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微小RNA-124-3p通过靶向抑制RIPK1的表达对缺氧/复氧心肌细胞损伤中的保护作用及其机制研究

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摘要:

目的 探讨微小RNA-124-3p(miR-124-3p)调控受体相互作用蛋白激酶1(RIPK1)对心
肌细胞缺氧/复氧(H/R)损伤的保护作用。方法 大鼠心肌细胞随机分为Con组、H/R组、H/R+miR-124-
3p组、H/R+miR-NC组、H/R+si-RIPK1组、H/R+siNC组、H/R+miR-124-3p+pcDNA组、H/R+miR-124-
3p+pcDNA-RIPK1组。采用qRT-PCR法检测miR-124-3p与RIPK1 mRNA表达水平,Western blot法检测
RIPK1蛋白表达情况,双荧光素酶报告基因检测验证miR-124-3p与RIPK1的靶向关系。流式细胞术检测
细胞凋亡能力变化,Western blot法检测Bcl-2、Bax蛋白表达情况。检测乳酸脱氢酶(LDH)活性及丙二
醛(MDA)、超氧化物歧化酶(SOD)含量。 结果 H/R组心肌细胞miR-124-3p表达水平显著低于Con
组(P<0.05),而RIPK1 mRNA及蛋白水平均显著升高(P<0.05);H/R组心肌细胞LDH活性、MDA
水平及Bax蛋白水平均显著高于Con组(P<0.05),细胞凋亡率显著升高(P<0.05),而SOD水平及
Bcl-2蛋白水平均显著低于Con组(P<0.05);miR-124-3p过表达与抑制RIPK1表达可降低细胞凋亡率、
LDH活性、MDA水平及Bax蛋白水平,而提高SOD水平及Bcl-2蛋白水平;双荧光素酶基因检测结果证实
RIPK1是miR-124-3p的靶基因;RIPK1过表达逆转了miR-124-3p过表达对H/R心肌细胞损伤的作用。结
论 MiR-124-3p过表达可通过下调RIPK1表达进而减轻心肌细胞H/R损伤进而对心肌细胞发挥保护作用。

Abstract:

Objective To investigate the preventive effect of microRNA-124-3p (miR-124-3p) on hypoxia/
reoxygenation (H/R) injury in cardiomyocytes through regulating receptor-interacting protein kinases1 (RIPK1).
Methods Rat cardiomyocytes were divided randomly into Con group, H/R group, H/R+miR-124-3p group,
H/R+miR-NC group H/R+si-RIPK1, H/R+siNC group, H/R+miR-124-3p+pcDNA group and H/R+miR-124-
3p+pcDNA-RIPK1 group. The mRNA expressions of miR-124-3p and RIPK1 were detected by using qRTPCR,
and protein expression of RIPK1 was detected by using Western blotting assay. The targeting relationship
between miR-124-3p and RIPK1 was detected by using dual-luciferase reporter gene assay. The apoptosis ability
was detected by using flow cytometry (FCM), and protein expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2-
associated X protein (Bax) were detected by using Western blotting assay. The activity of lactic dehydrogenase (LDH)
and content of malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. Results The expression
of miR-124-3p was significantly lower (P<0.05) and mRNA and protein expressions of RIPK1 were significantly
higher (P<0.05) in H/R group than those in Con group. LDH activity and levels of MDA and Bax were significantly
higher (P<0.05), apoptosis rate was significantly higher (P<0.05), and levels of SOD and Bcl-2 were significantly
lower (P<0.05) in H/R than those in Con group. The overexpression of miR-124-3p and inhibition of RIPK1
expression decreased apoptosis, LDH activity and levels of MDA and Bax, and increased levels of SOD and Bcl-2.
The results of dual-luciferase reporter gene assay showed that RIPK1 was the targeting gene of miR-124-3p, and
overexpression of RIPK1 reversed the damage effect of overexpression of miR-124-3p on H/R cardiomyocytes.
Conclusion The overexpression of MiR-124-3p can relieve H/R injury in cardiomyocytes through down-regulating
RIPK1 expression, and play a protective role to cardiomyocytes.

基金项目:

青海省卫生计生系统科研项目(2017wjzdx75)

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