猪圆环病毒2型通过外泌体miR-125a-5p靶向Bcl-2诱导淋巴细胞凋亡

Porcine circovirus type 2 induces apoptosis by exosomal miR-125a-5p targeting Bcl-2 in porcine lymphocytes

为研究miR-125a-5p在猪圆环病毒2型(porcine circovirus type 2,PCV2)诱导淋巴细胞凋亡中的作用及其作用机制,以PCV2感染PK-15细胞外泌体孵育的淋巴细胞为研究对象,采用流式细胞术、蛋白质免疫印迹试验(Western blotting)和实时荧光定量PCR,检测淋巴细胞凋亡率及凋亡相关miRNA表达;合成miR-125a-5p模拟物和抑制物转染PK-15细胞,检测miR-125a-5p过表达或抑制表达后细胞凋亡率;采用生物信息学方法预测miR-125a-5p的靶基因,双荧光素酶报告基因检测miR-125a-5p对靶基因的调控;Western blotting检测外泌体孵育淋巴细胞的线粒体凋亡信号通路相关蛋白Bcl-2、Bax、细胞色素C和caspase-3的表达。结果显示,感染PCV2的PK-15细胞分泌的外泌体极显著提高淋巴细胞凋亡率,在一定浓度范围内呈剂量依赖性;与PCV2诱导细胞凋亡相关的miRNA中,miR-125a-5p表达量极显著升高,miR-125a-5p模拟物转染细胞后极显著提高细胞凋亡率;利用TargetScan预测发现,miR-125a-5p与Bcl-23′UTR区有结合位点,miR-125a-5p模拟物极显著抑制pmir-Bcl-23′UTR-WT荧光素酶活性,对pmir-Bcl-23′UTR-MuT的荧光素酶活性无明显改变;外泌体孵育的淋巴细胞Bcl-2表达量显著降低,Bax、细胞色素C的释放和caspase-3表达量显著升高,Bcl-2/Bax的比值极显著降低。这表明,PCV2通过外泌体诱导淋巴细胞上调miR-125a-5p的表达,进而抑制Bcl-2 mRNA和蛋白表达,激活淋巴细胞线粒体凋亡通路诱导细胞凋亡。

In order to investigate the apoptosis triggered by porcine circovirus type 2(PCV2)in lymphocytes and the underlying mechanism,the levels of apoptosis and the expression levels of miRNA were examined by flow cytometry,Western blotting and real-time PCR(qPCR).The mimics or inhibitors of miR-125a-5p,an apoptosis-related miRNA,were transfected into PK-15 cells,and the apoptosis rate was examined upon overexpression or inhibition of mir-125a-5p.The target gene of mir-125a-5p was predicted by bioinformatics method,and the regulation of mir-125a-5p on the target gene was analyzed by luciferase reporter assay.The expressions of Bcl-2,Bax,cytochrome C and caspase-3 were detected by Western blotting.The results showed that exosomes secreted by PK-15 cells infected with PCV2 significantly increased the lymphocyte apoptosis rate,which was dose-dependent in certain concentration range.The expression of miR-125a-5p was dramatically increased.The apoptosis rate was increased significantly in the cells transfected with miR-125a-5p.It was predicted that there were binding sites of miR-125a-5p at Bcl-23′UTR by TargetScan.The luciferase activity of wild-type pmir-Bcl-23′UTR was inhibited significantly by miR-125a-5p mimics,but that of mutant pmir-Bcl-23′UTR was not changed.By Western blotting,Bcl-2 was reduced significantly,while Bax,cytochrome C and caspase-3 increased significantly,and the ratio of Bcl-2/Bax was significantly decreased.These results showed that PCV2 up-regulated the expression of miR-125a-5p through exosomes,then inhibited the expression of Bcl-2 at both mRNA and protein level,activated mitochondrial apoptosis pathway and induced apoptosis in lymphocytes.