The Role Of IL-10 And MicroRNA-92a In Regulation Of Autophagy And Apoptosis Of Host Cells With Human Cytomegalovirus Infection

Objectives We investigated whether IL-10 affects cell viability and authophay under the conditions of starvation and HCMV infection by using the MRC5 cell line.We also explored the role of IL-10-mediated autophagy on HCMV replication.;To investigate the expressions of miR-17~92 cluster,involved in the apoptosis in previous reports,in the peripheral blood from neonates with infection;To explore the expressions of miR-92 a in host cells with HCMV infections and the role of miR-92 a in regulating of apoptosis.Methods To study the effects of IL-10 on the human cytomegalovirus infection,MRC5 cells were treated with IL-10(100ng/ml)for 10 h,followed by the HCMV infection.And then,the autophagic protein LC3-II/LC3-I and p62 were testd by western blot,the autophagosomes were observed by the transmission electron microscope.Futhermore,to identify whether IL-10 impaired the replication of HCMV through regulation of autophagy induced by HCMV,the MRC5 cells were pretreated with Rapamycin,IL-10 and 1%BSA respectively,followed by HCMV infection.Then,the BECN1 overexpression cells were construced to further confirm the relationship between autophagy and replication of HCMV.;31 cases were enrolled in the patients groups,and according to their responses to the GCV treatment,they were classified the GCV(+)group 19 cases and GCV(-)group 12 cases.There were 20 healthy neoantes as control group.The sample were collected 2 times : before GCV treatment and the termination of treatment.The sample were extracted miRNA,followed by the q RT-PCR tests.The expressions of miR-17~92 cluster between differernt groups were analysed.p<0.05.;The expressions of miR-92 a was tested by RT-PCR when the different post time of infection with high/low MOI.The identification of target gene for miR-92 a was carried on by RT-PCR,Western blot and the dual luciferase reporter test.MRC5 cells were pretreated with miR-92 a inhibitors or miR NC,and then infected with HCMV.The mitochondria pathway apoptosis and ER stress were observed by the proteins respectively and by the JC-1 tests.Si RNA for Bim(Si Bim)were transfected into MRC cells,followed by HCMV infection.The hochest test and Annex V/PI test were used to investigate apoptosis of host cells.Results Our data showed that IL-10 inhibited the autophagic flux of the MRC5 cells irrespective of starvation or HCMV infection.and suppressed HCMV replication.The promotion of autophagy with either a phamacological inducer(rapamycin),or a technique to over-expression BECN1 gene reversed the effect of IL-10 on virus replication.Furthermore,the PI3K/Akt signal pathway was activated when the cells were pretreated with IL-10.;The level of miR-17 in patients groups was lower than that in control groups,however the level of miR-92 a was higher,compared with that in control group.The GCV(+)group had higher level of miR-17 than GCV(-)group.The expressions of miR-92 a in GCV(-)group were lower than that in GCV(+)group.And there were positive correlation of miR-17 and ALT and negative correlation of miR-92 a and ALT.The other 4 miRNAs with extremely low level had no obviously differences between the groups.;The level of miR-92 a was enhanced during the early stage of infection,either at high MOI or at low MOI.The target gene of miR-92 a is BCL2L11,encoding protein Bim,with evidence of Dual luciferase reporter test.MRC5 were trasfected with miR-92 a inhibitors of miR NC,followed by HCMV infection.Compared with control group(miR NC),miR-92 a inhibitors group had higher level of ER stress proteins,including GADD153,GRP78,caspase-12,and also with higher level of mitochondria pathway of apoptosis related proteins,including bim,bcl-2,Cyt-C.The jc-1 tests indicated that miR-92 a inhibitors group had more sever the mitochondria injury.Furthermore,MRC5 cells were transfected with si Bim Tthe data showed that the level of apoptosis,especially apoptosis at early stage of infection,were increased,evidenced by hochest and Annex V/PI tests.Conclusions Our results indicated that IL-10 can suprress HCMV replication by inhibiting autophagy in host cells during the early stages of infection.;The data indicated that miR-17 and miR-92 a from the cluster were involved in the HCMV infection and associated with liver injury.;The data indicated that miR-17 and miR-92 a from the cluster were involved in the HCMV infection and associated with liver injury.