MicroRNA-489 通过抑制 SPIN1 促进心肌缺血再灌注损伤诱导的心肌细胞凋亡。
MicroRNA-489 promotes cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion injury through inhibiting SPIN1.
机构信息
Department of Emergency, The First People's Hospital of Changzhou, The Third Affiliated Hospital of Soochow University, Changzhou, China.
出版信息
Eur Rev Med Pharmacol Sci. 2019 Aug;23(15):6683-6690. doi: 10.26355/eurrev_201908_18559.
OBJECTIVE
To investigate whether microRNA-489 could promote cardiomyocyte apoptosis through targeting inhibition of SPIN1, thus participating in the development of myocardial ischemia-reperfusion injury.
MATERIALS AND METHODS
MicroRNA-489 expression in H9c2 cells induced with hypoxia/reoxygenation (H/R) was determined by quantitative real-time polymerase chain reaction (qRT-PCR). With microRNA-489 overexpression in H/R H9c2 cells, activities of lactate dehydrogenase (LDH), methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were detected using relative commercial kits, respectively. The regulatory effect of microRNA-489 on the proliferation and apoptosis of H/R H9c2 cells was assessed through cell counting kit-8 (CCK-8) assay and flow cytometry (FCM), respectively. Through conducting a dual-luciferase reporter gene assay, we evaluated the binding condition between microRNA-489 and SPIN1. Protein expressions of apoptotic genes in H/R H9c2 cells with microRNA-489 overexpression were determined by Western blot. Finally, the regulatory role of microRNA-489 in PI3K/AKT pathway was detected through Western blot.
RESULTS
QRT-PCR data showed that microRNA-489 was highly expressed in H/R H9c2 cells than those in normoxic control. Overexpression of microRNA-489 in H/R H9c2 cells increased the activities of LDH, MDA and GSH-PX, while decreased the activities of SOD. MicroRNA-489 overexpression markedly inhibited the proliferative rate but accelerated apoptosis of H/R H9c2 cells. Western blot results indicated that protein expressions of pro-apoptotic genes Bax and cytochrome C upregulated, whereas anti-apoptotic gene Bcl-2 downregulated after overexpression of microRNA-489 in H/R H9c2 cells. We confirmed that microRNA-489 could target to SPIN1 and inhibit its expression. After overexpression of microRNA-489 in H/R H9c2 cells, PI3K/AKT pathway was inhibited, which was further reversed by PI3K/AKT pathway agonist SC79. Besides, SC79 treatment also reversed the regulatory effect of overexpressed microRNA-489 on cellular behaviors of H/R H9c2 cells.
CONCLUSIONS
MicroRNA-489 inhibits the proliferation and accelerates apoptosis of cardiomyocytes after MIRI by targeting inhibition of SPIN1 via inactivating PI3K/AKT pathway. MicroRNA-489 may be a potential therapeutic target for MIRI.
目的
探讨微小 RNA-489 是否可以通过靶向抑制 SPIN1 促进心肌细胞凋亡，从而参与心肌缺血再灌注损伤的发生。
材料与方法
采用实时定量聚合酶链反应（qRT-PCR）检测缺氧/复氧（H/R）诱导的 H9c2 细胞中微小 RNA-489 的表达。用微小 RNA-489 过表达 H/R H9c2 细胞，分别用相对商业试剂盒检测乳酸脱氢酶（LDH）、甲烷二羧酸醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-PX）的活性。通过细胞计数试剂盒-8（CCK-8）检测和流式细胞术（FCM）分别评估微小 RNA-489 对 H/R H9c2 细胞增殖和凋亡的调控作用。通过双荧光素酶报告基因检测，评估微小 RNA-489 与 SPIN1 的结合情况。用 Western blot 检测微小 RNA-489 过表达的 H/R H9c2 细胞中凋亡基因的蛋白表达。最后，通过 Western blot 检测微小 RNA-489 在 PI3K/AKT 通路中的调控作用。
结果
qRT-PCR 数据显示，微小 RNA-489 在 H/R H9c2 细胞中的表达高于正常氧对照。H/R H9c2 细胞中微小 RNA-489 的过表达增加了 LDH、MDA 和 GSH-PX 的活性，而降低了 SOD 的活性。微小 RNA-489 的过表达显著抑制 H/R H9c2 细胞的增殖率，但加速了其凋亡。Western blot 结果表明，过表达微小 RNA-489 后，促凋亡基因 Bax 和细胞色素 C 的蛋白表达上调，而抗凋亡基因 Bcl-2 的蛋白表达下调。我们证实微小 RNA-489 可以靶向 SPIN1 并抑制其表达。H/R H9c2 细胞中微小 RNA-489 过表达后，PI3K/AKT 通路被抑制，而 PI3K/AKT 通路激动剂 SC79 可进一步逆转这一作用。此外，SC79 处理还逆转了过表达微小 RNA-489 对 H/R H9c2 细胞细胞行为的调节作用。
结论
微小 RNA-489 通过靶向抑制 SPIN1 抑制 PI3K/AKT 通路的活性，抑制 MIRI 后心肌细胞的增殖并加速其凋亡。微小 RNA-489 可能是 MIRI 的潜在治疗靶点。
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