宫颈癌DExH-box解旋酶9抑制转录本来源的长链非编码RNA在高糖诱导的心肌细胞氧化应激损伤过程中的作用及其机制

Mechanisms of long non-coding RNA of cervical cancer DExH-box helicase 9 suppressive transcript in oxidative stress injury of cardiomyocytes induced by high glucose

目的探讨宫颈癌DExH-box解旋酶9抑制转录本来源的长链非编码RNA(lnc-CCDST)对高糖诱导的新生小鼠心肌细胞(NMC)中活性氧(ROS)生成和细胞凋亡的影响,以及微RNA-339-5p(miR-339-5p)负向调节lnc-CCDST的机制。方法①构建小鼠1型糖尿病模型后,检测其心肌细胞lnc-CCDST表达,体外实验采用高浓度葡萄糖(35 mmol/L)培养基刺激NMC后测定细胞内lnc-CCDST表达;②小干扰RNA(siRNA)抑制NMC中lnc-CCDST表达再予高糖刺激24 h,测定细胞内ROS含量、胱天蛋白酶-3(caspase-3)活性,以及DNA片段(DNA fragmentation);③过表达和抑制miR-339-5p,验证其参与lnc-CCDST的调节;④过表达miR-339-5p后再予高糖刺激,检测细胞内ROS生成、caspase-3活性,以及DNA fragmentation;⑤过表达lnc-CCDST和(或)miR-339-5p,分析miR-339-5p对心肌细胞氧化应激损伤的保护作用。结果糖尿病小鼠心肌组织中和高糖诱导后的NMC中lnc-CCDST的表达分别较相应的对照组均显著增加(P值均<0.01);高糖诱导后NMC的ROS生成、caspase-3活性和DNA fragmentation均显著增加(P值均<0.05),而抑制lnc-CCDST表达可减少高糖诱导的ROS生成、caspase-3活性和DNA fragmentation增加(P值均<0.05)。在NMC中过表达miR-339-5p,可显著抑制lnc-CCDST表达;抑制miR-339-5p可促进lnc-CCDST表达(P值均<0.05)。此外,过表达miR-339-5p能够显著抑制高糖诱导的ROS生成、caspase-3活性和DNA fragmentation增加(P值均<0.05),而此作用可被过表达lnc-CCDST所抵消。结论lnc-CCDST参与高糖诱导的心肌细胞氧化应激损伤过程,而这一作用受miR-339-5p负向调节。

Objective To investigate the effect of long non-coding RNA of cervical cancer DExH-box helicase 9 suppressive transcript(lnc-CCDST)on reactive oxygen species(ROS)production and apoptosis in neonatal mouse cardiomyocytes(NMCs)induced by high glucose,and the mechanisms of microRNA-339-5p(miR-339-5p)negatively regulating lnc-CCDST.Methods①The expression of lnc-CCDST in myocardium was detected after establishment of type 1 diabetes mellitus mouse model,and also in NMCs stimulated by high glucose(HG,35 mmol/L)medium in vitro.②The expression of lnc-CCDST in NMCs was inhibited by siRNA and then stimulated with high glucose for 24 h.Reactive oxygen species(ROS)production,caspase-3 activity and DNA fragmentation were measured.③The expression of lnc-CCDST in NMCs was measured after overexpression or inhibition of miR-339-5p in order to confirm the regulatory effect of miR-339-5p on lnc-CCDST.④Overexpression of miR-339-5p was followed by high glucose stimulation.Then ROS production,caspase-3 activity and DNA fragmentation were detected,respectively.⑤Overexpression of lnc-CCDST was employed to analyze the protective effect of miR-339-5p on oxidative stress injury of myocardial cells.Results Compared with normal control,the expression of lnc-CCDST was significantly increased in myocardial tissue of diabetic mice and NMCs induced by high glucose(both P<0.01).ROS production,caspase-3 activity and DNA fragmentation in NMCs were significantly increased with high glucose induction(all P<0.05),while inhibition of lnc-CCDST reversed these changes(all P<0.05).The overexpression of miR-339-5p in NMCs,obviously prevented lnc-CCDST,and the inhibition of miR-339-5p could promote lnc-CCDST.Furthermore,overexpression of miR-339-5p significantly decreased ROS production,caspase-3 activity and DNA fragmentation induced by high glucose(all P<0.05),while the role of miR-339-5p in reducing oxidative stress injury and apoptosis of NMCs could be offset by overexpression of lnc-CCDST.Conclusion lnc-CCDST is involved in oxidative stress injury of cardiomyocytes induced by high glucose,and this effect is negatively regulated by miR-339-5p.