Research On The Mechanism Of Apoptosis Of MDBK Cells Induced By BVDV

[Objective] Bovine viral diarrhea virus (BVDV) is a positive, single-stranded RNA molecule that belongs to the genus Pestivirus of the Flaviviridae family, the virus mainly lead to diarrhea、persistent infection、immunosuppression、abortion,and so on.At present,with the rapid development of cattle-feeding industry,Bovine viral diarrhea disease has bring great loss to our contry’s animal husbandry.BVDV strains exist as two biotypes, cytopathic (cp)and noncytopathic (ncp), according to their effects on tissue culture cells.cpBVDV can induce infected MDBK cells to undergo apoptosis for persistent infection.The research on the mechnism that cpBVDV induces infected MDBK cells to undergo apoptosis will help us develop efficient biological treatment methods of viral infection.[Method](1)The cDNA libararies for both MDBKCs and BMDBKCs were constructed,miRNA expression profiles were identified with solexa sequencing technique and bioinformatic analyses,in which miRNAs (miR-129-3p、miR-33a、miR-532、bno-miR-2、 bno-miR-69�'�bno-miR-160) that differentially-expressed significantally were validated.(2)Morphological changes of MDBK cells infected with cpBVDV were observated with fluorescence microscopy,and the apoptosis rate of cells was examined by Flow Cytometry (3)The expression level of miR-33a in MDBK cells infected with cpBVDV was examined by qRT-PCR.(4) The potential target gene of miR-33a was predicted by Targetscan software,the recombinant vector pMD18-MAP3K33’UTR and Dual-luciferase recombinant expression vector pmirGLO-MAP3K33’UTR were costructed and then degested by restriction enzyme SAC I/XHO I and sequenced, the target gene was validated with the method of Dual-luciferase gene detection.(5)The expression level of miR-33a in MDBK cells transfected with miR-33a NC (negative control) and miR-33a mimics was examined by qRT-PCR.(6)The transcriptional level and protein level of MAP3K3and BCL-2were examined respectivly by qRT-PCR and Western blot.(8) The apoptosis rate of transfected cells was examined by Flow Cytometry.[Result](1)Profiles of221known miRNAs and239novel miRNAs were identified,the expression level of miRNAs that validated by qRT-PCR was consistent with that of solexa sequencing technique;(2)Morphological changes of MDBK cells infected with cpBVDV were observed and the apoptosis rate of cells had dependence on time;(3)The expression level of miR-33a in MDBK cells infected with cpBVDV increased which had dependence on time;(4) The potential target MAP3K3of miR-33a was predicted,the recombinant vector pMD18-MAP3K33’UTR and Dual-luciferase recombinant expression vector pmirGLO-MAP3K33’UTR were costructed successfully,miR-33a can directly target MAP3K3;(5)The expression level of miR-33a in MDBK cells transfected with miR-33a mimics increased which had dependence on time;(7)Both the transcriptional level and the protein level of MAP3K3and BCL-2decreased significantly;(8)The apoptosis rate of cells that transfected with miR-33a mimics increased obviously.[Conclusion] These results shows that miRNA-33a can induces MDBK cells to undergo apoptosis by targeting MAP3K3and acting on antiapoptosis gene BCL-2.The study provides new ideas and development prospects for us to develop efficient biological treatment methods for viral infection.