Abstract:

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 Lipofectamine^TM 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.

Key words: Gastric neoplasm, MicroRNA-126, Target gene, A disintegrin and metalloprotease 9, Epithelial-mesenchymal transition