The Effect Of MiR-145 On Vascular Smooth Muscle Cells And Its Mechanism

PartⅠConstruction and function identification of recombinantlentivirus vector expressing rno-miR-145Objective To construct a lentivirus vector expressing microRNA (miRNA)rno- miR-145 and transfect the vector into VSMC.Methods The miR-145 shDNA double chain template sequence was synthesizedartificially and put this template sequence clone LV3 pGLV/H1/GFP+Puro-miRNALentivirus plasmid. 293FT cells were transfected. Lentivirus particles(virosome) wereharvested and concentrated, then infected primary cultured VSMCs of the rats.Construct Scramble-NC in the same way. VSMC was obtained by the adherencemethod of tissue culture. Blank control group, miR-145 group and miR-NC groupwere divided in this test. Fluorescence expression infected with VSMCs was observedby inverted fluorescence microscope. miR-145 expression condition was detectedwith Real- time PCR.Results microRNA-145 lentivirus plasmid was construted successfully.The viraltiter was 1×109TU/ml. microRNA-145 lentivirus expression plasmid was infectedsuccessfully.The best transfection efficiency when multiply of infection( MOI) was50 and on 3th day．Real-time PCR results revealed: overexpression of miR-145 andtransfect the vector into VSMC afer 72 hours, the expression of miR-145 was 72.5times than blank control group(P<0.01).Conclusions :1. The constructed lentivirus vector can express microRNA-145 in vitro.2. microRNA-145 lentivirus plasmid may infect rat VSMC efficiently. which is thebasis for further study on the function of microRNA-145.PartⅡEffect of miR-145 on the proliferation and phenotypetransformation of vascular smooth muscle cell in ratsObjective microRNA-145 lentivirus plasmid transfect into VSMC,and stimulationby PDGF to proliferate and phenotype transformation. Select the three VSMCphenotype marker genes, PCNA, c-Jun and SM22a, observation the effect of miR-145on VSMC phenotype transformation and proliferation in vitro.Methods The original generation culture VSMC and identifycation 10ng/mL PDGFinduction VSMC proliferate and phenotype transformation. VSMCs were divided into four groups: ( 1) control group, ( 2) miR-NC group, ( 3) PDGF-BB+ miR-145 group,( 4) PDGF-BB group. VSMC proliferation was measured by CCK-8, The mRNAexpression of C-Jun、PCNA and SM22a was detected by Real-time PCR，and theprotein expression level of C-Jun、PCNA and SM22a were assessed by Western blot.Results Overexpression of miR-145 inhibit the proliferation of primary culturedVSMC, PDGF-BB+miR-145 group vs PDGF-BB group(P<0.05); miR-145 canupgraded the expression of VSMC proliferation related genes and downgraded VSMCdedifferentiated related genes. PDGF-BB+miR-145 group vs PDGF-BB group(P<0.01); The protein expression of C-Jun、PCNA were increased in PDGF-BBgroup,but reduced in PDGF-BB+ miR-145 group, and SM22a were reduced inPDGF-BB group,but increased in PDGF-BB+ miR-145 group (P<0.01).Conclusions1. microRNA-145 may be inhibited VSMC proliferation.2. Overexpression of miR-145 can upgraded the expression of VSMC proliferationrelated genes and downgraded VSMC differentiation related genes, VSMCphenotype transformation may be inhibited by microRNA-145.PartⅢmiR-145 inhibits proliferation of VSMC mediated by theinhibition of MAPKObjective To explore the effects of VSMC protein expression level ofp-ERK/ERK,p-JNK/JNK and p-p38MAPK/p38MAPK, after miR-145 overexpressionand PDGF- induced proliferation on VSMC.Methods The original generation culture VSMC and identifycation 10ng/mL PDGFinduction VSMC proliferate and phenotype transformation. VSMCs were divided intofour groups: (1) control group, (2) miR-NC group, (3) PDGF-BB+ miR-145 group, (4)PDGF-BB group. the protein expression level of ERK1/2 and p-ERK1/2、JNK andp-JNK、p38MAPK and p-p38MAPK were assessed by Western blot.Results The protein expression of phosphor-ERK1/2、phosphor-JNK、phosphor-p38MAPK were increased in PDGF-BB group,but reduced in PDGF-BB+miR-145 group (P<0.05).Conclusions This study demonstrates that miR-145 inhibits PDGF-inducedproliferation of VSMC,which might be mediated by the inhibition of MAPK.