The Mechanism Of MiRNA-181a In PDGF-BB Induced Vascular Smooth Muscle Cells Proliferation And Migration

With the improvement of living standards, the incidence of vascular hyperplastic diseases increased year by year. It becomes a serious threat to human health, such as atherosclerosis(AS)and cardiovascular disease(CVD). The abnormal proliferation and migration of vascular smooth muscle cells(VSMCs) and neointimal formation are the important pathologic basis. Therefore, it is great significance for the pathogenesis of vascular hyperplastic diseases to explore the mechanism of VSMCs proliferation and migration mechanism.Platelet derived growth factor(PDGF) is found by Ross and other scholars in 1974. It is a growth factor that comes from the platelet and promotes cell proliferation. It has three forms:PDGF-AA, PDGF-BB and PDGF-AB. PDGF-BB can combine with the PDGF-? receptors on the surface of the VSMCs to mediate signaling pathways inducing cell proliferation and migration. MicroRNA(miRNA) is a kind of 20~25 nucleotides endogenous non-coding RNA molecules found in eukaryotic cells. It can participate in variety of biological function regulations. It can combine target mRNA by complementary base pairing and lead to mRNA degradation or deter translation. There are some literatures about mi RNA regulate the proliferation and migration of VSMCs by targeting genes in recent years. Many studies have confirmed that miR-181 a participated in tumorigenesis and inflammation process, and some target genes have been identified. Based on the bioinformatics software, we predict PDGFRA,14-3-3 ?(YWHAG), S1PR1 and CREBL2 which are associated with the cell proliferation and migration may be newtarget genes of miR-181 a. Gene expression analysis found that miR-181 a was negatively correlated with the VSMCs proliferation induced by PDGF-BB, which suggests that miR-181 a may participate in the VSMCs proliferation induced by PDGF-BB. Therefore, the project intends to explore the molecular mechanism of miR-181 a regulating VSMCs proliferation and migration by PDGF-BB.Human aortic VSMCs were used in the present study. The proliferation of VSMCs and expression of miR-181a-3p and miR-181a-5p which are two mature bodies of miR-181 a after different concentration PDGF-BB were detected by real-time fluorescent quantitative PCR(qRT-PCR) and MTT methods. Overexpression or inhibition miR-181 a stable VSMCs cell lines were built by lentivirus vectors, and then explored the effects of miR-181 a on PDGF-BB induced VSMCs proliferation and migration. The prediction and verification of miR-181 a target genes used bioinformatics and dual luciferase reporter gene methods. The influence of miR-181 a on neointimal formation was explored by rat carotid artery balloon injury model and local miR-181a-1 and mi R-181a-5p-inhibition lentivirus infection. The experimental results are as follows:1. The VSMCs proliferation showed in dose-dependent increase after induced by different concentrations PDGF-BB(0, 1, 5, 10, 20 ng/m L). MiR-181a-3p and miR-181a-5p expression were dose-dependent decline and miR-181a-3p lower 90~95% compared with the miR-181a-5p.It suggests that miR-181 a can participate in VSMCs proliferation, and miR-181a-5p is the main mature body.2. The negative control(NC), LV-miR-181 a and Anti-miR-181a-5p stable VSMCs cell lines were built by lentivirus infection(MOI value is 100) and screening by 2 ?g/mL puromycin. The miR-181a-5p expression of LV-miR-181 a raises about 12 times compared with the NC cells.mi R-181a-5p can significantly suppress the VSMCs proliferation induced by PDGF-BB used MTT and EdU methods, and Anti-miR-181a-5p can promote VSMCs proliferation significantly.Proliferating Cell Nuclear Antigen(PCNA) protein levels shows the same tendency.LV-miR-181 a can inhibit VSMCs migration induced by PDGF-BB by scratch damage and Transwell chambers method significantly, and Anti-miR-181a-5p can promote the migration of VSMCs. It suggests that miR-181a-5p can inhibit VSMCs proliferation and migration induced by PDGF-BB.3. The miR-181a-5p potential target genes PDGFRA, 14-3-3 ?, S1PR1 and CREBL2 which related to cell proliferation and migration predicted by bioinformatics software such as Targetscan. The luciferase-target gene 3'-UTR and mutant eukaryotic expression vectors were built and co-transfeced with miR-181a-5p mimics/inhibitors to 293 cells. The 14-3-3 ? and S1PR1 report gene plasmid luciferase activities are lower significantly. It indicates that the14-3-3 ? and S1PR1 are miR-181a-5p target genes. The 14-3-3 ? and S1PR1 protein levels in VSMCs by different concentrations PDGF-BB induction were detected by Western Blotting. The results showed that two protein expression gradually increased with increasing of PDGF-BBconcentration, and overexpression of miR-181a-5p could significantly reduce the protein levels.4. Carotid neointimal formation model with male Sprague Dawley(SD) rats were established by balloon injury. miR-181a-5p expression reduces about 80% in model group compared with the Sham group detected by qRT-PCR. It shows that miR-181a-5p can participate in the neointimal formation. The expression of miR-181a-5p was raised about 4.5 times compared with NC group after local mi R-181 a lentivirus infection. The intima/media area ratio(I/M) of LV-miR-181 a dropped about 40% compared with NC group by morphological observation, and Anti-miR-181a-5p could rise about 30%. These results showed that miR-181a-5p could inhibit the neointimal formation. The 14-3-3 ? protein expression in Model group was increased about 4.5 folds compared with the Sham group by Western Blotting, and was down-regulated about 35% after overexpression of miR-181 a.In general, the results showed that miR-181a-5p could inhibit proliferation and migration of VSMCs by PDGF-BB induction and carotid artery neointimal formation through the 14-3-3 ?target gene. It is great significance for elucidating the pathogenesis of vascular hyperplastic diseases.