MicroRNA-599对静脉曲张中血管平滑肌细胞去分化的影响及其机制研究

MicroRNA-599 inhibits vascular smooth muscle cell dedifferentiation and its mechanism in varicose veins

摘要:

目的  研究microRNA-599（miR-599）在曲张静脉中的表达，探讨miR-599是否通过下调血小板源性生长因子B（PDGF-BB），抑制曲张静脉中血管平滑肌细胞（VSMCs）去分化。方法  收集大隐静脉移植术患者中曲张静脉和正常静脉标本，通过实时荧光定量聚合酶链反应（qRT-PCR）检测42例曲张静脉组织及39例正常静脉组织中miR-599、miR-200、miR-145、miR-146b、miR-155的表达；Western blot检测两组标本中SM-
actin、SM-22、SM-MCH、OPN的表达；在细胞实验中，转染miR-599mimic和miR-NC至离体培养的VSMCs中，Western blot检测VSMCs中SM-actin、SM-22、SM-MCH、OPN、PDGF-BB的表达，噻唑蓝法测细胞增殖、划痕实验观察细胞迁移，利用荧光素酶试验验证PDGF是miR-599的靶基因。结果  相比于正常静脉组织，miR-599在曲张静脉中的表达降低（F =73.012，P =0.001）。两组miR-146b、miR-200、miR-30、miR-155的表达量比较，差异无统计学意义。相比于正常静脉组织，曲张静脉组织中SM-actin、SM-22、SM-MCH的表达降低（P <0.05），OPN的表达升高（F =56.414，P =0.009）；在细胞实验中，相比于对照组，miR-599组VSMCs中SM-actin、SM-22、SM-MCH的表达升高（P <0.05），OPN的表达降低（F =27.353，P =0.022），细胞增殖减少（F =
37.824，P =0.016），迁移减少（F =69.365，P =0.001），PDGF表达量减少（F =55.345，P =0.004）。荧光素酶试验结果显示，miR-599能够降低PDGF-3'-UTR质粒的荧光素活性（F =21.429，P =0.036）。结论  在曲张静脉组织中，miR-599低表达。在VSMCs中，miR-599可以通过下调PDGF-BB而抑制VSMCs去分化。

Abstract:

Objective To verify whether microRNA-599 (miR-599) could inhibit vascular smooth muscle cell (VSMC) dedifferentiation by targeting platelet-derived growth factor-BB(PDGF-BB) in varicose veins. Methods Varicose veins and normal samples were collected from patients undergoing great saphenous vein transplantation surgery. qRT-PCR was used to test the expressions of miR-599, miR-200, miR-145, miR-146b and miR-155 in the varicose veins. Western blot was used to test the expressions of SM-actin, SM-22, SM-MCH and OPN. For in vitro experiment, after miR-599 was transfected into VSMCs, MTT was used to investigate the proliferation ability of VSMCs, wound healing was employed to test the migratory ability of VSMCs, Western blot was used to investigate the expressions of SM-actin, SM-22, SM-MHC, OPN and PDGF-BB in VSMCs. Luciferase assay was used to confirm whether PDGF-3'-UTR was the target gene of miR-599. Results Compared with the healthy control tissue, the expressions of miR-599, SM-actin, SM-22  and SM-MHC in the varicose veins were significantly decreased (P < 0.05); meanwhile the expression of OPN increased (P < 0.01). The result of the in vitro experiment showed that compared with the control group, the expressions of SM-actin, SM-22 and SM-MHC were increased in the miR-599 group (P < 0.05), and the expression of OPN and PDGF-BB decreased (P < 0.05). The luciferase activity of the PDGF-3'-UTR plasmid was suppressed by miR-599 (P < 0.05). Conclusions In varicose veins, the expression of miR-599 decreases. miR-599 inhibits VSMC dedifferentiation by targeting PDGF-3'-UTR.