Research On The Role Of MiRNA Targeting Pig CLTC Gene In The Progress Of Japanese Encephalitis Virus Entering PK15 Cell

Japanese encephalitis virus(JEV)is a mosquito-borne flavivirus that belongs to the family Flaviviridae.JEV is one of the most important endemic encephalitides and can cause acute viral encephalitis.Pigs act as amplifying hosts of JEV;therefore,the domestic pig was considered to be a risk factor in the transmission of the disease to humans[1,2].JEV is also an important pathogen in swine and causes considerable economic losses in pork production.The primary symptoms of pigs infected with JEV are fetal abortion and stillbirth in infected sows and aspermia in boars [3,4].microRNAs(miRNAs)are a class of 20–23 nucleotide noncoding RNAs that regulate gene expression in multiple biological and pathological processes,more and more study shows that miRNA has a role of antiviral infection.Virus invade cells in a variety of package swallow ways,and clathrin-dependent endocytosis is one way we study clear.As early study shows that Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin-dependent endocytosis.so the project plan to screen out ssc-miRNA mimics targeting porcine CLTC genes,taking CLTC gene transcription regulation as the breakthrough point and explore the role of the miRNA-clathrin package in the process of JEV invading PK15 cells.To preliminary screen out ssc-miRNA mimics targeting porcine CLTC gene,6 ssc-miRNA mimics targeting CLTC gene were predicted by bioinformatics analysis.at the same time,the dual luciferase reporter assay system,Western Blotting and SYBR Green Realtime PCR were used to verifying the ssc-miRNA mimics that target CLTC gene,and 3'UTR of porcine CLTC gene containing the mutated ssc-miRNA mimics targeting sites by reversing the seed regions of targeting sites inserted into the psi-CHECK2 target reporter vector and transiently co-transfected with candidate miRNA mimics into PK15 cell,the dual luciferase reporter assay system was used to evaluate the activity of luciferase.RT-q PCR was used to detect the impact of miRNA mimics targeting CLTC gene on JEV invading PK15 cell.At last,Time-dependent expression of miRNA mimics in JEV-infected PK15 cell for 4h,12 h,24h,36 h were evaluated using qPCR..Results: the dual luciferase reporter assay system,Western Blot and SYBR Green Realtime PCR demonstrate that ssc-miR-1 and ssc-miR-129-5p could down-regulate the expression of CLTC gene.Point mutation test showed that ssc-miR-1 could down-regulate the luciferase activity by targeting the predicted seed region of 3'UTR of porcine CLTC gene,but ssc-miR-129-5p could down-regulate the luciferase activity,the reason of which is not clearly.qPCR showed that ssc-miR-1 and ssc-miR-129-5p targeting CLTC gene could inhibit the invasion of JEV on PK15 cell.Time-dependent expression of miRNA mimics in JEV-infected PK15 cell for 4 h,12 h,24 h,36 h showed that there is no significantly statistical difference of the expression of ssc-miRNA mimics in JEV-infected PK15 cell for 4h,12h,24h,36h.