微小RNA27a在利拉鲁肽改善心肌细胞凋亡中的作用

Role of microRNA-27a in the anti-apoptotic effects of liraglutide in cardiomyocytes

目的探讨微小RNA-27a（miR-27a）在利拉鲁肽改善心肌细胞凋亡中的作用。方法大鼠H9c2心肌细胞在不同培养条件下分为低糖培养组、高糖培养组、miR-27a过表达组、miR-27a抑制组、阴性miR-27a过表达组、阴性miR-27a抑制组、脂质体lipo2000组、高渗组、利拉鲁肽干预组、利拉鲁肽干预miR-27a过表达组、利拉鲁肽干预阴性miR-27a过表达组。采用实时定量聚合酶链反应检测及比较低糖、高糖培养和利拉鲁肽干预后H9c2心肌细胞miR-27a的表达；原位末端转移酶标记技术观察miR-27a模拟物或抑制剂转染心肌细胞后及利拉鲁肽干预前、后细胞凋亡变化并计算、比较细胞凋亡指数（AI），Western blotting法检测B细胞淋巴瘤/白血病2（Bcl-2）、细胞凋亡抑制蛋白1（cIAP-1）、Bcl-2相关X蛋白（Bax）、含半胱氨酸的天冬氨酸蛋白水解酶3（caspase-3）、凋亡蛋白酶活化因子1（Apaf-1）表达变化。采用单因素方差分析及t检验分析数据。结果与低糖组相比，高糖组H9c2心肌细胞miR-27a表达增加（35.8±4.9比1.0±0.0，t=12.199，P〈0.05）。与高糖组相比，miR-27a过表达组心肌细胞AI显著增加[78.5±6.6比22.9±1.7，最小显著性差异法（LSD法），P〈0.05]，同时Bcl-2、cIAP-1表达降低（LSD法，均P〈0.05），Bax、caspase-3、Apaf-1表达增高（LSD法，均P〈0.05）。与高糖组相比，miR-27a抑制组H9c2心肌细胞AI显著降低（2.6±0.4比22.9±1.7，LSD法，P〈0.05），同时Bcl-2、cIAP-1表达增加（LSD法，均P〈0.05），Bax、caspase-3、Apaf-1表达降低（LSD法，均P〈0.05）。与高糖组相比，利拉鲁肽干预组心肌细胞miR-27a表达明显被抑制（7.9±0.2比35.8±4.9，t=-9.785，P〈0.05），AI显著降低（4.9±0.1比22.9±1.7，LSD法，P〈0.05）。与利拉鲁肽组相比，利拉鲁肽干预miR-27a过表达组心肌细胞AI显著增高（61.3±5.1比4.9±0.1，LSD法，P〈0.05）。结论利拉鲁肽可改善高糖状态下心肌细胞凋亡，可能与其显著抑制心肌细胞miR-27a表达有关。

Objective To investigate the role of MieroRNA-27a （miR-27a） in the anti-apoptotic effects of liraglutide （LR） in cardiomyocytes. Methods The H9e2 rat eardiomyocytes were cultured in different environment and divided into low glucose group, high glucose group, miR-27a over-expression group, miR-27a inhibited group, miR-27a negative over-expression group, miR-27a negative inhibited group, liposome 2000 group, hypertonic solution group, LR intervention group, LR intervention and miR-27a over-expression group, LR intervention and miR-27a negative over-expression group. The expression of miR-27a in cardiomyocytes, which were cultured in low glucose solution, high glucose solution and interferenced by LR, was detected by fluorescence-based real-time quantitative polymerase chain reaction （PCR）. The apoptosis of cardiomyoeytes was detected by terminal-deoxynueleoitidyl trausferase mediated nick end labeling （TUNEL） and then the cell apoptosis index （AI） was computed and compared among the groups. The expression of apoptosis-related protein B-cell lymphoma/leukemia-2 （Bcl-2）, Bcl-2 associated X protein （Bax）, cysteine-containing aspartate-specific proteases-3 （caspase-3）, cellular inhibitors of apoptosis-1 （cIAP-1）, anti-apoptotic protease activating factor-1 （Apaf-1） was detected by Western blotting method. Data were analyzed with one-way analysis of variance test or t test. Results The expression of miR-27a in high glucose group was higher than that in low glucose group（35.8±4.9 vs 1.0±0.0, t=12.199, P〈 0.05）. Compared with the high glucose group, the AI of cardiomyocytes increased significantly in miR-27a over-expression group （78.5±6.6 vs 22.9± 1.7, least-significant difference （LSD）, P〈0.05）, and the protein expression of Bcl-2 and cIAP-1 decreased （LSD, both P〈0.05）, the protein expression of Bax, caspase-3 and Apaf-1 increased（LSD, all P〈0.05）. Compared with the high glucose group, the AI of cardiomyocytes decreased significantly in miR-27a inhibition group （2.6±0.4 vs 22.9± 1.7, LSD, P〈0.05）, and the protein expression level of Bcl-2 and cIAP-1 increased （LSD, both P〈0.05）, the protein expression level of Bax, caspase-3 and Apaf-1 significantly decreased（LSD, all P〈0.05）. Compared with the high glucose group, the expression level of miR-27a decreased significantly in LR intervention group （7.9 ± 0.2 vs 35.8± 4.9, t=-9.785, P〈0.05）, and the AI of cardiomyocytes decreased remarkably （4.9±0.1 vs 22.9± 1.7, LSD, P〈0.05）. The AI of cardiomyocytes increased in LR intervention and miR-27a over-expression group when compared with that in LR intervention group （61.3±5.1 vs 4.9±0.1, LSD, P〈0.05）. Conclusion LR may ameliorate the apoptosis of cardiomyocytes in the condition of high glucose by inhibiting the expression of miR-27a in H9c2 rat cardiomyocytes.