LncRNA-H19靶向miR-145-5p通过JAK2/STAT3调节大鼠脑血管平滑肌细胞增殖和迁移

LncRNA-H19 targeting miR-145-5p regulates rat cerebral vascular smooth muscle cell proliferation and migration through JAK2/STAT3

目的探讨LncRNA-H19靶向miR-145-5p通过JAK2/STAT3信号通路调节TNF-α诱导大鼠脑血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖和迁移。方法培养VSMC,对照组的细胞正常培养,TNF-α组的细胞用100 ng/mL TNF-α处理;将si-NC、si-H19、miR-NC、miR-145-5p mimics转染至TNF-α诱导的VSMC,分别记为TNF-α+si-NC组、TNF-α+si-H19组、TNF-α+miR-NC组、TNF-α+miR-145-5p组;将si-H19和anti-miR-NC、si-H19和anti-miR-145-5p共转染至TNF-α诱导的VSMC,记为TNF-α+si-H19+anti-miR-NC组、TNF-α+si-H19+anti-miR-145-5p组。RT-qPCR法检测LncRNA-H19和miR-145-5p表达水平;MTT法检测细胞增殖能力;Transwell法检测细胞迁移能力;荧光素酶报告实验检测LncRNA-H19和miR-145-5p的靶向关系;Western blot法检测细胞周期蛋白D1(Cyclin D1)、p21、基质金属蛋白酶2(matrix metallo proteinase 2,MMP-2)、基质金属蛋白酶9(matrix metallo proteinase 9,MMP-9)、磷酸化JAK2(phosphorylated JAK2,p-JAK2)、磷酸化STAT3(phosphorylated STAT3,p-STAT3)蛋白表达水平。结果与对照组比较,LncRNA-H19在TNF-α组中的表达水平显著升高,miR-145-5p在TNF-α组中的表达水平显著降低(P<0.01)。与TNF-α+si-NC组比较,TNF-α+si-H19组H19表达水平显著降低,OD值、Cyclin D1、细胞迁移数、MMP-2及MMP-9蛋白表达水平显著降低,P21蛋白表达水平显著升高(P<0.01)。与TNF-α+miR-NC组比较,TNF-α+miR-145-5p组miR-145-5p表达水平显著降低,OD值、Cyclin D1、细胞迁移数、MMP-2及MMP-9蛋白表达水平显著降低,P21蛋白表达水平显著升高(P<0.01)。与TNF-α+si-H19+anti-miR-NC组比较,TNF-α+si-H19+anti-miR-145-5p组OD值、Cyclin D1、细胞迁移数、MMP-2及MMP-9蛋白表达水平显著升高,P21蛋白表达水平显著降低(P<0.05)。与TNF-α+si-NC组比较,TNF-α+si-H19组p-JAK2、p-STAT3蛋白表达水平显著降低(P<0.05);与TNF-α+si-H19+anti-miR-NC组比较,TNF-α+si-H19+anti-miR-145-5p组p-JAK2、p-STAT3蛋白表达水平显著升高(P<0.05)。结论干扰LncRNA-H19表达、上调miR-145-5p表达可抑制TNF-α组的VSMC增殖和迁移,其与阻断JAK2/STAT3信号通路的磷酸化有关。

Objective To investigate the LncRNA-H19 targeting miR-145-5p through JAK2/STAT3 signaling pathway to regulate TNF-α-induced rat cerebral vascular smooth muscle cell(VSMC)proliferation and migration.Methods Culture VSMC,cells in the control group were cultured normally,cells in the TNF-αgroup were treated with 100 ng/mL TNF-α;si-NC,si-H19,miR-NC,miR-145-5p mimics were transfected into TNF-α-induced VSMC,which were marked as TNF-α+si-NC group,TNF-α+si-H19 group,TNF-α+miR-NC group,TNF-α+miR-145-5p group;si-H19 and anti-miR-NC,si-H19 and anti-miR-145-5p were co-transfected into TNF-α-induced VSMC,and denoted as TNF-α+si-H19+anti-miR-NC group,TNF-α+si-H19+anti-miR-145-5p group.RT-qPCR was used to detect the expression levels of LncRNA-H19 and miR-145-5p;MTT was used to detect cell proliferation;Transwell was used to detect cell migration;luciferase reporter assay was used to detect the targeting relationship between LncRNA-H19 and miR-145-5p;Western blot was used to detect Cyclin D1,p21,matrix metallo proteinase 2(MMP-2),matrix metallo proteinase 9(MMP-9),phosphorylated JAK2(p-JAK2),phosphorylated STAT3(p-STAT3)protein expression levels.Results Compared with the control group,the expression level of LncRNA-H19 was significantly increased while miR-145-5p was significantly decreased in TNF-αgroup(P<0.01).Compared with the TNF-α+si-NC group,the H19 expression level in the TNF-α+si-H19 group was significantly reduced,and the OD value,Cyclin D1,cell migration number,MMP-2 and MMP-9 protein expression levels were significantly reduced,and the P21 protein expression level was significantly increased(P<0.01).Compared with the TNF-α+miR-NC group,the expression level of miR-145-5p in the TNF-α+miR-145-5p group was significantly decreased,while levels of OD value,Cyclin D1,cell migration number,MMP-2,MMP-9 protein were significantly decreased,and the expression level of P21 protein increased significantly(P<0.01).Compared with TNF-α+si-H19+anti-miR-NC group,the expression levels of OD value,Cyclin D1,cell migration number,MMP-2,MMP-9 protein were increased significantly in TNF-α+si-H19+anti-miR-145-5p group,and the expression level of P21 protein decreased significantly(P<0.05).Compared with the TNF-α+si-NC group,the p-JAK2 and p-STAT3 protein expression in the TNF-α+si-H19 group were significantly reduced(P<0.05);compared with TNF-α+si-H19+anti-miR-NC group,p-JAK2 and p-STAT3 protein expression in TNF-α+si-H19+anti-miR-145-5p group were significantly increased(P<0.05).Conclusion Interfering the expression of LncRNA-H19 and up-regulating the expression of miR-145-5p can inhibit the VSMC proliferation and migration of TNF-αgroup,which is related to blocking the phosphorylation of JAK2/STAT3 signaling pathway.