Jiangxi Provincial Key Laboratory of Drug Targets and Drug Screening, School of Pharmaceutical Science, Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Institute of Geriatrics, Jiangxi Provincial People's Hospital, First Affiliated Hospital of Nanchang Medical College, Nanchang, Jiangxi 330006, P.R. China.
Mol Med Rep. 2023 Apr;27(4). doi: 10.3892/mmr.2023.12977. Epub 2023 Mar 10.
Long noncoding RNAs (lncRNAs) are related to the development of atherosclerosis (AS). However, the role of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in tumor necrosis factor‑α (TNF‑α)‑induced rat aortic endothelial cell (RAOEC) pyroptosis, as well as the underlying mechanisms, remain unclear. RAOEC morphology was assessed using an inverted microscope. The mRNA and/or protein expression levels of MALAT1, microRNA(miR)‑30c‑5p and connexin 43 (Cx43) were assessed using reverse transcription‑quantitative PCR (RT‑qPCR) and/or western blotting, respectively. The relationships among these molecules were validated by dual‑luciferase reporter assays. Biological functions, such as LDH release, pyroptosis‑associated protein levels and the proportion of PI‑positive cells, were evaluated using a LDH assay kit, western blotting and Hoechst 33342/PI staining, respectively. The present study demonstrated that compared with the control group, the mRNA expression levels of MALAT1 and protein expression levels of Cx43 were significantly up‑regulated, whereas miR‑30c‑5p mRNA expressions levels were significantly decreased in TNF‑α‑treated RAOEC pyroptosis. Knockdown of MALAT1 or Cx43 significantly attenuated the increase in LDH release, pyroptosis‑associated protein expression and PI‑positive cell numbers among RAOEC treated using TNF‑α, whereas an miR‑30c‑5p mimic exerted the opposite effect. Furthermore, miR‑30c‑5p was demonstrated to be a negative regulator of MALAT1 and could also target Cx43. Finally, co‑transfection with siMALAT1 and miR‑30c‑5p inhibitor could attenuate the protective effect of MALAT1 knockdown against TNF‑α‑mediated RAOEC pyroptosis by upregulation of Cx43 expression. In conclusion, MALAT1 might serve an important role in TNF‑α‑mediated RAOEC pyroptosis by regulating the miR‑30c‑5p/Cx43 axis, which would provide a potential novel diagnostic and therapeutic target for AS.
长链非编码 RNA(lncRNA)与动脉粥样硬化(AS)的发生发展有关。然而,lncRNA 转移相关肺腺癌转录物 1(MALAT1)在肿瘤坏死因子-α(TNF-α)诱导的大鼠主动脉内皮细胞(RAOEC)细胞焦亡中的作用及其潜在机制尚不清楚。倒置显微镜观察 RAOEC 形态。采用逆转录-定量 PCR(RT-qPCR)和/或 Western blot 分别检测 MALAT1、微小 RNA(miR)-30c-5p 和连接蛋白 43(Cx43)的 mRNA 和/或蛋白表达水平。采用双荧光素酶报告基因检测验证这些分子之间的关系。采用 LDH 试剂盒检测 LDH 释放、焦亡相关蛋白水平和 PI 阳性细胞比例,Western blot 和 Hoechst 33342/PI 染色分别评估细胞的生物功能。研究结果表明,与对照组相比,TNF-α处理的 RAOEC 细胞焦亡中 MALAT1 的 mRNA 表达水平和 Cx43 的蛋白表达水平显著上调,而 miR-30c-5p 的 mRNA 表达水平显著下调。MALAT1 或 Cx43 的敲低显著减弱了 TNF-α处理的 RAOEC 中 LDH 释放、焦亡相关蛋白表达和 PI 阳性细胞数量的增加,而 miR-30c-5p 模拟物则产生相反的效果。此外,miR-30c-5p 是 MALAT1 的负调节剂,也可以靶向 Cx43。最后,共转染 siMALAT1 和 miR-30c-5p 抑制剂可通过上调 Cx43 表达来减弱 MALAT1 敲低对 TNF-α介导的 RAOEC 细胞焦亡的保护作用。综上所述,MALAT1 可能通过调节 miR-30c-5p/Cx43 轴在 TNF-α介导的 RAOEC 细胞焦亡中发挥重要作用,为 AS 的诊断和治疗提供新的靶点。