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

摘要:

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-2 3'UTR区有结合位点，miR-125a-5p模拟物极显著抑制pmir-Bcl-2 3'UTR-WT荧光素酶活性，对pmir-Bcl-2 3'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。

Abstract:

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-2 3'UTR by TargetScan. The luciferase activity of wild-type pmir-Bcl-2 3'UTR was inhibited significantly by miR-125a-5p mimics, but that of mutant pmir-Bcl-2 3'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.