The Effect Of MiR-885 On Mitochondrial Damage Induced By TGEV In IPEC-J2

Transmissible gastroenteritis(TGE)is an acute,highly-contact intestinal infectious disease that causes huge economic losses in the pig industry.Its source is Transmissible Gastroenteritis Virus(TGEV),which infect Intestinal Porcine Epithelial Cell(IPEC-J2)can induce severe morphological changes,biochemical dysfunction and mitochondrial damage in cells.It has been reported that microRNA plays an important role in inducing mitochondrial damage in cells.Our previous study found that miR-885 is up-regulated significantly after TGEV infecting IPEC-J2.However,whether miR-885 regulates mitochondrial damage is still a problem.In this research,we study on miR-885,explore its effect and mechanism on mitochondrial damage induced by TGEV in IPEC-J2.Firstly,we test the change of miR-885 expression after TGEV infection.Secondly,we test the effect of miR-885 on mitochondrial damage induced by TGEV in IPEC-J2.At last,we verify the target gene of miR-885 and tset the effect of target gene on mitochondrial damage.The study obtained the following results.1.IPEC-J2 were infected with 1.0 MOI TGEV.The calcium ion concentration of the cell mitochondria was tested 24 h after the infection.The results showed that the concentration of calcium ions in the mitochondria of the infected cells was significantly higher than that of the control(p<0.01).Real-time PCR was used to test the expression of miR-885 after IPEC-J2 infected with TGEV.The results showed that after 24 h of TGEV infection,the expression of miR-885 was significantly higher that of control cells(p<0.01).2.IPEC-J2 were transfected with miR-885 mimics or miR-885 inhibitors,24 h after transfection,IPEC-J2 were infected with 1.0 MOI TGEV.24 h after infection,we test concentration of calcium ions in the mitochondria.The results showed that after overexpression of miR-885,the mitochondrial calcium ion concentration was significantly increased(p<0.01),and after miR-885 was inhibited,the mitochondrial calcium ion concentration was significantly reduced(p<0.05).3.Bioinformatics analysis found the target gene bcl-10 of miR-885.We tested bcl-10 via dual luciferase test and western blotting.In order to construct dual luciferase recombinant plasmid,we connected wild type 3′ UTR and mutation type 3′ UTR to psi CHECK-2 vector respectively.Subsequently,IPEC-J2 were transfected with miR-885 mimics or miR-885 inhibitors and wild type dual luciferase recombinant plasmid or mutation type dual luciferase recombinant plasmid.Then we tested the binding activity of miR-885 to 3′ UTR of target gene.The results showed,miR-885 can significant decline the activity of luciferase in which IPEC-J2 were transfected with bcl-10 3′ UTR wild type dual luciferase recombinant plasmid(p<0.01).Meanwhile,the luciferase activity showed no significant change in IPEC-J2 that transfected with bcl-10 3′ UTR mutant type dual luciferase recombinant plasmid.The miR-885 mimics and miR-885 inhibitors were transfected into IPEC-J2 respectively,then western blotting was used to test the effect of miR-885 to BCL-10 expression in protein levels,we found that the overexpression of miR-885 decreased the BCL-10 in protein levels.After inhibition of miR-885,the BCL-10 protein level was significantly increased.4.Design and synthesis of bcl-10 siRNA to inhibit the expression of bcl-10,and simultaneously construct recombinant bcl-10 eukaryotic overexpression plasmid p EGFP-N1-BCL-10 to overexpress bcl-10,then si BCL-10 or recombinant p EGFP-N1-BCL-10 plasmids were transfected into IPEC-J2.After 24 h,IPEC-J2 were infected with 1.0 MOI TGEV.24 h after infection,the calcium ion concentration was changed.The results showed that overexpression of bcl-10 can significantly decreased the mitochondrial calcium ion concentration(p<0.01).Conversely,inhibition of bcl-10 expression can increased it significantly(p<0.01).This research found that miR-885 can promote mitochondrial damage.The dual luciferase assay and western blotting results demonstrated that bcl-10 is the target gene of miR-885.In addition,we also found that miR-885 can promote mitochondrial damage induced by TGEV in IPEC-J2 mitochondrial damage by inhibiting its target gene bcl-10.The results of the research shed light on the role of miR-885 in the induction of IPEC-J2 mitochondrial damage induced by TGEV,and provide a theoretical basis for further study on the interaction between host cell miRNA and TGEV.