微小RNA-181d-5p靶向转导素β1X连锁受体蛋白1保护缺氧心肌细胞损伤的机制研究

Mechanism of miR-181d-5p in protecting hypoxic cardiomyocytes against injury by targeting transducer β1X-linked receptor 1

目的研究微小RNA(miR)-181d-5p是否通过靶向转导素β1X连锁受体蛋白1(TBL1XR1)保护缺氧诱导的心肌细胞损伤。方法选取H9C2心肌细胞分为对照组,缺氧组,缺氧+miR对照组(A组),缺氧+miR-181d-5p组(B组),缺氧+小干扰RNA对照组(C组),缺氧+TBL1XR1小干扰RNA组(D组),转染miR阴性对照组(E组),miR-181d-5p组(F组),转染miR抗体阴性对照组(G组),转染miR-181d-5p抗体组(H组),缺氧+miR-181d-5p+pcDNA对照组(I组),缺氧+miR-181d-5p+pcDNA-TBL1XR1组(J组)。检测细胞周期蛋白D1(CyclinD1)、活化的半胱氨酸天冬氨酸酶蛋白3(Caspase-3)、细胞存活率、细胞凋亡率、TNF-α、白细胞介素6(IL-6)水平。结果与对照组比较,缺氧组H9C2心肌细胞中miR-181d-5p(0.38±0.03 vs 1.00±0.11)下调,TBL1XR1 mRNA(2.17±0.17 vs 1.01±0.09)和TBL1XR1蛋白(0.89±0.08 vs 0.42±0.04)上调(P=0.000)。B组较A组及D组较C组H9C2细胞中CyclinD1蛋白、细胞存活率明显升高,活化的Caspase-3蛋白、细胞凋亡率、TNF-α、IL-6水平明显降低(P<0.05,P<0.01)。F组较E组及H组较G组TBL1XR1明显升高(P<0.05)。与I组比较,J组CyclinD1、细胞存活率明显减少,TBL1XR1、活化的Caspase-3、细胞凋亡率、TNF-α、IL-6水平明显升高,差异有统计学意义(P<0.05)。结论miR-181d-5p通过靶向TBL1XR1,促进缺氧诱导的心肌细胞存活,并抑制其凋亡和炎性因子分泌,从而保护缺氧诱导的心肌细胞损伤。

Objective To study whether miR-181d-5p protects cardiomyocytes against hypoxia-induced injury by targeting TBL1XR1.Methods H9C2 cardiomyocytes were divided into control group,hypoxia group,hypoxia+miR-con group(group A),hypoxia+miR-181d-5p group(group B),hypoxia+si-con group(group C),hypoxia+si-TBL1XR1 group(group D),miR-con group(group E),miR-181d-5p group(group F),antimiR-con group(group G),antimiR-181d-5p group(group H),hypoxia+miR-181d-5p+pcDNA-con group(group I),hypoxia+miR-181d-5p+pcDNA-TBL1XR1 group(group J).The expressions of CyclinD1 and Caspase-3 were detected.The survival rate and apoptosis rate of H9C2 cardiomyocytes and the serum levels of TNF-αand IL-6 were measured.Results The expression level of miR-181d-5p was significantly lower while that of TBL1XR1 mRNA and protein in H9C2 cardiomyocytes was significantly higher in hypoxia group than in control group(0.38±0.03 vs 1.00±0.11,2.17±0.17 vs 1.01±0.09,0.89±0.08 vs 0.42±0.04,P=0.000).The expression level of CyclinD1 protein in H9C2 cardiomyocytes and survival rate of H9C2 cardiomyocytes were significantly higher while the expression levels of Caspase-3,serum levels of TNF-αand IL-6 in H9C2 cardiomyocytes and survival rate of H9C2 cardiomyocytes were significantly lower in group B than in group A and in group D than in group C(P<0.05,P<0.01).The expression level of TBL1XR1 was significantly higher in group F than in group E and in group H than in group G(P<0.05).The expression level of CyclinD1 protein in H9C2 cardiomyocytes and survival rate of H9C2 cardiomyocytes were significantly lower while the expression levels of TBL1XR1 and Caspase-3 protein,serum levels of TNF-αand IL-6 in H9C2 cardiomyocytes and survival rate of H9C2 cardiomyocytes were significantly higher in group J than in group I(P<0.05).Conclusion miR-181d-5p prolongs the survival of hypoxia-induced cardiomyocytes,inhibits their apoptosis and secretion of inflammatory factors by targeting TBL1XR1,and can thus protect cardiomyocytes against hypoxia-induced injury.