Porcine MiR-124Regulates Cav1Gene Expression And Play Important Roles In JEV Infection

Diseases have become the greatest threat to modern pig industry, especially zoonotic impact on human health are considerably serious. It is urgent for breeders to carry out disease resistance breeding and identifying disease candidate genes. In the previous work in our laboratory, we classified the porcine Cavl gene into the candidate genes of general resistance to disease. We analyzed the gene structure and the transcription regulation of Cavl gene. In addition, we analyzed the Cavl gene expression changes in HPS infected pigs. In this study, post-transcription regulation of the porcine Cavl gene was studied on the basis of preliminary results. We chose the Japanese encephalitis virus (JEV) as infectious material to explore the mechanism of miRNA-mediated anti-virus pathway in PK15cells. The main results are as follows:1. The online software TargetScan was used to predict the miRNA targeting Cavl gene and we selected those miRNA containing conservative target site for validation. Dual luciferase reporter experimental results showed that miR-124, miR-199a-5p and miR-199b-5p can significantly reduce the renilla luciferase activity; In miR-124and miR-199a-5p transfected cells, the mRNA level of Cavl gene were significantly decreased, compare to the control. In addition, overexpression of miR-124and miR-499a-5p can decrease the protein level of Cavl gene, these results showed that miR-124and miR-199a-5p target porcine Cavl gene.2. We detected the expression profile of12tissue samples of adult Large White pig by qPCR method, the results showed that Cavl gene was high expression in heart, lung, fat, and leg muscle. And miR-124was brain-specific high expression, but Cavl gene was low expression in brain.3. We detected the expression of Cavl gene in PK15cell by IF A method, the results showed that caveolin-1was mainly localized in cell membrane. Thus, we speculated that there may be lots of caveolae present on the cell membrane of PK15cells.4. PK15cells were transfected with miR-124mimics,48h post-transfection, the ultra-thin sections were prepared. Caveolae was observed by transmission electron microscopy (TEM) and the number of caveolae was quantified, the results showed that the number of caveolae was significantly decreased in miR-124transfected cells, compared to control (P<0.05). These results showed that miR-124could reduce caveolar density in PK15cells.5. PK15cells were transfected with miR-124mimics and NC,48h post-transfection, the cells were infected with JEV,24h post-infection, the proliferation of JEV was detected by western blot and flow cytometry. The results showed that the proliferation of JEV was significantly decreased in miR-124transfected cells, compared to control (P<0.05).6. PK15cells were treated with M|3CD to remove the membrane cholesterol, then infected with JEV, the proliferation of JEV was detected by IFA and western blot. The results showed that the infection of JEV was significantly decreased in MβCD treated cells, compared to control, and the inhibition is dose-dependent. The cell activity did not change significantly when treated with MβCD at the concentration of10mM. These results showed that JEV enter PK15cells via a cholesterol-dependent pathway.7. siRNA against Cavl and clathrin heavy chain (CHC) were designed. Western blot results showed that the protein level of Cavl and CHC decreased markedly. PK15cells were transfected with siRNA,48h post-transfection, the cells infected with JEV,36h post-infection, the proliferation of JEV was detected by western blot and flow cytometry. The results showed that the JEV infection slightly decreased in siRNA against Cavl transfected cells, however, the JEV infection was not effected by gensitein, which was a tyrosine protein kinase inhibit caveola-mediated endocytosis. These results showed that JEV entry was not through a caveola-mediated pathway. The JEV infection was decreased significantly in siRNA against CHC cells (P＜0.05), the results demonstrated that JEV enter PK15cell through a clathrin-dependent pathway.In conclusion, this work lay the foundation for the study of post-transcription regulation of the porcine Cavl gene, and the research on the miR-124in JEV infection provide a potential therapeutic target against JEV.