摘要
目的探讨miR-702-3p对心肌细胞缺氧复氧(hypoxia reoxygenation,H/R)损伤后凋亡的影响。方法原代培养大鼠心肌细胞,缺氧3 h复氧4 h构建心肌细胞H/R损伤模型。心肌细胞被分为正常对照组(CON)、缺氧复氧+无转染组(H/R)、缺氧复氧+转染miR-702-3p inhibitor NC组(H/R+inhibitor NC)、缺氧复氧+转染miR-702-3p inhibitor组(H/R+inhibitor)。转染miR-NC和miR-702-3p inhibitor 24 h后,进行后续实验。采用CCK-8实验和流式细胞术测定各组细胞活力和凋亡率。qRT-PCR检测各组心肌细胞中miR-702-3p的表达水平。Western blot检测各组心肌细胞中乙醛脱氢酶2(aldehyde dehydrogenase 2,ALDH2)、Bcl-2、Bax、cleaved Caspase-3蛋白表达水平。采用双荧光素酶报告基因实验检测miR-702-3p与ALDH23′UTR之间的作用关系。结果与对照组相比,H/R组心肌细胞miR-702-3p表达量显著升高(P<0.05);与H/R组相比,H/R+inhibitor组miR-702-3p表达显著降低(P<0.05)。CCK-8实验和流式细胞术检测结果证实,与对照组相比,H/R组心肌细胞活力降低(P<0.05),凋亡率升高(P<0.05);与H/R组相比,H/R+inhibitor组细胞活力显著改善(P<0.05),凋亡率降低(P<0.05)。Western blot结果表明,与对照组相比,H/R组Bax/Bcl-2比值升高,cleaved Caspase-3表达升高,ALDH2蛋白表达量降低,差异均有统计学意义(P<0.05);与H/R组相比,H/R+inhibitor组Bax/Bcl-2比值降低,cleaved Caspase-3表达降低,ALDH2蛋白的表达升高,差异均有统计学意义(P<0.05)。荧光素酶活性检测结果表明,ALDH2是miR-702-3p的靶基因。结论本实验表明,ALDH2是miR-702-3p的靶基因,抑制miR-702-3p表达上调了ALDH2蛋白水平,降低了H/R处理后心肌细胞凋亡率。
Objective To explore the effect and mechanism of microRNA(miRNA,miR)-702-3p on cardiomyocyte apoptosis after hypoxia reoxygenation(H/R)injury.Methods The H/R injury model of primary rat cardiomyocytes was constructed after 3 h hypoxia and 4 h reoxygenation.Cardiomyocytes were divided into control group,hypoxia-reoxygenation group(H/R),hypoxia-reoxygenation+miR-702-3p inhibitor NC transfection group(H/R+inhibitor NC),and hypoxia-reoxygenation+miR-702-3p inhibitor transfection group(H/R+inhibitor).The following experiments were conducted 24 h after transfection with miR-NC and miR-702-3p inhibitor.CCK-8 assay and flow cytometry were used to determine the cell viability and the apoptosis rate of cardiomyocytes.The qRT-PCR was used to detect the expression level of miR-702-3p in each group.Western blot was used to detect the protein levels of aldehyde dehydrogenase 2(aldehyde dehydrogenase 2,ALDH2),Bcl-2,Bax,cleaved Caspase-3 in cardiomyocytes.The specific interaction of miR-702-3p with the 3′UTR of ALDH2 was examined by luciferase reporter assays.Results Expression of miR-702-3p in cardiomyocytes was higher in H/R group than in control group(P<0.05),and the expression level of miR-702-3p was significantly lower in H/R+inhibitor group than in H/R group(P<0.05).Compared with control group,the cell viability of cardiomyocytes was decreased(P<0.05),while the cell apoptosis rate was increased in H/R group(P<0.05).Compared with H/R group,the cell viability were significantly improved(P<0.05)while the apoptosis rate was decreased in H/R+inhibitor group(P<0.05).Besides,compared with control group,the ratio of Bax/Bcl-2 was increased(P<0.05),the protein level of cleaved Caspase-3 was increased(P<0.05),while the protein level of ALDH2 was decreased in H/R group(P<0.05).Compared with H/R group,the Bax/Bcl-2 ratio was decreased(P<0.05),the protein level of cleaved Caspase-3 was decreased(P<0.05),while the protein level of ALDH2 was increased in H/R+inhibitor group(P<0.05).The result of luciferase activity test showed that ALDH2 was the target gene of miR-702-3p.Conclusion These results indicate that ALDH2 is the target gene of miR-702-3p.Decreasing expression of miR-702-3p may up-regulate the ALDH2 protein level,and decrease the apoptosis rate of cardiomyocytes after H/R treatment.
作者
宫能静
姜玉玉
李小雨
高娇娇
程向阳
GONG Nengjing;JIANG Yuyu;LI Xiaoyu;GAO Jiaojiao;CHENG Xiangyang(Department of Anesthesiology,First Affiliated Hospital of Bengbu Medical College,Bengbu 233030,China)
出处
《山西医科大学学报》
CAS
2020年第7期641-646,共6页
Journal of Shanxi Medical University
基金
安徽省教育厅自然科学研究重点项目(K2018AO212)
蚌埠医学院研究生科研创新计划项目(Byycx1909)。
