Study on the mechanism involving the apoptosis of BGC-823 strain induced by miRNA-143
10.3969/j.issn.1009-0754.2015.06.012
- VernacularTitle:微小 RNA－143诱导胃癌细胞株 BGC－823凋亡机制的探讨
- Author:
Zhonghua HUO
1
;
Xiaoyan ZHANG
;
Yan QIU
;
Feifei LIU
Author Information
1. 解放军第四五四医院
- Keywords:
Bcl-2;
BGC-823;
miRNA-143;
Apoptosis
- From:
Journal of Navy Medicine
2015;(6):502-507
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the mechanism involving the apoptosis of BGC-823 strain induced by miRNA-143. Methods Fluorometry and Western blot were used to detect Bcl-2 mRNA and the protein contents in the gastric cancer strain ( BGC-823)and normal gastric mucosa strain (GES-1).We verified the binding site by a Lnciferase reporter method .MiRNA-143-mimics, in-hibitor and NC were chemically synthesized .Bcl-2 protein levels were measured by Western blotting 48 hours after transiently transfect-ed with either miRNA-143 mimics, inhibitors, or negative control miRNAs into BGC-823 cells.Apoptosis assays were employed to eval-uate cell apoptosis in BGC-823 strain, and miRNA-143 expression levels in BGC-823 were measured by quantitative PCR ( qPCR) sim-ultaneously at different time points after transfection .Results The study successfully predicted and verified the putative miRNA-143 binding site in the 3′UTR of human Bcl-2 gene.Bcl-2 level in BGC-823 cells was significantly increased and miRNA-143 level was de-creased, as compared with that of GES-1 cells, and significant differences could be noted , when comparisons were made between the groups(P<0.01).Research results indicated that miRNA-143-mimics/miRNA-143-inhibitor and the wild type luciferase reporter gene co-transfection significantly reduced and induced the activity of luciferase .Forty-eight hours after transfection , statistical significance could be noticed, when the co-transfection group was compared with the single transfection group (P<0.05).The transfection of miR-NA-143-mimics and miRNA-143-inhibitors had no significant effects on the activity of mutant luciferase reporter gene .Statistical signifi-cance could be seen, when the co-transfection group was compared with the single transfection group (P<0.05).Furthermore, the transfection of miRNA-143-mimics and miRNA-143-inhibitor could significantly inhibit or enhance the expression of Bcl-2 protein, and statistical significance could be found , when it was compared with the control group (P<0.05).miRNA-143-mimics and miRNA-143-inhibitor transfected cells could induce changes in miRNA-143 level.Forty-eight hours after transfection , miRNA-143 level in the miR-NA-143-mimics transfected cells was obviously elevated , while that in the miRNA-143-inhibitor was significantly reduced , and statisti-cal significance could also be noted , when it was compared with the control group (P<0.05).There were no significant differences in miRNA-143 levels, when the negative control group was compared with the transfected control group (P<0.05).Results of apoptosis detection indicated that the elevated expression level of miRNA-143 could significantly induce apoptosis of BGC-823, and statistical sig-nificance could be seen, when the gene intervention group was compared with the transfected control group (P<0.05).Conclusion Through the inhibition of Bcl-2 gene expression, miRNA-143 could induce the apoptosis of the gastric cancer BGC-823 strain.
