bta-miR-302c影响牛病毒性腹泻病毒复制的研究

Investigation on the effect of bta-miR-302c on the replication of Bovine viral diarrhea virus

为了探索bta-miR-302c通过调控天然免疫(JAK/STAT)信号通路影响牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)复制的作用机理,试验应用miRbase、Microcosm Targets、TargetScan等生物信息学平台预测与抗病毒JAK/STAT信号通路相关的bta-miR-302c靶基因及miRNA识别位点;将bta-miR-302c模拟物(Agomir)和阻抑物(Antagomir)以及阴性对照物(Control Agomir)转染至MDBK细胞中,采用Western-blot和实时荧光定量PCR检测SOCS3蛋白及mRNA表达情况;将SOCS3基因3′UTR miRNA识别位点及其突变体克隆至双荧光素酶报告载体pmiRGLO中,构建pmiRGLO-SOCS3-MRS和pmiRGLO-SOCS3-MRS-M质粒,再分别与bta-miR-302c Agomir共转染至HEK-293T细胞中,48 h后测定相对荧光素酶反应强度;分别将bta-miR-302c Agomir和Antagomir以及Control Agomir转染至MDBK细胞中,然后感染BVDV新疆分离株TC株,于感染后不同时间采用实时荧光定量PCR测定BVDV 5′UTR mRNA水平,Reed-Muench法测定BVDV的效价变化。结果表明:bta-miR-302c成熟序列中的UCGUGA能特异性结合SOCS3基因3′UTR的miRNA识别位点AGCACU;与pmiRGLO相比,转染pmiRGLO-SOCS3-MRS可极显著降低相对荧光素酶反应强度(P<0.01);与转染Control Agomir和bta-miR-302c Antagomir相比,转染bta-miR-302c Agomir极显著降低SOCS3蛋白表达和mRNA转录水平(P<0.01);与转染Control Agomir和bta-miR-302c Antagomir相比,BVDV感染bta-miR-302c Agomir转染的MDBK细胞12 h后其5′UTR mRNA水平和病毒效价显著或极显著降低(P<0.05或P<0.01)。说明bta-miR-302c靶向于SOCS3基因3′UTR的miRNA识别位点,抑制靶基因的mRNA转录并降低其蛋白表达,以及抑制BVDV的复制。

The aim was to explore the mechanisms of bta-miR-302 c in regulating the replication of Bovine viral diarrhea virus(BVDV) by affecting the innate immune signal pathway JAK/STAT,and the experiment used miRbase, Microcosm Targets, TargetScan and other bioinformatics platforms to predict bta-miR-302 c target genes and miRNA recognition sites related to the antiviral natural immune pathway;synthesis of bta-miR-302 c mimic(Agomir),inhibitor(Antagomir) and negative control(Control Agomir) were carried out, which were then transfected MDBK cells, and Western-blot and real-time fluorescent quantitative PCR were used to detect SOCS3 protein and mRNA expression. The miRNA recognition site in the 3′UTR region of SOCS3 and its mutant into the dual luciferase reporter vector pmiRGLO to construct pmiRGLO-SOCS3-MRS and pmiRGLO-SOCS3-MRS-M plasmids, which were co-transfected with bta-miR-302 c Agomir respectively into HEK-293 T cells, and the relative luciferase reaction intensity was measured after 48 h. Bta-miR-302 c Agomir, Antagomir and Control Agomir were transfected into MDBK cells, respectively, which were then infected with BVDV TC isolate from Xinjiang. Real-time fluorescent quantitative PCR was used to determine the level of BVDV 5′UTR mRNA at different times after infection, and the Reed-Muench method was used to determine the titer of BVDV. The results showed that UCGUGA in the mature sequence of bta-miR-302 c can specifically bind to the miRNAs recognition site AGCACU in the 3′UTR region of the SOCS3 gene. Compared with pmiRGLO, pmiRGLO-SOCS3-MRS extremely significantly reduced the relative luciferase reaction intensity(P<0.01);compared with transfection of Control Agomir and bta-miR-302 c Antagomir, transfection of bta-miR-302 c Agomir extremely significantly reduced the protein expression and mRNA transcription level of SOCS3(P<0.01);compared with Control Agomir and bta-miR-302 c Antagomir transfection, the 5′UTR mRNA level and virus titer were significantly or extremely significantly reduced(P<0.05 or P<0.01) in bta-miR-302 c Agomir transfected MDBK cells at 12 h after BVDV infection. The results suggested that bta-miR-302 c targeted the miRNA recognition site in the 3′UTR of SOCS3,inhibited the mRNA transcription of the target gene, and reduced its protein expression, as well as the inhibition of the replication of BVDV.