基于LncRNA MIAT/miR-384-5p研究九龙藤黄酮抑制自噬抗心肌缺氧损伤的作用机制

Anti-myocardial hypoxia injury effect of Bauhinia championii flavones by inhibiting autophagy based on LncRNA MIAT/miR-384-5p

目的 基于长链非编码RNA(long non coding RNA,LncRNA)心肌梗死相关转录本(myocardial infarction associated transcript,MIAT)/miR-384-5p探讨九龙藤黄酮(Bauhinia championii flavones,BCF)抑制自噬抗心肌缺氧损伤的作用及机制。方法 培养H9c2心肌细胞及敲低LncRNA MIAT稳转株,建立心肌细胞氧糖剥夺(oxygen-glucose deprivation,OGD)模型模拟心肌缺血缺氧,给予BCF预处理;选取SD雄性大鼠,沉默miR-384-5p基因,建立急性心肌梗死(acute myocardial infarction,AMI)模型,给予BCF预处理。以qRT-PCR法检测LncRNA MIAT、miR-384-5p及自噬相关基因表达;采用双荧光素酶报告基因实验及RNA反义纯化(RNA antisense purification,RAP)实验验证LncRNA MIAT与miR-384-5p的靶向关系;采用ELISA法检测细胞上清液及大鼠血清中肌钙蛋白-I(cardiac troponin-I,c Tn-Ⅰ)水平;采用CCK-8法检测细胞活力;采用透射电镜观察自噬小体数量;以自噬双标腺病毒Ad-mRFP-GFP-LC3感染细胞检测自噬流;采用TTC染色法检测大鼠心肌梗死面积;采用Western blotting法检测自噬相关蛋白表达。结果 与OGD组比较,BCF能够下调LncRNA MIAT并上调miR-384-5p基因表达(P<0.05、0.01),提高心肌细胞活力(P<0.01),降低c Tn-I水平(P<0.001),下调Beclin1、Cathepsin D、LC3基因及蛋白表达(P<0.05、0.01),减少自噬小体数量(P<0.01),减轻自噬流(P<0.01)。双荧光素酶报告基因及RAP实验结果显示,LncRNA MIAT靶向负调控miR-384-5p表达。敲低LncRNA MIAT可抑制自噬,减轻心肌缺氧损伤;与单纯BCF预处理相比,敲低LncRNA MIAT可显著增强BCF抑制自噬和保护心肌的作用(P<0.05、0.01、0.001)。与AMI+BCF组比较,沉默miR-384-5p后给予BCF处理,大鼠心肌梗死面积显著增加(P<0.01),血清中c Tn-I水平显著升高(P<0.01),心脏组织中自噬相关基因及蛋白表达均显著上调(P<0.01),心肌缺血损伤加重。结论 BCF通过LncRNA MIAT/mi R-384-5p抑制自噬,从而减轻心肌缺氧损伤。

Objective To investigate the effect of Bauhinia championii flavones(BCF) on myocardial hypoxia injury by inhibiting autophagy based on long non-coding RNA(LncRNA) myocardial infarction associated transcript(MIAT)/miR-384-5p. Methods H9c2 cardiomyocytes and stable LncRNA MIAT knockdown strain were cultured, and cardiomyocyte oxygen-glucose deprivation(OGD) model was established to simulate myocardial ischemia and hypoxia, cells were pretreated with BCF. Male SD rats were selected to establish acute myocardial infarction(AMI) model by silencing miR-384-5p gene expression, and were pretreated with BCF. qRT-PCR was used to detect the expressions of LncRNA MIAT, miR-384-5p and autophagy-related genes;Dual luciferase reporter gene experiment and RNA antisense purification(RAP) experiment were used to verify that LncRNA MIAT and miR-384-5p targeting relationship;ELISA method was used to detect cardiac troponin-I(cTn-I) levels in supernatant of cells and serum of rats;CCK-8method was used to detect cell viability;Transmission electron microscopy(TEM) was used to observe the number of phagosomes;Autophagy flux was detected by infecting cells with autophagy double-labeled adenovirus Ad-mRFP-GFP-LC3;Myocardial infarction size was detected by TTC staining;Expressions of autophagy-related proteins was detected by Western blotting. Results Compared with OGD group, BCF down-regulated LncRNA MIAT and up-regulated miR-384-5p gene expressions(P < 0.05, 0.01), increased myocardial cells viability(P < 0.01), decreased cTn-I level(P < 0.001), down-regulated Beclin1, Cathepsin D, LC3 gene and protein expressions(P < 0.05, 0.01), decreased the number of autophagosomes(P < 0.01), and reduced autophagic flux(P < 0.01). The results of dual-luciferase reporter gene and RAP assay showed that LncRNA MIAT targeted and negatively regulated the expression of miR-384-5p. Knockdown of LncRNA MIAT could inhibit autophagy and alleviate myocardial hypoxia injury;Compared with BCF pretreatment, knockdown of LncRNA MIAT significantly enhanced the effect of BCF on autophagy inhibition and myocardial protection(P < 0.05, 0.01, 0.001). Compared with AMI + BCF group, after silencing miR-384-5p and then giving BCF treatment, myocardial infarction area of rats was significantly increased(P < 0.01), cTn-I level in serum was significantly increased(P < 0.01), autophagyrelated gene and protein expressions in cardiac tissue were significantly up-regulated(P < 0.01), and myocardial ischemia injury was aggravated. Conclusion BCF alleviates myocardial hypoxic injury by inhibits autophagy through LncRNA MIAT/miR-384-5p.