Objective: To investigate the regulation and possible mechanism of microRNA (miR)-1249 on myocardial apoptosis in chronic intermittent hypoxia rats. Methods: A total of 16male SD rats aged 8 weeks were randomly divided into 2 groups by the random number table: normoxia control group and chronic intermittent hypoxia group (CIH) (n=8 each). The CIH group was exposed to intermittent hypoxia every day from 9: 00 to 17: 00 for 8 consecutive weeks, while the control group received the same frequency of pulse air. Hemodynamic values were measured via a cannula inserted into right common carotid artery. The expressions of miR-1249 and microtubule-associated protein light chain 3 (LC3) mRNA were observed by real-time PCR. The expressions of LC3 and Cleaved Caspase-3 were detected by Western bolt. TUNEL staining was performed to detect myocardial apoptosis. The rat cardiomyocyte cell H9C2 was divided into normoxia group, intermittent hypoxia (IH) group and miR-1249 inhibitor transfected and IH treatment group (inhibitor group). At the end of the experiment, the activation of LC3 protein in each group of cells was determined. Results: Compared with normoxia control group, left ventricle end diastolic pressure (LVEDP) increased [(4.6±0.4) vs (2.2±0.1) mmHg (1 mmHg=0.133 kPa)], left ventricular systolic pressure (LVSP) , maximal rate of pressure decline (-dp/dtmax), and maximal rate of pressure development (+ dp/dtmax) decreased in CIH group [(92.7±4.1) vs (135.3±3.2) mmHg, (4 247±108) vs (7 626±235) mmHg/s, and (3 168±105) vs (6 028±81) mmHg/s] (all P<0.001). The expression of miR-1249 and LC3 mRNA were significantly higher in CIH group than that in normoxia control group (all P<0.001), and a positive correlation was found between the expression of LC3 mRNA and miR-1249. The expression of LC3 and Cleaved Caspase-3 protein in myocardial tissue of CIH rats were significantly higher than that of the normoxia control group (all P<0.001). The proportion of myocardial cell apoptosis in CIH rats was significantly higher than that in the normoxia control group [(23.84±4.94)% vs (2.93±0.73)%] (P<0.001). The activation of LC3 in myocardial cells of inhibitor group was significantly lower than that of IH group, but higher than that in normoxia group. Conclusions: CIH could induce LC3 by raising the expression of miR-1249, and then induce the activation of apoptosis protein Caspase3. It ultimately induces myocardial apoptosis.
目的： 探讨微RNA(miR)－1249调控慢性间歇低氧(CIH)大鼠心肌细胞凋亡的可能机制。 方法： 采用随机数字表法将16只8周龄雄性SD大鼠分为常氧对照组、CIH组各8只。CIH组每天9：00－17：00时给予连续8周间歇低氧处理，常氧对照组同时给予相同频率的脉冲空气处理。实验结束时通过颈动脉插管测定大鼠血流动力学指标，实时荧光定量PCR测定心肌组织中miR－1249及微管相关蛋白轻链3(LC3)mRNA的相对表达量，Western印迹法检测心肌组织中LC3及活化型半胱氨酸天冬氨酸蛋白酶3(Caspase－3)的表达情况，TUNEL染色检测心肌凋亡情况。将大鼠心肌细胞株H9C2分为常氧组、间歇低氧(IH)组及转染miR－1249抑制剂并给予IH处理组(抑制剂组)，实验结束时测定各组细胞LC3蛋白活化情况。 结果： CIH大鼠左室舒张末压显著高于常氧对照组[(4.6±0.4)比(2.2±0.1)mmHg(1 mmHg＝0.133 kPa)]，而左室收缩压、左室内压最大下降速率、左室内压最大上升速率均显著低于常氧对照组[(92.7±4.1)比(135.3±3.2)mmHg、(4 247±108)比(7 626±235)mmHg/s、(3 168±105)比(6 028±81)mmHg/s](均P<0.001)。CIH大鼠心肌组织miR－1249相对表达量显著高于常氧对照组，LC3 mRNA相对表达量显著高于常氧对照组(均P<0.001)，且两者间呈正相关；CIH大鼠心肌组织中LC3及活化型Caspase－3蛋白相对表达量均显著高于常氧对照组(均P<0.001)；CIH大鼠心肌细胞凋亡比例显著高于常氧对照组[(23.84±4.94)%比(2.93±0.73)%](P<0.001)。抑制剂组心肌细胞LC3活化程度显著低于IH组，但高于常氧组。 结论： CIH可通过上调miR－1249表达诱导LC3蛋白表达，进而诱导凋亡蛋白Caspase－3活化，最终诱导大鼠心肌凋亡。.
Keywords: Apoptosis; Autophagy; Cell hypoxia; Micro RNAs; Rats.