lncRNA KCNQ1OT1调控miR-384/CACNA1C轴对心肌细胞增殖、凋亡的影响

Effect of lncRNA KCNQ1OT1 on cardiomyocyte proliferation and apoptosis by regulating miR-384/CACNA1C axis

目的探讨长链非编码RNA钾电压门控通道亚家族Q成员1反向转录物1(lncRNA KCNQ1OT1)调控miR-384/L型钙离子通道α1C亚基基因(CACNA1C)轴对心肌细胞增殖、凋亡的影响。方法实时荧光定量PCR(qRT-PCR)、Western blot分别检测正常培养的大鼠心肌H9c2、CP-R073细胞及缺氧/复氧(H/R)诱导的H9c2、CP-R073细胞中的KCNQ1OT1、miR-384、CACNA1C蛋白表达。将H9c2细胞分为Ct组、Model组、si-NC组、si-KCNQ1OT1组、mimic NC组、miR-384 mimic组、si-KCNQ1OT1+inhibitor NC组、si-KCNQ1OT1+miR-384 inhibitor组。除Ct组外,其他组H9c2细胞均需转染对应物质后构建H/R损伤模型。qRT-PCR检测细胞中KCNQ1OT1、miR-384表达;CCK-8法、EdU染色检测细胞增殖;流式细胞术检测细胞凋亡;ELISA法检测细胞中肌红蛋白(Mb)、肌钙蛋白-Ⅰ(cTnⅠ)水平;Western blot检测CACNA1C、增殖细胞核抗原(PCNA)、半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)、Bcl-2相关X蛋白(Bax)蛋白表达;双荧光素酶报告基因实验验证KCNQ1OT1与miR-384、miR-384与CACNA1C的关系。结果H/R诱导的H9c2、CP-R073细胞中KCNQ1OT1、CACNA1C蛋白表达升高,miR-384表达降低,且H/R诱导的H9c2细胞中KCNQ1OT1、CACNA1C蛋白表达最高,miR-384表达最低,因此,后续选择H9c2细胞为研究对象。与Ct组比较,Model组细胞中KCNQ1OT1、细胞凋亡率、Mb、cTnⅠ水平、CACNA1C、Caspase-3、Bax蛋白表达升高,miR-384表达、D 450值、EdU阳性率、PCNA蛋白表达降低(P<0.05);沉默KCNQ1OT1或过表达miR-384可促进H/R诱导的H9c2细胞增殖,抑制细胞凋亡;miR-384 inhibitor减弱了沉默KCNQ1OT1对H/R诱导的H9c2细胞增殖的促进作用,以及对细胞凋亡的抑制作用;KCNQ1OT1靶向调控miR-384/CACNA1C轴。结论沉默KCNQ1OT1可能通过上调miR-384来抑制CACNA1C表达,进而促进H/R诱导的H9c2细胞增殖,抑制细胞凋亡。

Objective To investigate the effect of long non-coding RNA potassium voltage-gated channel subfamily Q member 1 reverse transcript 1(lncRNA KCNQ1OT1)on cardiomyocyte proliferation and apoptosis by regulating miR-384/calcium channel,L type,alpha 1C subunit gene(CACNA1C)axis.Methods Real-time quantitative PCR(qRT-PCR)and Western blot were used to detect the protein expression of KCNQ1OT1,miR-384,and CACNA1C in normal cultured rat myocardial H9c2 and CP-R073 cells and in H9c2 and CP-R073 cells induced by hypoxia/reoxygenation(H/R).H9c2 cells were divided into the following groups:a Ct group,a Model group,a si-NC group,a si-KCNQ1OT1 group,a mimic NC group,a miR-384 mimic group,a si-KCNQ1OT1+inhibitor NC group,and a si-KCNQ1OT1+miR-384 inhibitor group.Except for the Ct group,H9c2 cells in other groups were transfected with the corresponding substances to construct a H/R injury model.Furthermore,qRT-PCR was applied to detect the expression of KCNQ1OT1 and miR-384 in cells.The CCK-8 assay and EdU staining were used to detect cell proliferation.Flow cytometry was used to detect apoptosis.ELISA method was used to detect the levels of myoglobin(Mb)and cardiac troponin-Ⅰ(cTnⅠ).Western blot was used to detect the protein expression of CACNA1C,proliferating cell nuclear antigen(PCNA),cysteine-containing aspartate-specific proteases-3(Caspase-3),Bcl-2-associated X protein(Bax).Dual-luciferase reporter gene assay was used to verify the relationship between KCNQ1OT1 and miR-384,and between miR-384 and CACNA1C.Results The protein expression of KCNQ1OT1 and CACNA1C in H/R-induced H9c2 and CP-R073 cells increased,and the expression of miR-384 decreased,and H/R-induced H9c2 cells had the highest protein expression of KCNQ1OT1 and CACNA1C,and the lowest expression of miR-384.Therefore,H9c2 cells were selected for the subsequent studies.Compared with the Ct group,the Model group showed increases in the expression of KCNQ1OT1,apoptotic rate,the levels of Mb and cTnⅠ,and the expression of CACNA1C,Caspase-3 and Bax,and decreases in the expression of miR-384,D 450 value,EdU positive rate,and the protein expression of PCNA(P<0.05).Silencing KCNQ1OT1 or overexpressing miR-384promoted H/R-induced H9c2 cell proliferation and inhibited cell apoptosis.Moreover,miR-384 inhibitor attenuated the promoting effects of KCNQ1OT1 silencing on H/R-induced H9c2 cell proliferation and the inhibitory effect on apoptosis.KCNQ1OT1 targeted and regulated the miR-384/CACNA1 C axis.Conclusions Silencing KCNQ1OT1 may inhibit the expression of CACNA1C by up-regulating miR-384,thereby promoting H/R-induced H9c2 cell proliferation and inhibiting cell apoptosis.