Jiao Kunli, Cheng Jiahao, Wang Qi, Hao Mingxiu
Department of Cardiology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200092, China.
Department of Geriatrics, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, No. 160 Pujian Road, Pudong New Area, Shanghai, 200127, P.R. China.
J Bioenerg Biomembr. 2024 Dec;56(6):607-617. doi: 10.1007/s10863-024-10045-8. Epub 2024 Nov 14.
To explore the regulatory mechanism of lncRNA UCA1 and NRF2 in cardiomyocyte aging. In this study, we explored how lncRNA UCA1 regulates NRF2 and its effect on cardiomyocyte aging. H9c2 cardiomyocytes were cultured and treated with H2O2 to simulate cardiomyocyte aging in vitro. The expression levels of lncRNA UCA1 and NRF2 in cells were detected using qRT-PCR. Cell viability was assessed using the CCK8 assay, and cell aging was detected via Sa-β-gal staining. The levels of oxidative stress markers (SOD, MDA, ROS) and the expressions of ferroptosis-related proteins (ACSL4, TFR1, FTH1, GPX4) were measured. The regulatory mechanism between UCA1 and NRF2 was investigated using RIP-qPCR. Additionally, changes in m6A modification levels and the expression of m6A modification-related proteins in cells after UCA1 overexpression were analyzed by western blot. Our results indicate that H2O2 treatment significantly downregulated the expression of lncRNA UCA1 and NRF2. UCA1 overexpression promoted H9c2 cell proliferation, inhibited cell aging, increased SOD activity and the expression of FTH1 and GPX4 proteins, and decreased MDA and ROS content as well as ACSL4 and TFR1 protein expression. RIP-qPCR verified that UCA1 can promote the expression of NRF2 in cells. Overexpression of UCA1 significantly increased the expression of the demethylase FTO, leading to a reduction in m6A modification levels. Furthermore, there was significant enrichment between FTO and NRF2, and overexpression of FTO improved the expression of NRF2 protein in cells. Taken together, lncRNA UCA1 inhibits oxidative stress and ferroptosis, thereby preventing cardiomyocyte aging. This protective effect is likely mediated by increasing the expression of demethylase FTO and reducing mA modification, which promotes the expression of NRF2.
探讨长链非编码RNA UCA1和NRF2在心肌细胞衰老中的调控机制。在本研究中，我们探究了长链非编码RNA UCA1如何调控NRF2及其对心肌细胞衰老的影响。培养H9c2心肌细胞并用H2O2处理以在体外模拟心肌细胞衰老。使用qRT-PCR检测细胞中长链非编码RNA UCA1和NRF2的表达水平。使用CCK8法评估细胞活力，并通过衰老相关β-半乳糖苷酶染色检测细胞衰老。检测氧化应激标志物（超氧化物歧化酶、丙二醛、活性氧）水平以及铁死亡相关蛋白（酰基辅酶A合成酶长链家族成员4、转铁蛋白受体1、铁蛋白、谷胱甘肽过氧化物酶4）的表达。使用RNA免疫沉淀-定量聚合酶链反应研究UCA1和NRF2之间的调控机制。此外，通过蛋白质免疫印迹分析UCA1过表达后细胞中m6A修饰水平的变化以及m6A修饰相关蛋白的表达。我们的结果表明，H2O2处理显著下调长链非编码RNA UCA1和NRF2的表达。UCA1过表达促进H9c2细胞增殖，抑制细胞衰老，增加超氧化物歧化酶活性以及铁蛋白和谷胱甘肽过氧化物酶4蛋白的表达，并降低丙二醛和活性氧含量以及酰基辅酶A合成酶长链家族成员4和转铁蛋白受体1蛋白表达。RNA免疫沉淀-定量聚合酶链反应证实UCA1可促进细胞中NRF2的表达。UCA1过表达显著增加去甲基化酶FTO的表达，导致m6A修饰水平降低。此外，FTO和NRF2之间存在显著富集，FTO过表达提高细胞中NRF2蛋白的表达。综上所述，长链非编码RNA UCA1抑制氧化应激和铁死亡，从而预防心肌细胞衰老。这种保护作用可能是通过增加去甲基化酶FTO的表达并减少m6A修饰来介导的，这促进了NRF2的表达。
