内含子源性27nt-microRNA对大鼠血管平滑肌细胞表型转换的影响及其机制研究

Effect and mechanism of intron-derived 27-nt microRNA on phenotypic switch of rat vascular smooth muscle cells

摘要:

目的 探讨内含子源性27nt-miRNA（27nt-miR）对血小板源性生长因子-BB（PDGF-BB）诱导大鼠血管平滑肌细胞（VSMCs）表型转换的影响及其分子机制。方法 用27nt-miR表达慢病毒转染培养的VSMCs，观察其对PDGF-BB诱导的VSMCs表型转换的影响；将细胞分为对照组、PDGF-BB诱导组、27nt-miR组、27nt-miR-sponge组及27nt-miR NC组（27nt-miR NC组）。雷帕霉素（100 nmol/L）与MHY-1485（10 μmol/L）作用于27nt-miR组、27nt-miR-sponge组及27nt-miR NC组，以验证27nt-miR是否参与mTOR与p70S6k通路的调控。用生物信息学预测27nt-miR与mTOR的靶向结合位点，用CCK-8法与EdU法分别测定VSMCs活力与增殖情况，划痕试验测定VSMCs迁移能力，实时荧光聚合酶链反应检测α-SMA、SM22α及OPN mRNA表达，Western blotting检测α-SMA、SM22α、OPN、mTOR、p-mTOR、p70S6k与p-p70S6k蛋白表达。结果 CCK-8法结果示，PDGF-BB诱导组细胞增殖率较正常值高（P <0.05），27nt-miR NC组与PDGF-BB诱导组比较，差异无统计学意义（P >0.05）；27nt-miR组细胞增殖率较27nt-miR NC组低（P <0.05），27nt-miR-sponge组与27nt-miR NC组比较，差异无统计学意义（P >0.05）。PDGF-BB诱导组细胞迁移率较对照组高（P <0.05），27nt-miR NC组与PDGF-BB诱导组比较，差异无统计学意义（P >0.05）；27nt-miR组细胞迁移率较27nt-miR NC组低（P <0.05），27nt-miR-sponge组细胞迁移率与27nt-miR NC组比较，差异无统计学意义（P >0.05）。EdU法结果示，PDGF-BB 诱导组细胞增殖率较对照组高（P <0.05），27nt-miR NC组与PDGF-BB诱导组细胞增殖率比较，差异无统计学意义（P >0.05）；27nt-miR组细胞增殖率较27nt-miR NC组低（P <0.05），27nt-miR-sponge组与27nt-miR NC组比较，差异无统计学意义（P >0.05）。PDGF-BB诱导组α-SMA mRNA相对表达量较对照组低（P <0.05），两组SM22α与OPN mRNA相对表达量比较，差异无统计学意义（P >0.05）。27nt-miR NC组与PDGF-BB诱导组α-SMA、SM22α、OPN mRNA相对表达量比较，差异无统计学意义（P >0.05）；27nt-miR组α-SMA、SM22α mRNA相对表达量较27nt-miR NC组高（P <0.05），OPN mRNA相对表达量较27nt-miR NC组低（P <0.05）；27nt-miR-sponge组与27nt-miR NC组α-SMA、SM22α mRNA相对表达量比较，差异均无统计学意义（P >0.05），27nt-miR-sponge组OPN mRNA相对表达量较27nt-miR NC组高（P <0.05）。PDGF-BB诱导组α-SMA、SM22α蛋白相对表达量较对照组低（P <0.05）；27nt-miR NC组、PDGF-BB 诱导组OPN蛋白相对表达量较27nt-miR组高（P <0.05）；27nt-miR组α-SMA、SM22α蛋白相对表达量较27nt-miR NC组高（P <0.05），OPN蛋白相对表达量较27nt-miR NC组低（P <0.05）；27nt-miR组p70S6k、p-p70S6k与p-mTOR蛋白相对表达量较27nt-miR NC组低（P <0.05）；各组经雷帕霉素处理后，27nt-miR-sponge组与27nt-miR NC组各蛋白相对表达量比较，差异均有统计学意义（P <0.05）。27nt-miR组p70S6k、p-p70S6k、mTOR、p-mTOR蛋白相对表达量较27nt-miR NC组低（P <0.05），27nt-miR-sponge组与27nt-miR NC组p-p70S6k、mTOR、p-mTOR蛋白相对表达量比较，差异均有统计学意义（P <0.05）。各组经MHY-1485处理后，27nt-miR组p70S6k、p-p70S6k、mTOR、p-mTOR蛋白相对表达量较27nt-miR NC组低（P <0.05）；27nt-miR-sponge组与27nt-miR NC组各蛋白相对表达量比较，差异均有统计学意义（P <0.05）。结论 27nt-miR可能通过靶向mTOR/p70S6k信号通路从而调节大鼠血管平滑肌细胞增殖活力、增殖、迁移与表型转换。

Abstract:

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.