LncRNAGAS5通过miR-137/SIRT6轴抑制糖尿病大鼠心肌细胞凋亡的机制研究

Mechanism of long non-coding RNA growth arrest-specific transcripts 5 inhibiting the apoptosis of cardiomyocytes in diabetic rats via miR-137/silent information regulator 6 axis

目的探讨长链非编码RNA生长停滞特异性转录因子5(LncRNA GAS5)对糖尿病心肌病(DCM)大鼠心肌细胞凋亡的影响及其分子机制。方法通过高糖高脂饮食联合链脲佐菌素腹腔注射构建大鼠DCM模型,分为假手术(Sham)组和DCM组。苏木素-伊红(HE)染色观察心肌组织的病理变化;Masson染色检测大鼠心肌纤维化程度;实时荧光定量聚合酶链式反应(qRT-PCR)检测心肌组织LncRNA GAS5的表达情况。大鼠心肌细胞(H9c2)随机分为对照组、高糖组、高糖+GAS5组、高糖+miR-137 mimics组、高糖+miR-137 mimics+GAS5组、高糖+沉默调节蛋白6(SIRT6)组、高糖+SIRT6+miR-137 mimics组并进行相应处理。qRT-PCR检测LncRNA GAS5、miR-137的表达量;Western blot检测B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关X蛋白(Bax)、SIRT6蛋白表达量;流式细胞术检测细胞凋亡情况;细胞计数试剂盒8检测细胞活力;双荧光素酶报告基因实验验证LncRNAGAS5与miR-137、miR-137与SIRT6的靶向作用。结果HE染色和Masson染色结果显示,与Sham组相比,DCM组心肌细胞肥大,排列紊乱,胶原纤维增多,大量炎性细胞浸润。与Sham组比较,DCM组LncRNAGAS5表达降低(P<0.01)。与对照组比较,高糖组LncRNAGAS5表达水平随高糖培养时间延长逐渐降低(P<0.01)。与高糖组比较,高糖+GAS5组LncRNAGAS5表达量、细胞活力、Bcl-2表达增加(P<0.05);miR-137、细胞凋亡比例、Bax表达降低(P<0.05)。与高糖组比较,高糖+miR-137 mimics组miR-137、Bax蛋白表达量、细胞凋亡率增加(P<0.05);细胞活力、SIRT6、Bcl-2蛋白表达量降低(P<0.05)。与高糖+miR-137 mimics组相比,高糖+GAS5+miR-137 mimics组细胞活力、Bcl-2表达增加(P<0.01);细胞凋亡比例、Bax表达降低(P<0.05)。与高糖组比较,高糖+SIRT6组细胞活力、Bcl-2、SIRT6蛋白表达增加(P<0.01);细胞凋亡比例、Bax表达降低(P<0.01)。与高糖+SIRT6组比较,高糖+SIRT6+miR-137 mimics组细胞凋亡率、Bax蛋白表达量升高;细胞活力、Bcl-2蛋白表达量降低(P<0.01)。双荧光素酶报告基因实验证实LncRNA GAS5和miR-137、miR-137和SIRT6存在靶向关系。结论过表达LncRNAGAS5可通过内源性竞争机制减少miR-137与SIRT6的结合,促进SIRT6表达抑制DCM大鼠心肌细胞凋亡。

Objective To investigate the effect of long non-coding RNA growth arrestspecific transcripts 5(LncRNA GAS5)on the apoptosis of cardiomyocytes and its molecular mechanism in diabetic cardiomyopathy(DCM)rats.Methods DCM rat models were constructed by high-glucose high-fat diet combined with streptozotocin intraperitoneal injection,and were devided into Sham group and DCM group.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of myocardial tissues;Masson staining was used to detect the degree of myocardial fibrosis in rats.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of LncRNA GAS5 in myocardial tissues.Rat cardiomyocytes(H9c2)were randomly divided into the control group,the high glucose group,the high glucose+GAS5 group,the high glucose+miR-137 mimics group,the high glucose+miR-137 mimics+GAS5 group,the high glucose+SIRT6 group,and the high glucose+SIRT6+miR-137 mimics group for corresponding treatment.QRT-PCR detected the expression levels of LncRNA GAS5 and miR-137.Western blot detected the protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2 associated x(Bax)and silent information regulator 6(SIRT6).Flow cytometry detected apoptosis.Cell counting kit 8 detected cell viability.Dual luciferase reporter gene assay verified the targeting relationship between LncRNA GAS5 and miR-137,miR-137 and SIRT6.Results HE staining and Masson staining results showed that compared with the Sham group,cardiomyocytes in the DCM group were hypertrophic,disorganized,with increased collagen fibers and a large number of inflammatory cells infiltrated.Compared with the Sham group,LncRNA GAS5 expression decreased in the DCM group(P<0.01).Compared with the control group,the expression level of LncRNA GAS5 in the high glucose group gradually decreased with the prolongation of high glucose incubation time(P<0.01).Compared with the high glucose group,LncRNA GAS5 expression,cell viability,and Bcl-2 expression increased in the high glucose+GAS5 group(P<0.05),while miR-137,cell apoptosis ratio,and Bax expression decreased(P<0.05).Compared with the high glucose group,miR-137,Bax protein expression,and cell apoptosis ratio increased in the high glucose+miR-137 mimics group,while cell viability,SIRT6,and Bcl-2 protein expressions decreased(P<0.05).Compared with the high glucose+miR-137 mimics group,cell viability,Bcl-2 expression increased in the high glucose+GAS5+miR-137 mimics group,while cell apoptosis ratio and Bax expression decreased(P<0.05).Compared with the high glucose group,cell viability,Bcl-2,and SIRT6 protein expressions increased in the high glucose+SIRT6 group(P<0.01),while cell apoptosis ratio and Bax expression decreased(P<0.05).Compared with the high glucose+SIRT6 group,cell apoptosis ratio and Bax protein expression increased in the high glucose+SIRT6+miR-137 mimics group(P<0.01),but cell viability and Bcl-2 protein expression decreased(P<0.01).Dual luciferase reporter gene assay confirmed the targeting relationship between LncRNA GAS5 and miR-137,miR-137 and SIRT6.Conclusion Overexpression of LncRNA GAS5 reduces the binding of miR-137 and SIRT6 through an endogenous competitive mechanism,and promotes SIRT6 expression to inhibit cardiomyocytes apoptosis in DCM rat.