Résumé
Objective To investigate the role of microRNAs (miRs) in the proliferation of vascular smooth muscle cells （VSMCs）induced by endothelial insulin-like growth factor-1 (IGF-1) under low shear stress (LowSS). Methods Endothelial cells (ECs) and VSMCs were co-cultured and exposed to normal shear stress (NSS, 1.5 Pa) and LowSS (0.5 Pa) for 12 h with parallel plate flow chamber system, respectively. Real-time PCR was used to examine the expression levels of miRs. The target genes of miR-133b were predicted by multiple algorithms. The expression of polypyrimidine tract binding protein 1 (Ptbp1) and N-myc downstream regulated 1 (Ndrg1) in VSMCs was detected by Western blotting. The VSMC proliferation was detected by EdU flow cytometry assay. Results After treated with recombinant IGF-1, the expression of both miR-133b and miR-378a in VSMCs was increased. Compared with NSS, LowSS significantly induced the expression of miR-133b in the co-cultured VSMCs, but had no obvious effect on miR-378a. In VSMCs, the protein and mRNA levels of Ptbp1 and Ndrg1 were down-regulated by miR-133b mimics. miR-133b inhibitor up-regulated the mRNA levels of Ptbp1 and Ndrg1. miR-133b overexpression promoted the proliferation of VSMCs significantly. Conclusions IGF-1 secreted by ECs in response to LowSS can upregulate the expression of miR-133b in the co-cultured VSMCs, which subsequently depresses the expression of Ptbp1 and Ndrg1, and induces the proliferation of VSMCs eventually. The research findings provide a potential new target for cardiovascular disease therapy.
Résumé
Objective To investigate the role of microRNA-34a (miR-34a) in the proliferation of vascular smooth muscle cells (VSMCs) induced by low shear stress (LowSS). Methods Using co-culture parallel plate flow chamber system, endothelial cells (ECs) and VSMCs were co-cultured and applied with normal shear stress (1.5 Pa) and LowSS (0.5 Pa) for 12 h. The expression of proliferating cell nuclear antigen (PCNA) in the co-cultured VSMCs was detected by Western blotting to determine the proliferation capacity of VSMCs. Real-time PCR was used to examine the miR levels of miR-34a in the co-cultured VSMCs. The target proteins of miR-34a were predicted by TargetScan, miRWalk and some other websites. Western blotting was used to detect expression of Forkhead box j2 (Foxj2) in the co-cultured VSMCs. Mimics and inhibitor were used to up-regulate or inhibit the expression of miR-34a, and then the expression of Foxj2 and PCNA was detected by Western blotting to verify the regulation relationship between miR 34a and Foxj2. Results Compared with NSS, LowSS promoted the PCNA expression and significantly up-regulated the miR-34a expression in the co-cultured VSMCs. Foxj2 was predicted to be the downstream target protein of miR-34a by TargetScan, miRWalk and some other websites. Foxj2 expression decreased significantly in the co-cultured VSMCs under LowSS application. Under static condition, the expression of Foxj2 obviously decreased and the expression of PCNA obviously increased by up-regulating miR-34a expression in VSMCs. While inhibiting the expression of miR-34a in VSMCs would result in a significant increase in the expression of Foxj2 and a significant decrease in the expression of PCNA. Conclusions LowSS can promote the proliferation of VSMCs by regulating miR-34a and target protein Foxj2 in the co-cultured VSMCs. This research finding will provide new mechanobiological experimental reference for further illustrating the pathogenesis of atherosclerosis and finding the therapeutic targets for drugs.