Cai Jiumei, Zhang Zhiwei, Chen Lingling, Wang Xiaoping, Zhong Yiming, Xie Dongyang, Liao Wei
Department of Cardiology, First Affiliated Hospital of Gannan Medical University, Ganzhou, China.
Department of Pediatric Cardiology, Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangzhou, China.
J Biochem Mol Toxicol. 2024 Dec;38(12):e70085. doi: 10.1002/jbt.70085.
To investigate the impact of LncRNA 93358 on ischemia-reperfusion induced myocardial cell apoptosis and the underlying mechanism. After being subjected to hypoxia for 4 h, three models of hypoxia-reoxygenation (H/R) with reoxygenation times of 8, 16, and 24 h were established. The expression of LncRNA 93358 was detected by qPCR, and the most suitable conditions were selected for subsequent experiments. The LncRNA 93358 knockout rat myocardial cells were established by transfecting with shRNA-93358 and identified by the RT-PCR assay, followed by constructing the in vitro H/R model. H/R myocardial cells were treated with blank medium (Model), shRNA-NC (LncRNA 93358 NC), shRNA-93358 (LncRNA 93358 knock), and shRNA-93358 + LY294002 (LncRNA 93358 knockout+LY294002), respectively. Normal myocardial cells treated with blank medium was taken as the control group. The cell cycle and apoptosis were analyzed by the flow cytometry. The level of cellular SOD and MDA was measured by the ELISA assay. The expression level of LncRNA 93358 was determined by the RT-PCR assay and Western blot assay was utilized to evaluate the expression level of AKT1, p-AKT1, mTOR, p-mTOR, bcl-2, and Bax. Compared to control, the expression of LncRNA 93358 in H9C2 cells was significantly increased under hypoxic conditions for 4 h followed by reoxygenation for 8 h/16 h. Moreover, the expression of LncRNA 93358 was relatively higher under hypoxic conditions for 4 h followed by reoxygenation for 16 h. Compared to control, significantly lower p-mTOR/mTOR and p-AKT1/AKT1 level was observed in the model group, accompanied by the elevated MDA level, declined SOD level, increased apoptotic rate, enhanced arrest at S phase, upregulated Bax, and downregulated Bcl-2. Compared to the model and LncRNA 93358 NC group, the expression level of p-mTOR/mTOR and p-AKT1/AKT1 was significantly promoted in the LncRNA 93358 knock group, accompanied by the declined MDA level, increased SOD level, reduced apoptotic rate, increased arrest at G0/G1 phase, downregulated Bax, and upregulated Bcl-2, which were dramatically reversed in the LncRNA 93358 knockout+LY294002 group. LncRNA 93358 aggravated the apoptosis of myocardial cells after ischemia-reperfusion by mediating the PI3K/AKT/mTOR pathway.
探讨长链非编码RNA 93358对缺血再灌注诱导的心肌细胞凋亡的影响及其潜在机制。在缺氧4小时后，建立再氧合时间分别为8、16和24小时的三种缺氧复氧（H/R）模型。通过qPCR检测长链非编码RNA 93358的表达，并选择最适宜的条件用于后续实验。通过转染shRNA-93358建立长链非编码RNA 93358基因敲除大鼠心肌细胞，并通过RT-PCR检测进行鉴定，随后构建体外H/R模型。H/R心肌细胞分别用空白培养基（模型组）、shRNA-NC（长链非编码RNA 93358阴性对照）、shRNA-93358（长链非编码RNA 93358敲除组）和shRNA-93358 + LY294002（长链非编码RNA 93358基因敲除+LY294002）处理。用空白培养基处理的正常心肌细胞作为对照组。通过流式细胞术分析细胞周期和凋亡情况。采用ELISA法检测细胞内超氧化物歧化酶（SOD）和丙二醛（MDA）水平。通过RT-PCR法测定长链非编码RNA 93358的表达水平，利用蛋白质免疫印迹法评估AKT1、p-AKT1、mTOR、p-mTOR、bcl-2和Bax的表达水平。与对照组相比，在缺氧4小时后再氧合8小时/16小时的条件下，H9C2细胞中长链非编码RNA 93358的表达显著增加。此外，在缺氧4小时后再氧合16小时的条件下，长链非编码RNA 93358的表达相对较高。与对照组相比，模型组中p-mTOR/mTOR和p-AKT1/AKT1水平显著降低，同时MDA水平升高、SOD水平下降、凋亡率增加、S期阻滞增强、Bax上调、Bcl-2下调。与模型组和长链非编码RNA 93358阴性对照组相比，长链非编码RNA 93358敲除组中p-mTOR/mTOR和p-AKT1/AKT1的表达水平显著升高，同时MDA水平下降、SOD水平升高、凋亡率降低、G0/G1期阻滞增加、Bax下调、Bcl-2上调，而在长链非编码RNA 93358基因敲除+LY294002组中这些变化显著逆转。长链非编码RNA 93358通过介导PI3K/AKT/mTOR信号通路加重缺血再灌注后心肌细胞的凋亡。
