抑制lncRNA LUCAT1表达通过靶向调控miR-204-3p减轻高糖诱导的心肌H9c2细胞损伤

Knock-down of lncRNA LUCAT1 expression targets miR-204-3p to at⁃tenuate high glucose-induced injury in H9c2 cardiomyocytes

目的:探讨敲减长链非编码RNA肺癌相关转录本1(LUCAT1)表达减轻高糖(HG)诱导的心肌H9c2细胞损伤是否与其靶向调控微小RNA-204-3p(miR-204-3p)表达有关。方法:将体外培养的大鼠心肌H9c2细胞分为对照组、HG组、HG+siRNA-NC组、HG+siRNA-LUCAT1组、HG+siRNA-LUCAT1+inhibitor-NC组和HG+siR⁃NA-LUCAT1+miR-204-3p inhibitor组,采用生物信息学软件预测和双萤光素酶报告基因实验验证LUCAT1和miR-204-3p的靶向关系,RT-qPCR法检测细胞中LUCAT1和miR-204-3p的表达水平,CCK-8实验检测细胞活力,乳酸脱氨酶(LDH)试剂盒检测LDH漏出量,流式细胞术检测细胞凋亡,Western blot检测细胞中Bcl-2和Bax蛋白的表达水平,比色法检测超氧化物岐化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量。结果:生物信息学软件预测到LUCAT1和miR-204-3p之间存在互补的结合位点,双萤光素酶报告基因实验证实LUCAT1可与miR-204-3p靶向结合。与对照组比较,HG刺激后H9c2细胞中LUCAT1表达水平、LDH漏出量、细胞凋亡率、细胞中Bax蛋白表达水平和MDA含量均明显升高,而细胞中miR-204-3p表达水平、细胞活力、细胞中Bcl-2蛋白的表达水平及SOD和GSH-Px活性均明显降低(P<0.05)。与HG组比较,转染siRNA-LUCAT1可明显逆转HG引起的上述变化(P<0.05),但转染siRNA-NC对H9c2细胞无明显影响(P>0.05);与HG+siRNA-LUCAT1组比较,转染inhibitor-NC后H9c2细胞各指标无明显改变(P>0.05),但转染miR-204-3p inhibitor可明显逆转siRNA-LUCAT1对H9c2细胞的作用(P<0.05)。结论:抑制LUCAT1表达可通过靶向调控miR-204-3p表达抑制心肌H9c2细胞的凋亡和氧化应激,减轻HG诱导的心肌H9c2细胞损伤。

AIM:To investigate the effect of knock-down of long non-coding RNA(lncRNA)lung cancer-as⁃sociated transcript 1(LUCAT1)expression on the injury of H9c2 cardiomyocytes induced by high glucose(HG)and its re⁃lationship with targeted regulation of microRNA-204-3p(miR-204-3p)expression.METHODS:Rat H9c2 cardiomyo⁃cytes were divided into control group,HG group,HG+siRNA-NC group,HG+siRNA-LUCAT1 group,HG+siRNALUCAT1+inhibitor-NC group and HG+siRNA-LUCAT1+miR-204-3p inhibitor group.Bioinformatics software prediction and dual-luciferase reporter assay were used to verify the targeting relationship between LUCAT1 and miR-204-3p.The ex⁃pression of LUCAT1 and miR-204-3p was detected by RT-qPCR.The cell viability was measured by CCK-8 assay,lactate dehydrogenase(LDH)leakage was detected by LDH kit,and the apoptosis was analyzed by flow cytometry.The protein expression of Bcl-2 and Bax was determined by Western blot,and the activity of superoxide dismutase(SOD)and glu⁃tathione peroxidase(GSH-Px),and the content of malondialdehyde(MDA)were detected by colorimetry.RESULTS:Bioinformatics software prediction showed that there were complementary binding sites between LUCAT1 and miR-204-3p,and dual-luciferase reporter assay confirmed that LUCAT1 was able to match miR-204-3p for targeted binding.Com⁃pared with control group,the expression level of LUCAT1,LDH leakage,apoptotic rate,protein expression level of Bax and MDA content in H9c2 cells were significantly increased after HG stimulation,while miR-204-3p expression level,the cell viability,Bcl-2 protein expression level,and the activity of SOD and GSH-Px were significantly reduced(P<0.05).Compared with HG group,transfection with siRNA-LUCAT1 significantly reversed the above-mentioned changes caused by HG(P<0.05),and no significant effect on the H9c2 cells after transfection with siRNA-NC was observed(P>0.05).Compared with HG+siRNA-LUCAT1 group,no significant change in various indicators of the H9c2 cells after transfection with inhibitor-NC was found(P>0.05).However,transfection with miR-204-3p inhibitor significantly reverse the effects of siRNA-LUCAT1 on the H9c2 cells(P<0.05).CONCLUSION:Knock-down of LUCAT1 expression inhibits the apop⁃tosis and oxidative stress of H9c2 cardiomyocytes by targeting miR-204-3p to reduce the HG-induced injury.