MiR-195 Inhibits Esophageal Cancer Cell Proliferation Via Targeting Cyclin D1 And Cdc42

Background:Esophageal cancer is a common malignant tumor originating in the epithelial or glandular mucosa of the esophagus,which is the sixth leading cause of cancer-related deaths and the seventh most common cancer in the world.Although some progress has been made in the diagnosis and treatment of esophageal cancer in recent years,the therapeutic effect of esophageal cancer is still unsatisfactory,and the 5-year survival rate is only about 10-15%.Therefore,the study on the mechanism of the occurrence and development of esophageal cancer provides experimental basis for finding new therapeutic targets to improve the prognosis of esophageal cancer in clinic.miRNA is a highly conserved endogenous non-coding small RNA,which is considered to be an important factor in regulating the expression level of post-transcriptional genes.Previous studies have shown that mir-195 is abnormally expressed in gastric cancer,liver cancer,colorectal cancer and other gastrointestinal tumors,and extensively regulates important physiological and pathological processes such as cell proliferation,differentiation,migration and apoptosis.Recent study has shown that mir-195 can target Cdc42 3'UTR,inhibit the expression of Cdc42 and Cyclin D1,inhibit cell cycle transition from G1 to S,and inhibit the proliferation,invasion and metastasis of esophageal cancer,but the specific mechanism is still unclear.This study was to investigate the effect of mir-195 and its target gene Cyclin D1 on the proliferation of esophageal carcinoma cellsMethods:1.qPCR was used to detect the expression level of miR-195 in esophageal cancer cell lines,esophageal cancer tissues and paired adjacent tissues,and the relationship between its expression level and the clinicopathological characteristics of the patients was analyzed.2.The effect of miR-195 transfection mimics and inhibitors on the proliferation of esophageal cancer cells was detected by MTT assay,cloning formation assay and flow cytometry3.Expression level of CCND1 mRNA in esophageal cancer and paracancer tissues were detected by qPCR,and its correlation with miR-1 95 expression level was analyzed4.The downstream target genes of miR-195 were verified by qPCR and Western bolt through biological information prediction5.The effect on esophageal cancer proliferation induced by miR-195 was detected by MTT assay,cloning formation assay and flow cytometry after restoring Cyclin D16.The effect on esophageal cancer proliferation induced by miR-195 was detected by MTT assay,cloning formation assay and Western bolt after restoring Cyclin D1 and Cdc42.Result:1.miR-195 expression was down-regulated in esophageal squamous cell carcinoma tissues and cell lines.The decreased expression level was associated with later disease stage,worse differentiation,intraspinal tumor thrombus and nerve invasion.2.Compared with normal esophageal epithelial cells,the expression level of miR-195 in esophageal cancer cells was significantly down-regulated,among which the expression levels of ECA109 and TE-8 were the lowest.3.Transfection of miR-195 mimics inhibited the cell proliferation of esophageal cancer cell lines ECA109 and TE-8 while transfection of miR-195 inhibitor enhanced the proliferation.4.Cyclin D1 is the target gene of miR-195.5.Co-transfection of miR-195 mimics with Cyclin D1 significantly increased the proliferation of esophageal cancer cells.Co-transfection of mir-195 inhibitor and Cyclin D1 further increased the proliferation of esophageal cancer cells6.Co-transfection of Cyclin dl-wt/mut and Cdc42 showed differences in reversing the expression of corresponding proteins which was suppressed by miR-195,but it showed no differences in effect of proliferation of esophageal cancer cells.Conclusion:1.miR-195 expression was down-regulated and Cyclin D1 expression was increased in esophageal cancer tissues,showing a negative correlation.2.miR-195 can promote cell cycle arresting in G1 phase and inhibit the proliferation of esophageal cancer cells by targeting Cyclin D1 3'UTR3.Cyclin D1 can reverse the effect on the proliferation of esophageal cancer cells induced by miR-195.4.Cyclin D1 can competitively bind miR-195 with Cdc42,reducing the inhibitory effect of miR-195 on the latter.At the same time,increased expression of Cyclin D1 inhibited the expression of Cdc42 by negative feedback.5.miR-195 inhibits esophageal cancer cell proliferation via targeting cyclin D1 and Cdc42 respectively.