miR-193a在MDBK细胞中诱导细胞凋亡的研究

本试验旨在研究miR-193a在致细胞病变型牛病毒性腹泻病毒(cp BVDV)感染MDBK细胞过程中诱导细胞凋亡的分子机制。试验利用TargetScan和Microcosm Targets等生物信息学在线软件预测凋亡相关的miR-193a靶基因Bax,并利用双荧光素酶报告基因系统验证miR-193a的靶基因;用过表达miR-193a的慢病毒pre-miR-193a-lv和抑制miR-193a表达的慢病毒pre-miR-193a-inhibitor-lv分别侵染MDBK细胞,48 h后收集细胞,然后用实时荧光定量PCR、Western blotting和流式细胞仪检测凋亡通路中Bax的表达水平及MDBK细胞凋亡率。结果预测并验证了miR-193a的靶基因为Bax;双荧光素酶报告基因分析结果显示miR-193a能直接结合到Bax 3'UTR区域中的miRNA反应位点,并极显著下调Bax的表达水平(P<0.01);流式细胞仪检测凋亡率结果显示,感染pre-miR-193a-lv慢病毒的MDBK细胞凋亡率极显著升高(P<0.01)。结果表明miR-193a能直接靶向凋亡通路中Bax基因,从而促进凋亡的发生。

Study on the Inductive Effects of miR-193a on Apoptosis in MDBK Cells

The study was aimed to investigate the molecular mechanisms of miR-193a inducing apoptosis in cytopathogenic bovine virus diarrhea virus (cp BVDV) infected MBDK cells. Prediction of apoptosis-related miR-193a target gene Bax with bioinformatics online applications,TargetScan and Microcosm Targets,and verification of the interaction between miR-193a and Bax by dual-luciferase reporter assay were carried out to investigate the interaction between miR-193a and Bax. miR-193a overexpressing lentivirus pre-miR-193a-lv and lentivirus pre-miR-193a-inhibitor-lv inhibiting miR-193a expression were used to infect MDBK cells,respectively;Bax expression levels and apoptosis rates of MDBK cells were detected by Real-time quantitative PCR,Western blotting and flow cytometry,48 h later. According to the prediction and verification,we convinced that apoptosis-related gene Bax was the target of miR-193a;the results of Real-time quantitative PCR,Western blotting and dual-luciferase reporter assay showed that miR-193a directly targeted the miRNA response site of Bax 3'UTR and Bax expression levels were extremely significantly downregulated after miR-193a overexpression (P<0.01);meanwhile,the results of flow cytometry assay showed that apoptosis rates were extremely significantly decreased in MDBK cells infected with pre-miR-193a-lv (P<0.01). In a word,miR-193a could directly target Bax gene in pathway of apoptosis and promot apoptosis.