Effect of miR-34a-5p on apoptosis of gastric adenocarcinoma

ZHU Jinfeng; ZENG Wei; WANG Haijiang

doi:10.3969/j.issn.1002-266X.2017.36.006

Abstract：Objective To investigate the effect and mechanism of miR-34a-5p on the apoptosis of gastric adenocarcinoma.-Methods ①Bioinformatics tools were used to predict the potential target gene of miR-34a-5p.②Normal gastric mucosa epithelial cells RGM-1 and gastric adenocarcinoma cells SGC7901 were cultured,and qRT-PCR was performed to detect the expression of miR-34a-5p and Bcl-2 mRNA in these two cell lines.③SGC7901 cells were assigned into two groups:the observation group was transfected with miR-34a-5p mimic plasmid and the control group with scramble plamid for 48 h,and then qRT-PCR was performed to detect Bcl-2 mRNA,and Western blotting was used to detect the Bcl-2 protein.④SGC7901 cells were assigned into three groups:the negative control group was co-transfected with miR-34a-5p mimic and pRL-TK;the Bcl-2 WT group was co-transfected with miR-34a-5p mimic,Bcl-2 WT plasmid,and pRL-TK;the Bcl-2 MT group was co-transfected with miR-34a-5p mimic,Bcl-2 MT plasmid,and pRL-TK.The relative luciferase activity was measured by dual luciferase reporter assay at 48 h.⑤SGC7901 cells were assigned into four groups:the negative control group was co-transfected with miRNA scramble and pcDNA3.1 empty vector,the miR-34a-5p mimic group was transfected with miR-34a-5p mimic,the pcDNA3.1-Bcl-2 group was transfected with pcDNA3.1-Bcl-2,and the miR-34a-5p mimic + pcDNA3.1-Bcl-2 group was co-transfected with miR-34a-5p mimic and pcDNA3.1-Bcl-2.The apoptotic rate of the four groups was measured by flow cytometry after 48 h of transfection.Results ①Bcl-2 was predicted to be the potential target gene of miR-34a-5p.② Compared with RGM-1 cells,SGC7901 cells had relatively low expression of miR-34a-5p and high expression of Bcl-2 mRNA (both P ＜ 0.01).③ The relative expression of Bcl-2 mRNA and protein in the observation group was lower than that in the control group (P ＜ 0.01).④The relative luciferase activity of the Bcl-2 WT group was lower than that of the negative control group and Bcl-2 MT group (P ＜ 0.01).⑤The apoptotic rate of the miR-34a-5p mimic group was higher than that of the negative control group,and the apoptotic rates of the pcDNA3.1-Bcl-2 group and miR-34a-5p mimic + pcDNA3.1-Bcl-2 group were lower than those of the negative control group and miR-34a-5p mimic (P ＜ 0.01).Conclusion The miR-34a-5p can inhibit the apoptosis of gastric adenocarcinoma by up-regulating the expression of Bcl-2 and thus be involved in the occurrence and development of gastric adenocarcinoma.