Effect Of SERPIN A3 Targeting By MiR-944 On Proliferation Of Ges1 Cells

Objective: The previous studies of our group found that SERPIN A3 was lower expressed in gastric cancer tissues,which could inhibit tumor growth.This study will aim to explore the regulatory mechanism of SERPIN A3 expression in gastric cancer,and further to observe the mechanism of its effect on the proliferation of gastric cancer cells,so as to provide experimental basis for elucidating the molecular mechanism of early occurrence and development of gastric cancer.Methods:1.The miRNAs targeting SERPIN A3 gene were analyzed by online bioinformatics software.2.The expression of miR-944 was detected by Real time RT-PCR in immortalized gastric epithelial GES-1cells.3.The 3’-UTR binding site of miR-944 and SERPIN A3 was predicted by Targetscan software.4.Mi R-944 mimics(miR-control as control)were transfected into GES-1 cells,and the binding of miR-944 with SERPIN A3 was detected by luciferase reporter gene detection system.5.Mi R-944 mimics were transfected into GES-1 cells,and the expression level of SERPIN A3 was detected.6.After miR-944 mimics(miR-control as control)was transfected into GES-1 cells,the effects of miR-944 expression on the growth and proliferation of GES-1 cells were detected and observed by cell growth curve and cell plate clone formation experiment.7.The effect of miR-944 mimics(miR-control as control)on Wnt1/β-catenin signaling pathway was detected by Western blotting in GES-1 cells.Results: 1.The results of bioinformatics analysis showed that miR-944 was targeted to SERPIN A3.2.The results of real time RT-PCR showed that miR-944 was down regulated in GES-1 cells(P<0.05).3.There was a binding site between miR-944 and SERPIN A3 3 ’-UTR.4.The results of luciferase reporter gene assay showed that the luciferase activity of GES-1 cells transfected with miR-944 mimics(miR-control as control)decreased significantly.5.After miR-944 mimics(miR-control as control)was transfected into GES-1 cells,the results of real-time PCR and Western blotting showed that the expression levels of SERPIN A3 mRNA and protein were both decreased(P<0.05).6.After miR-944mimics(miR-control as control)was transfected into GES-1 cells,the cell growth curve showed that the growth and proliferation rate of GES-1cells were significantly increased.The results of plate clone formation experiment results showed that the number of clones was significantly increased,and the clone volume became larger,indicating that the proliferation ability of GES-1 cells was enhanced(P<0.05).7.After miR-944 mimics(miR-control as control)was transfected into GES-1cells,the results of Western blotting showed that the expression of Wnt1 and β-Catenin proteins were increased,but the phosphorylation level ofβ-Catenin protein was decreased(P< 0.05).Conclusions: miR-944 can inhibit the expression of SERPIN A3,and activate Wnt1/β-catenin pathway to promote the cellular proliferation in GES-1 cells.