MiR-206在低氧诱导心肌细胞凋亡过程中的作用及其机制研究

发布时间：2018-07-17 19:50

【摘要】：背景与目的:心肌缺氧是冠心病的关键病理过程,常常导致心肌细胞的损伤和调亡,进而使心肌细胞失去生理功能。近年来研究发现,mi RNA在心肌细胞凋亡中的作用及其表达异常引起了广泛关注,mi R-206便是其中之一,它在心肌低氧应激下的表达会发生明显变化。我们实验室的前期研究发现,mi R-206在冠心病人循环血中的水平高于非冠心病人。然而,mi R-206在低氧诱导心肌细胞中的作用及其机制仍不清楚。因此,深入研究mi R-206在低氧诱导心肌细胞凋亡过程中的作用及其机制,将为临床靶向延缓心肌损害提供新的视角。方法:将心肌细胞H9c2置于1%O2、94%N2、5%CO2的三气培养箱内,模拟心肌缺氧;在缺氧环境下分别转染mi R-206模拟物agomir及其阴性对照agomirNC,mi R-206抑制剂antagomir及其阴性对照antagomir-NC;MTT法检测干扰后低氧环境下各组心肌细胞活力;Annexin V/PI双标法流式细胞术检测干扰后低氧环境下各组心肌细胞凋亡率;q RT-PCR法检测干扰后低氧环境下各组mi R-206的靶基因EGFR、HIF-1α和IGF-1的m RNA水平变化;Western Blot检测干扰后低氧环境下各组EGFR、HIF-1α和IGF-1蛋白表达水平变化。双荧光素酶报告基因法验证mi R-206与其靶基因EGFR和HIF-1α的m RNA 3’UTR的结合。结果:MTT法检测显示agomir组较agomir-NC组细胞活力增高,而antagomir组较antagomir-NC组细胞活力下降;流式细胞术检测显示agomir组较agomirNC组细胞凋亡率降低,而antagomir组较antagomir-NC组细胞凋亡率升高;q RT-PCR法检测发现干扰后各组EGFR、HIF-1α和IGF-1的m RNA水平无变化,但Western Blot结果显示agomir组较agomir-NC组EGFR和HIF-1α蛋白表达水平降低,而antagomir组较antagomir-NC组EGFR和HIF-1α蛋白表达水平升高。双荧光素酶报告基因检测显示mi R-206可以直接与EGFR和HIF-1α的m RNA 3’UTR结合。结论:Micro RNA-206在低氧诱导的心肌细胞损伤和凋亡过程中,发挥了保护心肌细胞的作用。这种抗损伤和凋亡的作用正是通过转录后水平负性调控EGFR和HIF-1α的表达发挥作用,这为延缓心肌损害提供了一个新的潜在的药物作用靶点。
[Abstract]:Background & AIM: myocardial hypoxia is a key pathological process of coronary heart disease. In recent years, it has been found that the role and abnormal expression of TMI RNA in cardiomyocyte apoptosis is one of them, and its expression will change obviously under hypoxic stress. Our previous laboratory study found that MMI R-206 levels in circulating blood were higher in patients with coronary heart disease than in patients with non-coronary heart disease. However, the role of MMI R-206 in hypoxia-induced cardiomyocytes and its mechanism remain unclear. Therefore, the further study of the role and mechanism of mi R-206 in hypoxia induced cardiomyocyte apoptosis will provide a new perspective for clinical targeted delay of myocardial damage. Methods: the cardiomyocytes H9c2 were placed in the three-air incubator of 1O _ 2, N _ 2 and N _ 2O _ 2 to simulate myocardial anoxia. Transfection of miR-206 analogue agomir and agomir-NCncncnmc-mi R-206 inhibitor antagomir in anoxic environment and antagomir-NCX assay in negative control group for detecting myocardial viability in hypoxic environment after interference and flow cytometry with Annexin V / Pi double labeled flow cytometry to detect the low level of myocardial cell viability after interference Apoptosis rate of cardiomyocytes in each group was determined by Q RT-PCR after hypoxia. The mRNA levels of EGFR- HIF-1 伪 and IGF-1 mRNA were detected by Western blot. The expression levels of HIF-1 伪 and IGF-1 protein were detected by Western blot. The binding of mi R-206 to its target genes EGFR and HIF-1 伪 was confirmed by double luciferase reporter gene method. Results the cell viability of agomir group was higher than that of agomir-NC group, while that of antagomir group was lower than that of antagomir-NC group, and the apoptosis rate of agomir group was lower than that of agomirNC group by flow cytometry. The expression of EGFR and HIF-1 伪 in antagomir group was lower than that in agomir-NC group, but the mRNA levels of HIF-1 伪 and IGF-1 in agomir group were lower than those in agomir-NC group. The expression of EGFR and HIF-1 伪 in antagomir group was higher than that in antagomir-NC group. The detection of double luciferase reporter gene showed that mi R-206 could directly bind to EGFR and HIF-1 伪 mRNA 3UTR. Conclusion\%\ This action of anti-injury and apoptosis is played by the negative regulation of EGFR and HIF-1 伪 expression at post-transcriptional level, which provides a new potential drug target for delaying myocardial injury.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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