| Objective:Gastric cancer(GC)is one of the most common malignant tumors of digestive system.Early diagnosis was thought to be improved the prognosis.The development of GC is the result of the interaction of many factors,including genetics,epigenetics and environmental factors.Epigenetics refers to changes in gene expression without changes in the DNA sequence of organisms,and this change is hereditary and can be steadily passed to future generations during cell proliferation and biological development.Current research on epigenetics includes methylation of DNA,histone modification and Micro RNAs.Micro RNAsare a class of small molecule RNAs that can identify and combine target genes to precisely regulate the development,differentiation,proliferation and apoptosis of organisms.The methylation state of Micro RNA gene promoter can affect its expression level,which in turn affects the expression of multiple target genes.And itultimately has an important influence on the occurrence and development of tumors.The miR-320 a gene is located on chromosome 8p21.3,and miR-320 a expression was decreased in a variety of hematologic and solid malignancies.However,the expression pattern,regulatory mechanism,biological function and possible target gene of miR-320 a in gastric cancer were not reported.At present,it has been found that miR-638 was downregulated in a variety of tumors,suggesting that miR-638 may act as an anti-cancer gene.However,the anti-tumor mechanism of miR-638 in gastric cancer is not yet clear.The purpose of this study is to explore the expression of miR-320 a and miR-638 in gastric cancer tissue and cell lines,and to analyze the clinicopathologic significance of miR-320 a and miR-638.We investigated the role of miR-320 a and miR-638 methylation and expression in GC cell lines and tumor tissues,the clinicopathological and prognostic values of miR-638 methylation and expression in patients with GC,the regulation of the target genes and the functional impact of ectopic miR-320 a and miR-638 expression on GC cell invasion,cell cycle,apoptosis,proliferation and angiogenesis.Method: Real-time fluorescence quantitative PC R(q RT-PCR)is used to detect the miR-320 a and miR-638 levels in gastric cancer tissue and cell lines(SGC-7901,MKN-45,BGC-823,AGS).MSP method is used to detect methylation levels in the gene promoter region of miR-320 a and miR-638.MTT is used to analyze the effect of different miR-638 and miR-320 a levels on the proliferation in gastric cancer cells,flow cytometry is used to analyze the effects of different miR-320 a and miR-638 levels on apoptosis in gastric cancer cells,transwell invasion assay is used to analyze different miR-320 a and miR-638 levels on the metastasis ability in gastric cancer cells,Wound healing assay is used fo analyze different miR-320 a and miR-638 levels on the ability of migratation in gastric cancer cells.Westernblot was used to examine the relationship between miR-320 a and miR-638 and their target genes.Result: 1.MiR-320 a expression levels in four GC cell lines(SGC-7901,MKN-45,BGC-823,AGS)were significantly down-regulated compared with levels in normal gastric mucosal epithelial cells(GES-1).miR-320 a levels were significantly downregulated in GC tissues compared with paired adjacent non-tumor tissues.Low miR-320 a expression was significantly associated with lymph node metastasis(P=0.011),well differentiated(P=0.026)and advanced TNM stage(P=0.001).2.MSP method is used to detect the methylation level of miR-320 a in tumor and healthy tissue,the results showed that high methylation state in 5/6 cases of tumor samples.The methylation level of miR-320 a in the gastric cancer cell lines(SGC-7901,MKN-45,BGC-823,AGS)is higher than that in normal gastric mucosa epithelial cells GSE-1.The expression of miR-320 a is increasedafter 5-Aza-Cd R treatment in gastric cancer cells.3.miR-320 a levels in mimics-treated cells were significantly higher than those in control cells.PBX3 expression levels were signi ficantly reduced after miR-320 a mimics treatment,indicated by western blot analysis.Cell proliferation of gastric cells was significantly reduced by miR-320 a compared with NC and pare ntal cells.The apoptosis rates of NC and mimics-treated cells indicating that restoration of miR-320 a expression significantly increased apoptosis of GC cells.The migration and invasive capacities of gastric cancer cells,assessed by Transwell assays,respectively,were significantly reduced following restoration of miR-320 a expression.miR-320 a inhibitor treatment showed the opposited effect on cell proliferation,migration,invasion and apoptosis.4.MiR-638 expression levels in four GC cell lines(SGC-7901,MKN-45,BGC-823,AGS)were significantly down-regulated compared with levels in normal gastric mucosal epithelial cells(GES-1).miR-638 levels were significantly downregulated in GC tissues compared with paired adjacent non-tumor tissues.Low miR-638 expression was significantly associated with venous invasion(P=0.038),lymph node metastasis(P=0.036),poor p T stage(P=0.040)and poor TNM stage(P=0.000).We also analyzed the association between miR-638 expression and survival duration using Kaplan-Meier analysis with the log-rank test.High miR-638 expression was significantly associated with better survival.5.DNA methylation levels in GC cell lines and tissues were analyzed by MSP.Hypermethylation and hypomethylation of PCR products were detected by electrophoresis.All four GC cell lines showed hypermethylation.Among the 104 GC tissue samples,58 samples showed hypermethylation(56%).Hypermethylation of the miR-638 gene promoter region was associated with lymph node metastasis(P=0.023),poor p T stage(P=0.007)and poor TNM stage(P=0.001).Kaplan-Meier estimation indicated that hypomethylation of miR-638 was significantly associated with better survival.6.miR-638 levels in mimics-treated cells were significantly higher than those in control cells.VEGF expression levels were significantly reduced after miR-638 mimics treatment,indicated by western blot analysis.Cell proliferation of gastric cells was significantly reduced by miR-638 compared with NC and parental cells.The apoptosis rates of NC and mimics-treated cells indicating that restoration of miR-638 expression significantly increased apoptosis of GC cells.The migration and invasive capacities of gastric cancer cells,assessed by Transwell assays,respectively,were significantly reduced following restoration of miR-638 expression.miR-638 inhibitor treatment showed the opposited effect on cell proliferation,migration,invasion and apoptosis.Conclusion: 1.MiR-320 a was significantly downregulated in gastric cancer cells and tissues and its low expression was related to lymph node metastasis,well differentiation and advanced TNM stage.2.MiR-320 a low expression might due to gene promoter hypermethylation.MiR-320 a methylation inhibition partially restores the expression level of miR-320 a in gastric cancer cel s.3.MiR-320 a may be silenced by promoter hypermethylation and functions as a tumor suppressor and play a role in inhibited tumor cell migration,invasion,proliferation,and promoted apoptosis in GC cells through targeting PBX3.4.MiR-638 was significantly downregulated in gastric cancer cells and tissues and its low expression was related to venous invasion,lymph node metastasis,poor p T stage and poor TNM stage.High miR-638 expression was significantly associated with better survival.5.MiR-638 low expression might due to gene promoter hypermethylation and hypermethylation status of miR-638 gene promoter was related to lymph node metastasis,poor p T stage and poor TNM stage.Hypomethylation of miR-638 was significantly associated with better survival.6.MiR-638 may be silenced by promoter hypermethylation and functions as a tumor suppressor and play a role in inhibited tumor cell migration,invasion,proliferation,and promoted apoptosis in GC cells through targeting VEGF. |
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