared with the NC cells,the mi R-634 mimic-treated cell group showed inhibited cell proliferation,migration,invasion,and EMT.Additionally,overexpression of mi R-634 inhibited the growth and metastasis of tumors in vivo.Taken together,our data showed that mi R-634 was associated with GC EMT,and regulated the Notch signaling pathway directly through its downstream target gene JAG1. |
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