miR-339通过靶向调控IGF2BP1抑制肺动脉平滑肌细胞的增殖

miR-339 suppresses proliferation of pulmonary arterial smooth muscle cells via regulating IGF2BP1

目的:本研究旨在探讨微小RNA-339(miR-339)在肺动脉平滑肌细胞(PASMCs)增殖中的作用并探讨其潜在机制。方法:利用不同浓度的血管紧张素Ⅱ处理PASMCs 48 h,通过转染miR-339模拟物(miR-339mimic)和miR-339抑制物(miR-339 inhibitor)分别使miR-339过表达或低表达,利用CCK-8法和活细胞计数检测细胞的增殖能力;利用RT-qPCR检测miR-339和PCNA mRNA的表达水平;利用Western blot检测蛋白水平;萤光素酶报告试验分析证实miR-339和IGF2BP1之间的相互作用。结果:血管紧张素II浓度依赖性地增加PASMCs的增殖和PCNA的mRNA表达,并降低miR-339的表达(P <0. 05)。miR-339过表达抑制PASMCs增殖和PCNA mRNA表达(P <0. 05),而miR-339表达下调则促进PASMCs的增殖和PCNA的mRNA表达(P <0. 05)。miR-339表达上调可以抑制血管紧张素II处理后的PASMCs的增殖(P <0. 05)。生物信息学分析以及萤光素酶报告试验检测证实胰岛素样生长因子2 mRNA结合蛋白1(IGF2BP1)的3’-UTR为miR-339靶点,进一步用RT-qPCR及Western blot实验发现miR-339负调控PASMCs中IGF2BP1的表达(P <0. 05)。IGF2BP1的过表达减弱了miR-339对PASMCs增殖和PCNA mRNA表达的抑制作用(P <0. 05)。miR-339 mimic转染可以抑制磷酸化p38蛋白的水平(P <0. 05),对p38蛋白的水平没有影响。结论:miR-339对PASMCs的增殖起抑制作用,这种抑制作用可能是部分通过调控IGF2BP1以及p38 MAPK信号通路来实现的。

AIM:The present study aimed to investigate the role of microRNA-339 miR-339 in the proliferation of pulmonary artery smooth muscle cells(PASMCs).METHODS:After treating with angiotensinⅡof different concentrations for 48 h,miR-339 mimic and miR-339 inhibitor were transfected into PASMCs,respectively.CCK-8 assay and viable cell counting were performed to determine cell proliferation.The expression levels of miR-339 and PCNA mRNA were measured by RT-qPCR.The protein levels were detected by Western blot.The interaction between miR-339 and insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1)was confirmed by luciferase reporter assay.RESULTS:Angiotensin II concentration-dependently increased cell proliferation and mRNA expression of PCNA,and decreased miR-339 expression in the PASMCs.Over-expression of miR-339 inhibited cell proliferation and mRNA expression of PCNA in the PASMCs,while mutation of miR-339 promoted cell proliferation and mRNA expression of PCNA in the PASMCs.In addition,miR-339 inhibited cell proliferation in angiotensin II-treated PASMCs.Bioinformatics analysis showed that miR-339 targeted the IGF2BP1 3’-UTR,which was confirmed by luciferase reporter assay,and miR-339 negatively regulated the expression of IGF2BP1 in the PASMCs.More importantly,over-expression of IGF2BP1 attenuated the inhibitory effects of miR-339 on cell proliferation and mRNA expression of PCNA in the PASMCs.miR-399 over-expression suppressed phosphorylated p38 protein level but not p38 protein level.CONCLUSION:miR-339 suppresses anti-proliferative effects in PASMCs partly via regulating IGF2BP1 and p38 MAPK signaling pathway.