Effects Of MiR-200c Overexpression On The Expression Of AHNAK And Invasion Of Pancreatic Cancer Cell

Objective: To investigate the effects of miR-200 c overexpression on the expression of AHNAK and invasion of pancreatic cancer cell.Methods: The expression of miR-200 c was detected by real-time quantitative PCR after transfected with miR-200 c mimics and its negative control(Scramble)for 24 hours in pancreatic cancer cells SW1990 and PANC-1.Then,Transwell invasion assay was used to detect invasiveness of SW1990 and PANC-1 cells after miR-200 c overexpression.TargetScan,PicTar and miranda databases were used to predict miR-200 c target genes.The miranda database was used to predict the site of binding of miR-200 c to the 3 'untranslated region(3'UTR)of the target gene AHNAK.Construction of AHNAK3'UTR(wild-type 3'UTR)sequence and AHNAK mutated sequence(mutant 3'UTR),which were the site of binding to miR-200 c.In SW1990 and PANC-1 cells,transfection experiments were performed according to the above groups,and the changes in the activity of firefly and Renilla luciferase were examined according to the instructions of the Dual-luciferase kit,verified that the 3'-untranslated region of miR-200 c and AHNAK 3'UTR.Finally,the effect of miR-200 c overexpression on the expression of AHNAK protein in SW1990 and PANC-1 cells was detected by Western Blotting.Results: In SW1990 and PANC-1 cells,the expression of miR-200 c was significantly increased after transfected with miR-200 c mimics for 24 hours compared with the negative control group(Scramble)(P <0.05).Compared with the control group(Scramble),the invasion ability of SW1990 and PANC-1 cells was significantly decreased after overexpression of miR-200 c in human pancreatic cancer cell lines SW1990 and PANC-1.Bioinformatics results show that miR-200 c specifically binds to the AHNAK 3'UTR region.AHNAK 3'UTR(wild-type 3'UTR)and(mutant 3'UTR)target sequence was constructed thathybridizes to the miR-200 c binding site.In SW1990 and PANC-1 cells,transfection experiments were performed in the above groups.Firefly and Renilla luciferase assays were performed according to the Dual-luciferase kit instructions.Renilla luciferase activity was as an internal control.Our experimental results show that miR-200 c mimics and pmirGLOAHNAK-wt were co-transfected into SW1990 and PANC-1 cells with significantly reduced luciferase activity compared to Scramble and pmirGLO-AHNAK-wt co-transfection.However,miR-200 c mimics and pmirGLO-AHNAK-mut were co-transfected into SW1990 and PANC-1 cells with essentially unchanged luciferase activity compared to co-transfection with Scramble and pmirGLO-AHNAK-mut.The above results indicate that miR-200 c binds to the 3'UTR of the AHNAK gene.In human pancreatic cancer cells SW1990 and PANC-1,overexpression of miR-200 c significantly inhibited AHNAK protein expression compared to control Scramble(P<0.05).Conclusion: miR-200 c may play a role in inhibiting the invasion of pancreatic cancer cells by targeting the AHNAK gene.