The Role Of MicroRNA-596 In Gastric Cancer And Its Regulatory Mechanisms

Introduction: Globally,the incidence of gastric cancer is fifth of malignant tumors,with a mortality rate of second;According to statistics,the incidence rate of gastric cancer in China in 2013 was 13.31/100,000,second only to lung cancer.Due to the low early diagnosis rate of gastric cancer in China,it has been found to be in the advanced stage,resulting in a higher mortality rate.Therefore,it is necessary to further study the molecular mechanism of gastric cancer and find more effective biomarkers for diagnosis and treatment of gastric cancer,which play an important role in early diagnosis and treatment of gastric cancer and improve prognosis.Recent studies have shown that endogenous non-coding RNA plays an important role in individual growth,development,differentiation and the occurrence and development of malignant tumors.Many studies have found that miRNA play an important role in the pathogenesis,progression and prognosis of gastric cancer.In addition,recent studies have also shown that abnormal methylation of many micro RNAs within or near the CPG islands leads to abnormal expression of micro RNAs,which may play an important role in the regulation of tumorigenesis and development.MicroRNA-596(miR-596)is located on human chromosome 8p23.3 and is a non-coding RNA between genes.In recent years,studies have found that miR-596 plays a role in tumor suppressor gene expression in various tumors such as hepatocellular carcinoma,cholangiocarcinoma,oral cancer,endometrial cancer,bladder cancer,and pancreatic cancer.However,the abnormal regulation and mechanism of expression in gastric cancer is still unclear.Objective: This study aimed to investigate the expression level of miR-596 in gastric cancer cells and tissues,its relationship with clinicopathological characteristics and its role in the occurrence and development of gastric cancer.The effects of DNA hypermethylation on the expression of miR-596 and the downstream target genes of miR-596 were also studied.The first part was to text the expression of miR-596 in gastric cancer tissues and gastric cancer cell lines,and to analyze the relationship between clinicopathological characteristics and miR-596 expression.We observed the effects of over-expression of miR-596 on the proliferation,migration and invasion of gastric cancer cell lines MKN-45 and MGC-803 in vitro.At the same time,the methylation level of miR-596 promoter region in gastric cancer cell lines was detected,and the effects of demethylation drug 5-aza-2-deoxycytidine(5-Aza-d C)on the expression and proliferation of miR-596 in gastric cancer MKN-45 and MGC-803 cell lines were observed.The second part studies the downstream target genes regulated by miR-596,and further explores the molecular mechanism of miR-596 in the biological function of gastric cancer.Methods: 1.Real-time fluorescence quantitative PCR was used to text the expression of miR-596 in normal gastric mucosal cell lines GES-1 and four gastric cancer cell lines.Meanwhile,the expression levels of miR-596 in 55 matched pairs of gastric cancer and paracancerous tissues were detected and analyzed statistically.2.In vitro experiments,chemical synthesis of miR-596 mimics and miR-NC,using logarithmic growth phase gastric cancer cell lines MKN-45,MGC-803,respectively,according to the experimental group using Lipofectamine3000 for transfection,through CCK-8 experiments,scratch healing experiments and Transwell chamber analysis of the effect of overexpression of miR-596 on the biological function of gastric cancer cells.3.Methylation status of promoter region of miR-596 gene in normal gastric mucosal cell line GES-1 and four gastric cancer cell lines was detected by methylation-specific PCR(MSP).Different concentrations of demethylation drug 5-aza-2-deoxycytidine(5-Aza-d C)were used to treat gastric cancer cell line MGC-803 in vitro.q RT-PCR was used to detect the change of miR-596 expression level,and MSP detected the methylation status of miR-596 promoter region.At the same time,the effect of proliferation on gastric cancer cells was detected by CCK-8 assay.4.Using database software for miR-596 potential target gene prediction;The expression of PRDX1 in gastric cancer tissues and cells was detected by q RT-PCR and Western blot,and the expression of target genes was detected after transfected with miR-596.The dual luciferase reporter assay then further identified the target gene binding site;The expression of PRDX1 was silenced by interfering RNA assay;The effects of silencing PRDX1 on the biological functions of gastric cancer cell proliferation,migration and invasion were researched by CCK-8 assay,scratch healing assay and Transwell chamber assay;Finally,the gastric cancer cell lines MKN-45 and MGC-803 were treated with 5-Aza-2-deoxycytidine(5-Aza-d C),and the expression of PRDX1 was detected by q RT-PCR and Western blot.Result: 1.The results of qRT-PCR showed that compared with GES-1,the expression of miR-596 in 4 gastric cancer cell lines decreased in varying degrees,and the expression level of miR-596 in 55 gastric cancer tissues was obviously lower than that in paired adjacent tissues(P < 0.001),the low expression of miR-596 is closely related to the degree of differentiation and TNM stage of tumor cells.2.In vitro transfection of 50 n M miR-596 mimics to gastric cancer cell lines MKN-45 and MGC-803,CCK-8 results showed that overexpression of miR-596 inhibited gastric cancer cell proliferation;scratch healing experiments and Transwell chamber experiments showed miR-596 overexpression inhibits migration and invasion of MKN-45 and MGC-803 cells.3.The results of MSP showed that miR-596 showed methylation bands with different brightness in the promoter region of Cp G island in 4 gastric cancer cell lines,and the degree of methylation was correlated with its expression level;After 72 hours of intervention with5-Aza-2-deoxycytidine(5-Aza-d C),the results of MSP showed that 5-Aza-d C could reverse the hypermethylation of the miR-596 promoter region,and the expression level of miR-596 was significantly increased by q RT-PCR;At the same time,the results of CCK-8 showed that the cell proliferation activity was negatively correlated with drug concentration and duration of action.4.Through the online prediction of PRDX1 target gene prediction software may be one of the target genes of miR-596;The results of q RT-PCR and Westernblot showed that the expression level of PRDX1 in gastric cancer tissues was higher than that in the matched adjacent tissues,and the difference was statistically significant;High expression of PRDX1 is closely related to the degree of differentiation of tumor cells and Borrmann classification;The expression levels of PRDX1 at m RNA and protein levels were significantly decreased after transfection of miR-596 mimics;The dual luciferase reporter assay showed that PRDX1 is a direct binding site for miR-596;Moreover,the biological function of PRDX1 silenced by si RNA was consistent with that of miR-596 mimics;After 5-Aza-d C treatment,the expression of PRDX1 protein was significantly decreased.Conclusion: 1.miR-596 is significantly down-regulated in gastric cancer cell lines and gastric cancer tissues.Overexpression of miR-596 can inhibit the proliferation,migration and invasion of gastric cancer cells,and its low expression may be regulated by hypermethylation of its gene promoter region.2.PRDX1 is up-regulated in gastric cancer and is one of the direct regulatory targets of miR-596.