干扰STX18-AS1通过靶向miR-204保护心肌细胞

郭春棉; 廉坤; 刘彬

doi:10.12125/j.chj.202207070

摘要:

目的探讨干扰长链非编码RNA STX18-AS1（long non-coding RNA, LncRNA STX18-AS1）是否通过上调微小RNA-204（miR-204）的表达从而影响心肌细胞缺氧损伤。

方法体外培养心肌细胞H9C2，采用二氯化钴（CoCl₂）处理心肌细胞建立细胞缺氧损伤模型，采用qRT-PCR检测CoCl₂处理不同时间点STX18-AS1、miR-204的表达量；实验设置对照组、模型组、si-NC组、si-STX18-AS1组、miR-NC组、miR-204组。采用甲基噻唑基四唑（MTT）检测细胞增殖活性；流式细胞术与TUNEL检测细胞凋亡率；采用生化试剂盒检测乳酸脱氢酶（LDH）活性、丙二醛（MDA）水平、超氧化物歧化酶（SOD）活性、过氧化氢酶（CAT）活性；双荧光素酶报告实验验证STX18-AS1、miR-204的靶向关系。

结果与对照组比较，在CoCl₂处理6 h、12 h、18 h、24 h时模型组和si-NC组STX18-AS1表达水平升高（P<0.01），miR-204表达水平降低（P<0.01）；与模型组、si-NC组比较，在CoCl₂处理6 h、12 h、18 h、24 h时si-STX18-AS1组STX18-AS1的表达水平降低（P<0.01），miR-204的表达水平升高（P<0.01）；与对照组比，模型组、si-NC组细胞增殖活性降低（P<0.01），细胞凋亡率及LDH活力、MDA的水平升高（P<0.01），SOD、CAT的活力降低（P<0.01）；与模型组、si-NC组比较，si-STX18-AS1组细胞增殖活性升高（P<0.01），细胞凋亡率及LDH活力、MDA的水平降低（P<0.01），SOD、CAT的活力升高（P<0.01）；转染miR-204 mimics对心肌细胞增殖活性、凋亡及氧化应激的作用与转染si-STX18-AS1的作用相同；双荧光素酶报告实验证实STX18-AS1可靶向结合miR-204。

结论干扰STX18-AS1表达可能通过上调miR-204，抑制细胞凋亡、促进增殖，减轻CoCl₂诱导的心肌细胞氧化损伤。

Abstract:

AIM To investigate whether interfering with LncRNA STX18-AS1 can up-regulate the expression of miR-204 to affect the myocardial cell hypoxia injury.

METHODS The cardiomyocytes H9C2 were cultured in vitro and the cardiomyocytes were treated with CoCl₂ to establish a model of cell hypoxia injury. The expression levels of STX18-AS1 and miR-204 at different time points of CoCl₂ treatment were detected by qRT-PCR. The experiment set up control group, model group, si-NC group, si-STX18-AS1 group, miR-NC group and miR-204 group. MTT was used to detect cell proliferation activity and flow cytometry and TUNEL were used to detect the apoptosis rate. The LDH activity, MDA level, SOD activity and CAT activity were detected using a biochemical kit and the targeting relationship of STX18-AS1 and miR-204 was verified by the dual luciferase report experiment.

RESULTS Compared with those in control group, the expression level of STX18-AS1 in model group and si-NC group was increased (P<0.01) and the expression level of miR-204 was decreased (P<0.01) at 6 h, 12 h, 18 h and 24 h treated with CoCl₂. Compared with those in si-NC group and si-NC group, the expression level of STX18-AS1 in si-STX18-AS1 group decreased (P<0.01) and the expression level of miR-204 increased (P<0.01) at 6 h, 12 h, 18 h and 24 h treated with CoCl₂. Compared with those in control group, the cell proliferation activity in model group and si-NC group decreased (P<0.01), apoptosis rate, LDH activity and MDA level were significantly increased (P<0.01) and the activity of SOD and CAT were significantly decreased (P<0.01). Compared with those in model group and si-NC group, the cell proliferation activity was significantly increased (P<0.01), cell apoptosis rate and the LDH activity and MDA level were notably reduced (P<0.01) and the activity of SOD and CAT were significantly increased (P<0.01). The effect of transfection of miR-204 mimics on the proliferation activity, apoptosis and oxidative stress of cardiomyocytes was the same as the effect of transfection of si-STX18-AS1. The dual luciferase report experiment confirmed that STX18-AS1 could target miR-204.

CONCLUSION Interfering with STX18-AS1 expression could up-regulate miR-204, inhibit cell apoptosis, promote proliferation and alleviate the oxidative damage of cardiomyocytes induced by CoCl₂.

干扰STX18-AS1通过靶向miR-204保护心肌细胞

Interfering with STX18-AS1 protects cardiomyocytes by targeting miR-204