miR-335-5p inhibits the proliferation of gastric cancer cells by tagerting Oct4

Abstract:

Objective：To investigate the effect of miR-335-5p on the proliferation of gastric cancer cells and its underlying mecha－nism. Methods：The expression of miR-335-5p in gastric cancer cell lines BGC-823，MKN-28，MKN-45 and normal gastric mucosal epithelial cell line GES-1 were tested by qRT-PCR. SGC-7901 and BGC-823 cell lines were transfected by overexpression and antagonist lentivus respectively and the experimental groups were divided into：miR-335-5p group（transfected with miR-335-5p overexpression lentivus） and negative control group（transfected with empty lentivus，NC） in SGC-7901 cell line，and antago-miR-335-5p group（transfected with antagonist lentivus） and negative control group（transfected with empty lentivus，NC） in BGC-823 cell line. The effect of miR-335-5p on the proliferation of gastric cancer cells was tested by CCK-8 assay，colony formation assay and EdU assay. The target gene of miR-335-5p was predicted by bioinformatics software. Western blot was selected to test the expression level of Oct4，AKT and pAKT. Results：The expression level of miR-335-5p was downregulated in gastric cancer cell lines compared with that in normal gastric mucosal epithelial cell line. CCK-8 assay，colony formation assay and EdU assay showed that the absorbance（A） value，colony formation number and EdU positive cell in overexpression miR-335-5p group were lower than those in NC group while antago-miR-335-5p group had higher A value，colony formation number and EdU positive cell than NC group. Oct4 was pre－dicted as a target gene of miR-335-5p. The protein expression level of Oct4 was lower in miR-335-5p group（0.469±0.054） than in NC group（0.670±0.045）（P=0.008）；the protein ex－pression level of Oct4 was higher in antago-miR-335-5p group（0.804±0.046） than in NC group（0.600±0.073）（P=0.015），and dual luciferase reporter assay was conducted to prove that miR-335-5p could target Oct4. The expression level of pAKT was lower in overexpression miR-335-5p group（0.525±0.046） than in its NC group（0.847±0.053）（P=0.001）；the expression level of pAKT was higher in antago-miR-335-5p group（0.841±0.069） than in NC group（0.563±0.063）（P=0.007）. Conclusion：miR-335-5p works as a tumor suppressor to inhibit the proliferation of gastric cancer cells by targeting Oct4 to inhibit the AKT pathway.