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龚翠兰,杨仁义,傅馨莹,周德生.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[J].湖南中医药大学学报,2022,42(3):373-379[点击复制] |
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| 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 |
| 龚翠兰,杨仁义,傅馨莹,周德生 |
| (湖南中医药大学, 湖南 长沙 410208;湖南中医药大学附属常德医院, 湖南 常德 415000;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 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。 |
| 关键词: LncRNA-H19 miR-145-5p JAK2/STAT3信号通路 TNF-α 血管平滑肌细胞 增殖 迁移 |
| DOI：10.3969/j.issn.1674-070X.2022.03.005 |
| 投稿时间：2021-04-20 |
| 基金项目:国家自然科学基金项目（81874463）；湖南省科技厅科技创新平台与人才计划（2017SK4005）；湖南省财政中医药项目名院工程（rsk-010-013/006-09）；湖南省教育厅一般项目（20C1405）；湖南省卫健委科研项目（202103071190）；湖南省中医药管理局资助项目（202038,202046,2021218）；湖南省研究生创新项目（CX20190587）；湖南中医药大学中西医结合一流学科开放基金项目（2019ZXYJH08,2018ZXYJH10）；湖南中医药大学研究生培养质量工程专项（2020CX62）。 |
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| LncRNA-H19 targeting miR-145-5p regulates rat cerebral vascular smooth muscle cell proliferation and migration through JAK2/STAT3 |
| GONG Cuilan,YANG Renyi,FU Xinying,ZHOU Desheng |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Changde Hospital Affiliated to Hunan University of Chinese Medicine, Changde, Hunan 415000, China;The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| 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-α group 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-α group (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-α group, which is related to blocking the phosphorylation of JAK2/STAT3 signaling pathway. |
| Key words: LncRNA-H19 miR-145-5p JAK2/STAT3 signaling pathway TNF-α vascular smooth muscle cell prolif?鄄eration migration |
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