miR-126过表达和ADAM9基因沉默对胃癌SGC-7901细胞生物学行为的影响及其机制OA北大核心CSTPCD

Objective:To discuss the effects of microRNA-126(miR-126)over-expression and disintegrin and metalloproteinase 9(ADAM9)gene silencing on the biological behavior of the gastric cancer cells,and to clarify the mechanism.Methods:The human poorly differentiated gastric adenocarcinoma SGC-7901 cells and human normal gastric mucosa epithelial NGEC cells were cultured in vitro.The total RNA was extracted from the cells,and the expression levels of miR-126 and ADAM9 mRNA in both types of cells were detected by real-time fluorescence quantitative PCR(RT-qPCR)method.The SGC-7901 cells at logarithmic growth phase were divided into miR-126 over-expression group(miR-126-OE group)and ADAM9 gene silencing group(ADAM9 siRNA group).The transfection with miR-126 mimics(miR-126)mimics and ADAM9 RNA oligonucleotides were conducted by LipofectamineTM 2000,and the corresponding negative control group was established;MTT assay was used to detect the proliferation activities of the cells in various groups;cell wound assay was used to detect the migration rate of the cells in various groups;Transwell chamber assay was used to detect the the numbers of migration and invasion cells in various groups;Western blotting method was used to detect the expression levels of E-cadherin,N-cadherin,and vimentin proteins in the cells in various gorups.The miR-126 target genes were predicted by TargetScan website,and the targeting regulatory relationship between miR-126 and ADAM9 was confirmed by dual-luciferase reporter assay.The expression levels of ADAM9 mRNA and protein in the SGC-7901 cells after transfected with miR-126 mimics were detected by RT-qPCR and Western blotting methods.Results:The RT-qPCR results showed that compared with human normal gastric mucosa epithelial NGEC cells,the expression level of miR-126 in the gastric cancer SGC-7901 cells was significantly decreased(P<0.05),while the expression level of ADAM9 mRNA was significantly increased(P<0.05).The MTT assay results showed that after 48 and 72 h of over-expressing miR-126 or silencing the ADAM9 gene in the SGC-7901 cells,compared with the corresponding negative control group,the proliferation activities of the cells in both miR-126-OE and ADAM9 siRNA groups were significantly decreased(P<0.05 or P<0.01).The cell wound assay results indicated that compared with the corresponding negative control group,the migration rates of the cells in both miR-126 OE and ADAM9 siRNA groups 48 h after transfection were significantly decreased(P<0.05).The Transwell chamber assay results showed that the numbers of migration and invasion cells in both miR-126-OE and ADAM9 siRNA groups were significantly lower than those in corresponding negative control group(P<0.05 or P<0.01).The Western blotting method results showed that compared with the corresponding negative control groups,the expression level of E-cadherin protein in the cells in miR-126-OE and ADAM9 siRNA groups were significantly increased(P<0.05 or P<0.01),while the expression levels of N-cadherin and vimentin proteins were significantly decreased(P<0.05 or P<0.01).The target prediction results showed that the 3′-UTR of ADAM9 contains nucleotide sequences complementary to miR-126-3p.The dual-luciferase reporter assay results showed that ADAM9 was a downstream target gene negatively regulated by miR-126.Compared with mimics NC group,the expression levels of ADAM9 mRNA and protein in the SGC-7901 cells after transfected with miR-126 mimics for 48 h were decreased(P<0.05 or P<0.01).Conclusion:The gastric cancer SGC-7901 cells are characterized by low expression of miR-126 and high expression of ADAM9 gene.Over-expression of miR-126 can inhibit the proliferative activity,migration,and invasion capabilities of the gastric cancer SGC-7901 cells;the mechanism may be related to the negative regulation of ADAM9 by miR-126 and the inhibition of epithelial-mesenchymal transition(EMT)process in the gastric cancer cells.