环状RNA在缺氧/复氧诱导的大鼠H9c2细胞焦亡中的作用及其机制

ROLE AND MECHANISM OF CIRCULAR RNA IN HYPOXIA/REOXYGENATION-INDUCED PYROPTOSIS OF RAT H9c2 CELLS

目的探讨环状RNA(circRNA)在缺氧/复氧(H/R)诱导的大鼠心肌细胞焦亡中的作用及其机制。方法将大鼠心肌细胞H9c2分为A~F组,分别进行缺氧0、1、3、6、12、24 h处理后,再进行6 h复氧,采用乳酸脱氢酶(LDH)试剂盒及Western blot方法检测H/R处理的H9c2细胞上清液中LDH活性和焦亡相关蛋白表达水平,确定诱导H9c2细胞焦亡的最佳缺氧时间。将H9c2细胞分为对照组和实验组,对照组H9c2细胞置于细胞培养箱中培养12 h,实验组H9c2细胞置于厌氧培养箱进行H/R处理,采用实时荧光定量PCR(RT-qPCR)方法检测两组H9c2细胞中circRNA的表达水平。将H9c2细胞分为G~I组,G组H9c2细胞置于细胞培养箱中培养12 h,H组转染对照siRNA(NC),I组转染mmu_circ_0000723特异性siRNA(siRNA-mmu_circ_0000723)。转染24 h后,采用RT-qPCR方法检测各组H9c2细胞中mmu_circ_0000723的表达水平。将H9c2细胞分为J~M组,J组H9c2细胞置于细胞培养箱中培养12 h,K组H9c2细胞置于厌氧培养箱进行H/R处理,L组和M组H9c2细胞分别转染对照NC和siRNA-mmu_circ_0000723,并于转染24 h后进行H/R处理。分别采用碘化丙啶(PI)染色、LDH试剂盒和Western blot方法检测J~M组的PI阳性细胞比率、细胞上清液中LDH活性,以及焦亡相关蛋白的表达水平。结果A~F组H9c2细胞上清液中LDH活性和焦亡相关蛋白的表达水平差异有显著性(F=24.86~2153.00,P<0.05)。与A组相比,D组H9c2细胞中LDH活性及焦亡相关蛋白的表达显著性差异最明显(t=4.14~62.88,P<0.05)。与对照组相比,实验组H9c2细胞中mmu_circ_0000723的表达水平明显降低(t=9.78,P<0.05)。G~I组H9c2细胞中mmu_circ_0000723的表达差异有显著性(F=348.20,P<0.05);与G组相比,I组H9c2细胞中mmu_circ_0000723相对表达水平显著降低(t=22.88,P<0.05)。J~M组PI阳性细胞比例、细胞上清液中LDH活性和焦亡相关蛋白的表达水平差异均有显著性(F=34.39~8528.00,P<0.05);与K组相比,M组H9c2细胞上述5个指标均明显升高(t=4.99~54.00,P<0.05),而L组细胞上述5个指标差异无显著性(P>0.05)。结论mmu_circ_0000723通过调控焦亡相关蛋白的表达,在H/R诱发的H9c2细胞焦亡中发挥重要作用。

Objective To investigate the role and mechanism of circular RNA(circRNA)in hypoxia/reoxygenation(H/R)-induced pyroptosis of rat cardiomyocytes.Methods Rat cardiomyocytes H9c2 were divided into groups A-F,which were exposed to hypoxia for 0,1,3,6,12,and 24 h,respectively,followed by reoxygenation for 6 h.Lactate dehydrogenase(LDH)kit and Western blot were used to measure LDH activity in the supernatant of H/R-treated H9c2 cells and the expression levels of pyroptosis-related proteins,and the optimal hypoxia time for inducing the pyroptosis of H9c2 cells was determined.H9c2 cells were divided into control group and experimental group;H9c2 cells in the control group were placed in a cell incubator for 12 h,and those in the experimental group were placed in an anaerobic incubator for H/R treatment.Quantitative real-time PCR was used to measure the expression level of circRNA in both groups.H9c2 cells were divided into groups G-I;H9c2 cells in group G were placed in a cell incubator for 12 h,those in group H were transfected with control siRNA(NC),and those in group I were transfected with mmu_circ_0000723-specific siRNA(siRNA-mmu_circ_0000723).At 24 h after transfection,quantitative real-time PCR was used to measure the expression level of mmu_circ_0000723 in each group.H9c2 cells were divided into groups J-M;H9c2 cells in group J were placed in a cell incubator for 12 h,those in group K were placed in an anaerobic incubator for H/R treatment,and those in groups L and M were transfected with NC and siRNA-mmu_circ_0000723,respectively,followed by H/R treatment at 24 h after transfection.Propidium iodide(PI)staining,LDH kit,and Western blot were used to measure the percentage of PI-positive cells,LDH activity in supernatant,and the expression levels of pyroptosis-related proteins in groups J-M.Results There were significant differences in LDH activity in the supernatant of H9c2 cells and the expression levels of pyroptosis-related proteins between groups A-F(F=24.86-2153.00,P<0.05),and the most significant differences in LDH activity and the expression levels of pyroptosis-related proteins were observed in group D compared with group A(t=4.14-62.88,P<0.05).Compared with the control group,the experimental group had a significant reduction in the expression level of mmu_circ_0000723(t=9.78,P<0.05).There was a significant difference in the expression level of mmu_circ_0000723 between groups G-I(F=348.20,P<0.05),and compared with group G,group I had a significant reduction in the relative expression level of mmu_circ_0000723(t=22.88,P<0.05).There were significant differences between groups J-M in the percentage of PI-positive cells,LDH activity in cell supernatant,and the expression levels of pyroptosis-related proteins(F=34.39-8528.00,P<0.05);compared with group K,group M had significant increases in the above five indices(t=4.99-54.00,P<0.05),while there were no significant differences in these indices between group K and group L(P>0.05).Conclusion This study shows that mmu_circ_0000723 plays an important role in H/R-induced H9c2 cell pyroptosis by regulating the expression of pyroptosis-related proteins.