LncRNA KCNQ1OT1调控miR-10a-5p加重缺氧复氧H9c2细胞损伤的机制

Effects of LncRNA KCNQ1OT1 on the hypoxia reoxygenation injury of H9c2 cells by regulating miR-10a-5p

目的探讨长非编码RNA KCNQ1重叠转录本1(KCNQ1OT1)调控缺氧复氧(H/R)心肌细胞(H9c2)增殖、凋亡的作用机制。方法成功建立H/R H9c2细胞模型;采用实时荧光定量反转录聚合酶链式反应(RT-qPCR)、细胞计数试剂盒(CCK8)、膜联蛋白V-异硫氰酸荧光素和碘化丙锭(Annexin V-FITC/PI)凋亡检测试剂盒、蛋白免疫印迹(Western blot)检测细胞中KCNQ1OT1、miR-10a-5p的表达、细胞活性、细胞凋亡率及P65、PHB、Bcl-2、Bax的蛋白表达;双荧光素酶报告基因检测实验检测细胞的荧光素酶活性。结果与H9c2组比较,H/R H9c2组细胞KCNQ1OT1表达显著升高,miR-10a-5p表达显著降低(P<0.05);过表达KCNQ1OT1或抑制miR-10a-5p具有相同的加重H/R对H9c2细胞的活性抑制、凋亡促进及P65、Bcl-2下调,PHB、Bax上调作用。miR-10a-5p明显抑制野生型KCNQ1OT1细胞的荧光活性,并负向调控KCNQ1OT1的表达。过表达miR-10a-5p部分逆转KCNQ1OT1对H/R H9c2细胞的活性抑制和凋亡促进作用。结论长链非编码RNA KCNQ1OT1抑制H/R H9c2细胞增殖,促进凋亡,其机制与靶向miR-10a-5p有关。

Objective To investigate the effects of long noncoding RNA KCNQ1 overlapping transcript 1(KCNQ1OT1)regulating the proliferation and apoptosis of hypoxia reoxygenation(H/R)cardiomyocytes(H9c2),and to explore its action mechanism.Methods The H/R H9c2 cell model was successfully established.Real time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR),cell counting Kit(CCK8),annexin V-fluorescein isothiocyanate and annexin V-FITC/PI apoptosis detection kit,Western blot(WB)were used to detect the expression levels of KCNQ1OT1,miR-10a-5p,cell activity,apoptosis rate and the expression levels of p65,PHB,Bcl-2 and Bax protein.Moreover Dual luciferase reporter gene assay was used to detect the luciferase activity of cells.Results Compared with those in H9c2 group,the expression levels of KCNQ1OT1 were significantly increased and the expression levels of miR-10a-5p were significantly decreased in H/R H9c2 group(P<0.05).The overexpression of KCNQ1OT1 or inhibition of miR-10a-5p could aggravate the inhibition of H/R activity,promotion of apoptosis,down-regulation of p65 and Bcl-2,and up-regulation of PHB and Bax in H9c2 cells.MiR-10a-5p significantly inhibited the fluorescence activity of wild-type KCNQ1OT1 cells and negatively regulated the expression of KCNQ1OT1.In addition overexpression of miR-10a-5p partially reversed the effects of KCNQ1OT1 on the activity inhibition and apoptosis promotion in H/R H9c2 cells.Conclusions Long chain noncoding RNA KCNQ1OT1 can inhibite the proliferation,and promote the apoptosis of H/R H9c2 cells,and its mechanism may be related to targeting miR-10a-5p.