长链非编码RNA ZFAS1/miR-150/ROCK1调控血管平滑肌细胞增殖和迁移

Long noncoding RNA ZFAS1/miR-150/ROCK1 axis regulates proliferation and migration of vascular smooth muscle cells

目的:探讨长链非编码RNA ZFAS1是否通过调控微小RNA-150(miR-150)/ROCK1促进血管平滑肌细胞(VSMCs)增殖和迁移,及其参与动脉粥样硬化发生发展的机制。方法:用血小板源性生长因子BB(PDGF-BB)诱导VSMCs增殖和迁移。用real-time PCR法检测VSMCs中ZFAS1的表达水平。进一步用小干扰RNA(siRNA)下调ZFAS1表达后,采用MTT和EdU法检测VSMCs的活力和增殖能力,采用Transwell法检测VSMCs的迁移能力,采用real-time PCR检测miR-150和ROCK1的表达水平,Western blot检测ROCK1蛋白的表达水平。萤光素酶报告基因实验验证RCOK1为miR-150的靶基因。最后,抑制miR-150表达,检测下调ZFAS1表达后VSMCs的增殖、迁移及ROCK1表达。结果:PDGF-BB上调VSMCs中ZFAS1的表达。下调ZFAS1表达后,VSMCs的增殖和迁移受到抑制(P<0.05),miR-150表达水平上升(P<0.05),ROCK1表达水平下降(P<0.05)。萤光素酶报告基因实验结果显示miR-150能直接靶向调控ROCK1。抑制miR-150表达后,能减弱下调ZFAS1表达导致的VSMCs增殖和迁移的抑制作用(P<0.05),上调ROCK1的表达水平(P<0.05)。结论:ZFAS1通过调控miR-150/ROCK1促进PDGF-BB诱导的VSMCs增殖和迁移。

AIM: To investigate whether long noncoding RNA ZNFX1(zinc finger NFX1-type containing 1) antisense RNA 1(ZFAS1) promotes the proliferation and migration of vascular smooth muscle cells(VSMCs) by regulating microRNA-150(miR-150)/ROCK1, and the involving mechanism of atherosclerosis. METHODS: Platelet-derived growth factor-BB(PDGF-BB) was used to induce proliferation and migration of VSMCs. Real-time PCR was used to detect the content of ZFAS1 in the VSMCs. After further down-regulating the expression of ZFAS1 by siRNA, the viability of VSMCs was detected by MTT assay, and the proliferation was measured by EdU staining. The migration ability of VSMCs was detected by Transwell method. The expression levels of miR-150 and ROCK1 were detected by RT-qPCR, and the protein level of ROCK1 was determined by Western blot. Luciferase reporter assay was used to confirm that ROCK1 was the target gene of miR-150. Finally, miR-150 expression was inhibited, and the proliferation and migration ability of VSMCs and expression of ROCK1 after down-regulation of ZFAS1 expression were examined. RESULTS: PDGF-BB up-regulated the expression of ZFAS1 in the VSMCs. After down-regulating the expression of ZFAS1, the proliferation and migration abilities of VSMCs were inhibited(P<0.05), the expression level of miR-150 was increased(P<0.05), and the expression level of ROCK1 was decreased(P<0.05). The results of luciferase reporter assay showed that miR-150 directly targeted ROCK1. Inhibition of miR-150 expression attenuated the inhibition of proliferation and migration of VSMCs by ZFAS1 expression knock-down(P<0.05) and up-regulated the expression level of ROCK1(P<0.05). CONCLUSION: ZFAS1 promotes the proliferation and migration of VSMCs induced by PDGF-BB by regulating miR-150/ROCK1.