Objective To investigate the effect and mechanism of intron-derived 27-nt microRNA(27nt-miR)on the phenotypic switch of rat vascular smooth muscle cells(VSMCs)induced by platelet-derived growth factor-BB(PDGF-BB).Methods VSMCs were transduced with 27nt-miR-expressing lentiviruses and their effects on phenotypic switch of VSMCs induced by PDGF-BB was observed.The cells were divided into control group,PDGF-BB induction group,27nt-miR group,27nt-miR-sponge group and 27nt-miR negative control(27nt-miR NC)group.The 27nt-miR group,27nt-miR-sponge group and 27nt-miR NC group were subjected to rapamycin(100 nmol/L)and MHY1485(10 μmol/L)to verify the involvement of 27nt-miR in the regulation of mTOR and p70S6k pathways.The target binding sites of 27nt-miRNA and mTOR were predicted by bioinformatics.The viability and proliferation of VSMCs were determined by CCK-8 and EdU cell proliferation assays.The migration ability of VSMCs was determined by the scratch assay.The relative mRNA expressions of α-SMA,SM22α and OPN were detected by qRT-PCR.The relative protein expressions of α-SMA,SM22α,OPN,mTOR,p-mTOR,p70S6k and p-p70S6k were detected by Western blotting.Results The CCK-8 assay revealed that the cell proliferation rate in the PDGF-BB induction group was higher than normal(P＜0.05),while there was no difference in the cell proliferation rate between the 27nt-miR NC group and the PDGF-BB induction group(P＞0.05).The cell proliferation rate in the 27nt-miR group was lower than that in the 27nt-miR NC group(P＜0.05),while there was no difference in the cell proliferation rate between the 27nt-miR-sponge group and the 27nt-miR NC group(P＞0.05).The cell migration rate in the 27nt-miR group was lower than that in the 27nt-miR NC group(P＜0.05),while there was no difference in the cell migration rate between the 27nt-miR-sponge group and the 27nt-miR NC group(P＞0.05).The EdU cell proliferation assay demonstrated that the cell proliferation rate in the PDGF-BB induction group was higher than that in the control group(P＜0.05),while there was no difference in the cell proliferation rate between the 27nt-miR NC group and the PDGF-BB induction group(P＞0.05).The cell proliferation rate in the 27nt-miR group was lower than that in the 27nt-miR NC group(P＜0.05),while there was no difference in the cell proliferation rate between the 27nt-miR-sponge group and the 27nt-miR NC group(P＞0.05).The relative mRNA expression of α-SMA in the PDGF-BB induction group was lower than that in the control group(P＜0.05),whereas there was no difference in the relative mRNA expressions of SM22α and OPN between the two groups(P＞0.05).The relative mRNA expressions of α-SMA,SM22α and OPN were not different between the 27nt-miR NC group and the PDGF-BB induction group(P＞0.05).The relative mRNA expressions of α-SMA and SM22α in the 27nt-miR group were higher than those in the 27nt-miR NC group(P＜0.05),while the relative mRNA expression of OPN in the 27nt-miR group was lower than that in the 27nt-miR NC group(P＜0.05).There was no difference in the relative mRNA expressions of α-SMA and SM22α between the 27nt-miR-sponge group and the 27nt-miR NC group(P＞0.05).The relative mRNA expression of OPN in the 27nt-miR-sponge group was higher than that in the 27nt-miR NC group(P＜0.05).The relative protein expressions of α-SMA and SM22α in the PDGF-BB induction group were lower than those in the control group(P＜0.05).The relative protein expression of OPN in the 27nt-miR NC group and the PDGF-BB induction group was higher than that in the 27nt-miR group(P＜0.05).The relative protein expressions of α-SMA and SM22α in the 27nt-miR group were higher than those in the 27nt-miR NC group(P＜0.05).The relative protein expression of OPN in the 27nt-miR group was lower than that in the 27nt-miR NC group(P＜0.05).The relative protein expressions of p70S6k,p-p70S6k and p-mTOR in the 27nt-miR group were lower than those in the 27nt-miR NC group(P＜0.05).After treatment with rapamycin,the relative expressions of all the proteins were different among the groups(P＜0.05).The relative protein expressions of p70S6k,p-p70S6k,mTOR and p-mTOR in the 27nt-miR group were lower than those in the 27nt-miR NC group(P＜0.05).There were differences in the relative protein expressions of p-p70S6k,mTOR and p-mTOR between the 27nt-miR-sponge group and the 27nt-miR NC group(P＜0.05).After treatment with MHY-1485,the relative protein expressions of p70S6k,p-p70S6k,mTOR and p-mTOR in the 27nt-miR group were lower than those in the 27nt-miR NC group(P＜0.05).The relative expressions of all the proteins were different between the 27nt-miR-sponge group and the 27nt-miR NC(P＜0.05).Conclusion The 27nt-miR may regulate the viability,proliferation,migration and phenotypic switch of rat VSMCs by targeting the mTOR/p70S6k signaling pathway.
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