LncRNA FGD5-AS1靶向miR-16-5p/CREB1轴减轻缺氧/复氧诱导大鼠H9c2心肌细胞凋亡的机制研究

LncRNA FGD5-AS1 Targeting miR-16-5p/CREB1 Axis Alleviates Hypoxia/Reoxygenation Induced Apoptosis of Rat H9c2 Cardiomyocytes

目的:探究长链非编码RNA(LncRNA)FGD5反义RNA1(FGD5-AS1)对缺氧/复氧(H/R)诱导的大鼠H9c2心肌细胞凋亡的影响以及对微小RNA-16-5p/cAMP响应元件结合蛋白1(miR-16-5p/CREB1)轴的调节机制。方法:将H9c2细胞分为对照组和H/R组(缺氧6 h,复氧6 h),然后将H/R组细胞分别进行转染,分为oe-NC组、oe-FGD5-AS1组、oe-FGD5-AS1+miR-16-5p mimic-NC组、oe-FGD5-AS1+miR-16-5p mimic组。实时荧光定量逆转录聚合酶链式反应法(RT-qPCR)检测细胞中FGD5-AS1、miR-16-5p、CREB1的mRNA水平;蛋白免疫印迹(Western Blot)法测定裂解凋亡蛋白酶-3(cleaved Caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达;CCK-8法测定细胞存活率;流式细胞术检测细胞凋亡率;双荧光素酶活性实验分别验证miR-16-5p和FGD5-AS1、CREB1的靶向关系。结果:与对照组比较,H/R组细胞中FGD5-AS1和CREB1的mRNA水平、细胞存活率、Bcl-2蛋白表达降低(P<0.05),miR-16-5p mRNA水平、细胞凋亡率及cleaved Caspase-3、Bax蛋白表达升高(P<0.05);与H/R组和oe-NC组比较,转染过表达FGD5-AS1基因的H9c2细胞中FGD5-AS1和CREB1的mRNA水平、细胞存活率、Bcl-2蛋白表达增高,凋亡率及miR-16-5p、cleaved Caspase-3、Bax水平下降(P<0.05)。双荧光素酶活性实验验证了FGD5-AS1、CREB1均与miR-16-5p有结合位点。结论:过表达FGD5-AS1可能通过靶向下调miR-16-5p表达,上调CREB1表达,抑制H/R诱导的H9c2心肌细胞凋亡。

Objective:To explore the effect of long non-coding RNA(LncRNA)FGD5 antisense RNA1(FGD5-AS1)on the apoptosis of rat H9c2 cardiomyocytes induced by hypoxia/reoxygenation(H/R)and the regulatory mechanism of microRNA-16-5p/cAMP response element-binding protein 1(miR-16-5p/CREB1)axis.Methods:H9c2 cardiomyocytes were divided into control group and H/R group(hypoxia for 6 h,reoxygenation for 6 h).H/R group cells were transfected separately,then divided into oe-NC group,oe-FGD5-AS1 group,oe-FGD5-AS1+miR-16-5p mimic-NC group,and oe-FGD5-AS1+miR-16-5p mimic group.Real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the mRNA levels of FGD5-AS1,miR-16-5p and CREB1 in cells.The expression of cleaved Caspase-3,B-cell lymphoma/leukemia-2(Bcl-2)and Bcl-2 associated X protein(Bax)were measured by Western Blot.Cell survival rate was measured by CCK-8 method.The apoptosis rate was detected by flow cytometry.The targeting relationship between miR-16-5p mRNA,FGD5-AS1 and CREB1 was verified by double luciferase activity assay.Results:Compared with the control group,mRNA levels of FGD5-AS1 and CREB1,cell survival rate,and Bcl-2 protein expression in H/R group decreased(P<0.05),miR-16-5p mRNA levels,apoptosis rate,and cleaved Caspase-3 and Bax protein expressions increased(P<0.05).Compared with H/R group and oe-NC group,mRNA levels of FGD5-AS1 and CREB1,cell survival rate,Bcl-2 protein expression increased in H9c2 cells transfected with FGD5-AS1 gene overexpression,the apoptosis rate,levels of miR-16-5p,cleaved Caspase-3,and Bax decreased(P<0.05).Dual luciferase activity test verified that FGD5-AS1 and CREB1 had binding sites with miR-16-5p.Conclusion:Overexpression of FGD5-AS1 may inhibit H/R-induced apoptosis of H9c2 cardiomyocytes through targeted down-regulation of miR-16-5p expression and up-regulation of CREB1 expression.