| Objective:To investigate the role and mechanism of miR-1929-3p differential expression induced by murine cytomegalovirus(MCMV)infection in the proliferation and remodeling of mouse vascular smooth muscle cells.Methods:(1)MCMV Smith strain was used to intraperitoneally infect mouse vascular smooth muscle cells(MOVAS),and the experiment was divided into Control group and MCMV infection group(MCMV group:MOI of infection complex was 0,0.001,0.01,0.1,1,and the infection time was 8h,12h,24h,48h,respectively).MCMV-DNA was detected by PCR technique on MOVAS to establish the model of vascular smooth muscle cells infected by MCMV in mice.(2)20nM miR-1929-3p mimic(miR-1929-3p mimic)and 30nM inhibitor(miR-1929-3p inhibitor)were transfected for 24h,and the mice were divided into six groups again:The intervention model of miR-1929-3p was established in the Control group,MCMV group,MCMV+IMIC-NC group,MCMV+miR-1929-3p mimic group,MCMV+inhibitor-NC group and MCMV+miR-1929-3p inhibitor group.(3)Real-time quantitative PCR(qRT-PCR)was used to verify the mRNA relative expression levels of differentially expressed miR-1929-3p and target gene endothelin type A receptor(ETAR).(4)By transfection of ETAR overexpressing adenovirus vectors with transfection time of 48h and MOI of 1000,and six groups were divided again:Control group,MCMV group,MCMV+mimic-NC group,MCMV+mimic-NC group,MCMV+mimic-Ad-NC group,MCMV+mimic-Ad-NC group and MCMV+mimic-Ad-ETAR group.(5)After adding 10nM ETAR inhibitor(BQ123)or 100nM NLRP3 inflammasome inhibitor(MCC950),the patients were divided into Control group,MCMV group,MCMV+BQ123 group,MCMV+ inhibitor NC,MCMV+inhibitor NC group and MCMV+inhibitor MCC950 group.(6)EDU test was used to detect the effect of miR-1929-3p on the proliferation of mouse vascular smooth muscle cells;Effect of flow cytometry on apoptosis.(7)The concentrations of IL-18 and IL-1β in the supernatant of the culture medium of vascular smooth muscle cells of mice were determined by ELISA.(8)The effects of miR-1929-3p on the expression of vascular proliferative remodeling proteins(a-SMA,PCNA,OPN)and related proteins activated by NLRP3 inflammasome(NLRP3,caspase-1,IL-1β,IL-18)and ETAR were determined by Western blot.(9)The experimental data were statistically processed by SPSS 20.0 statistical software.Results:(1)Model establishment:the vascular smooth muscle cells of mice were infected with MCMV,and the expression of MCMV DNA was detected,which indicated that the MCMV infection model was established successfully.(2)CCK8 detection of cell proliferation rate in different MCMV multiplicity of infection and time of infection:the results showed that when MOI was 0.01 and 48h,VSMC proliferation was the most obvious.(3)MCMV regulation of miR-1929-3p and target gene ETAR:qRT-PCR results showed that the expression of miR-1929-3p in MCMV group was decreased compared with the Control group(P<0.05).The mRNA expression of ETAR was significantly increased(P<0.05).Compared for MCMV+mimic NC group,the expression of miR-1929-3p have been fundamentally expanded(P<0.001),and the mRNA expression of ETAR might have been diminished(P<0.05).There was no huge distinction in miR-1929-3p expression between MCMV+miR-1929-3p inhibitor group and MCMV+inhibitor NC group(P>0.05),while the outflow for ETAR mRNA might have been altogether expanded(P<0.05).Western blot results demonstrated that the statement from claiming ETAR protein in MCMV group might have been higher over that the control group(P<0.05).Compared for MCMV+mimic NC group,the outflow about ETAR protein in MCMV+miR-1929-3p mimic group might have been diminished(P<0.05).Compared with MCMV+inhibitor NC group,those statement for ETAR protein in MCMV+miR-1929-3p inhibitor group was expanded(P<0.05).(4)MCMV-induced down-regulation of miR-1929-3p on cell proliferation and remodeling:EdU and Western blot results indicated that the proliferation rate of MCMV group might have been fundamentally higher over that from Control group(P<0.05),the protein expressions from claiming OPN and PCNA were increased,and the protein outflow for α-SMA might have been diminished(P<0.05).Compared for the MCMV+mimic NC group,the positive rate for EdU and the statement of OPN furthermore PCNA protein in the MCMV+miR-1929-3p mimic group were diminished(P<0.05),and the protein expression about α-SMA might have been expanded(P<0.05).Compared with MCMV+inhibitor NC group,the positive rate of EdU and the expression of OPN and PCNA protein in MCMV+ miR-1929-3p inhibitor group were increased(P<0.05),and the expression of α-SMA protein might have been diminished(P<0.05).(5)MCMV-induced down-regulation about miR-1929-3p has an effect on cell apoptosis:Flow cytometry and Western blot results showed that the apoptosis rate and Bax protein expression were increased and Bcl-2 protein expression was decreased in MCMV group compared with Control group(P<0.05).Compared with MCMV+mimic NC group,apoptosis rate and Bax expression in MCMV+miR-1929-3p mimic group were diminished(P<0.05),while Bcl-2 statement might have been expanded(P<0.05).Compared with MCMV+inhibitor NC group,apoptosis rate and Bax protein in MCMV+miR-1929-3p inhibitor group were increased(P<0.05),while Bcl-2 protein expression was decreased(P<0.05).(6)The role of ETAR in MCMV-induced miR-1929-3 down-regulation and cell proliferation and remodeling:EdU and Western blot results showed that compared with the Control group,the proliferation rate of the MCMV group increased(P<0.05),OPN and PCNA protein expression increased,α-SMA protein expression decreased(P<0.05);compared with MCMV+mimic NC group,the proliferation rate of MCMV+miR-1929-3p mimic group was significantly reduced(P<0.05),OPN and PCNA Protein expression decreased and α-SMA protein expression increased(P<0.05);compared with MCMV+miR-1929-3p mimic+Ad-NC group,the proliferation rate of MCMV+miR-1929-3p mimic+Ad-ETAR group was significantly increased(P<0.05),the expression of OPN and PCNA protein increased,and the expression of α-SMA protein decreased(P<0.05).(7)The role of ETAR in MCMV-induced miR-1929-3p down-regulation and cell apoptosis:Flow cytometry and Western blot results showed that compared with the Control group,the apoptosis rate of the MCMV group was increased(P<0.05),Bax protein expression increased,Bcl-2 protein expression decreased(P<0.05).Compared with MCMV+miR-1929-3p mimic+Ad-NC group,the apoptosis rate of MCMV+miR-1929-3p mimic+Ad-ETAR group Increased(P<0.05),Bax protein expression increased,Bcl-2 protein expression decreased(P<0.05).(8)MCMV-induced down-regulation of miR-1929-3p affects the expression of NLRP3 inflammasome-associated proteins:Western blot results showed that compared with the MCMV group,the MCMV+BQ123 group inhibited the decreased expression of NLRP3,Caspasel,and IL-1β(P<0.05),there might have been no huge distinction in the outflow of IL-18(P<0.05).Compared with the MCMV+ inhibitor NC group,the statement about NLRP3,Caspase-1 and IL-1β in the MCMV+miR-1929-3p inhibitor group expanded(P<0.05),there was no contrast on IL-18 expression(P<0.05).Compared with the MCMV+miR-1929-3p inhibitor group,the MCMV+miR-1929-3p inhibitor+MCC950 group had NLRP3,Caspase1,IL-1β The expression might have been clearly smothered(P<0.05),and there might have been no contrast on the expression of IL-18(P<0.05).Conclusion:MCMV infection promoted the proliferation and apoptosis of vascular smooth muscle cells in mice,which may be achieved by down-regulating the expression of miR-1929-3p,promoting the upregulation of target gene ETAR and the activation of NLRP3 inflammasome. |
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