miR-34a在人H9C2心肌细胞缺氧复氧损伤中的保护作用及机制

Protective effect of miR-34a on human H9C2 cardiomyocytes during hypoxia-reoxygenation injury and its mechanism

目的探讨miR-34a对缺氧复氧损伤人H9C2心肌细胞凋亡的影响及可能机制。方法对数生长期人H9C2心肌细胞,随机分为正常对照组、缺氧复氧组、miR-34a激动剂组、PI3K通道阻滞组。缺氧复氧组、miR-34a激动剂组、PI3K通道阻滞组缺氧培养3 h、复氧培养4 h制备缺氧复氧模型;在缺氧培养前48 h,miR-34a激动剂组于培养基中加入摩尔浓度为50 nmol/L的miR-34a激动剂agomir,PI3K通道阻滞组在培养基中加入摩尔浓度为50 nmol/L的agomir和摩尔浓度为10μmol/L的PI3K特异性阻断剂LY294002,缺氧复氧组不作处理。正常对照组不制备模型,不进行药物干预。复氧培养4 h,4组采用TUNEL法检测细胞凋亡率;实时荧光定量PCR法检测miR-34a mRNA、p-PI3K mRNA相对表达量;Western blot法检测心肌细胞caspase-3、B淋巴细胞瘤-2(B-cell lymphoma-2, Bcl-2)蛋白、p-PI3K、p-Akt相对表达量。结果复氧培养4 h,正常对照组、miR-34a激动剂组、PI3K通道阻滞组、缺氧复氧组人H9C2心肌细胞凋亡率[(20.00±2.14)%、(34.26±3.82)%、(58.58±5.69)%、(80.25±7.76)%]依次升高(P<0.05);miR-34a激动剂组、PI3K通道阻滞组、正常对照组、缺氧复氧组人H9C2心肌细胞miR-34a mRNA(3.214±0.321、2.165±0.224、1.000±0.002、0.496±0.051)、p-PI3K mRNA(2.161±0.232、1.446±0.153、1.000±0.006、0.501±0.052)、p-PI3K(0.316±0.031、0.204±0.021、0.189±0.024、0.095±0.086)、p-Akt(0.586±0.062、0.306±0.032、0.182±0.021、0.071±0.008)蛋白相对表达量依次降低(P<0.05);miR-34a激动剂组、PI3K通道阻滞组、缺氧复氧组人H9C2心肌细胞Bcl-2蛋白相对表达量(0.385±0.042、0.381±0.045、0.179±0.018)均低于正常对照组(0.832±0.062),caspase-3蛋白相对表达量(0.526±0.061、0.544±0.059、0.854±0.094)均高于正常对照组(0.518±0.021)(P<0.05);缺氧复氧组人H9C2心肌细胞Bcl-2蛋白相对表达量低于miR-34a激动剂组、PI3K通道阻滞组,caspase-3蛋白相对表达量高于miR-34a激动剂组、PI3K通道阻滞组(P<0.05);miR-34a激动剂组与PI3K通道阻滞组人H9C2心肌细胞Bcl-2、caspase-3蛋白相对表达量比较差异无统计学意义(P>0.05)。结论 miR-34a可抑制缺氧复氧损伤的人H9C2心肌细胞凋亡,其机制可能与促进PI3K磷酸化,活化PI3K/Akt信号通路,抑制细胞凋亡蛋白表达有关。

Objective To investigate the effect of miR-34 a on the apoptosis of human H9 C2 cardiomyocytes induced by hypoxia and reoxygenation and its possible mechanism. Methods Human H9 C2 cardiomyocytes in logarithmic growth phase were divided into normal control group, hypoxia-reoxygenation group, miR-34 a agonist group and PI3 K channel block group. The hopoxia-reoxygenation models were established after 3 h of hopoxia culture and 4 h of reoxygenation culture in hypoxia-reoxygenation group, miR-34 a agonist group and PI3 K channel block group. miR-34 a agonist group was added with 50 nmol/L of miR-34 a agonist agomir to the culture medium, PI3 K channel block group was added with 50 nmol/L of miR-34 a agonist agomir and 10 nmol/L of LY294002 to the culture medium 48 h before hypoxia culture, and hypoxia-reoxygenation group received no treatment. Normal control group did not establish models and received no treatment. After 4 h of reoxygenation, MTT method was used to detect the apoptotic rate in 4 groups. Real-time PCR technique was used to detect the relative expressions of miR-34 a mRNA and PI3 K mRNA. Western blot method was used to detect the relative expressions of caspase-3, B-cell lymphoma-2(Bcl-2), p-PI3 K and p-Akt. Results After 4 h of reoxygenation culture, the apoptotic rate of H9 C2 cardiomyocytes increased gradually in turn in normal control group, miR-34 a agonist group, PI3 K channel block group and hypoxia-reoxygenation group((20.00±2.14)%,(34.26±3.82)%,(58.58±5.69)%,(80.25±7.76)%)(P<0.05). The relative expressions of miR-34 a mRNA(3.214±0.321, 2.165±0.224, 1.000±0.002, 0.496±0.051), p-PI3 K mRNA(2.161±0.232, 1.446±0.153, 1.000±0.006, 0.501±0.052), p-PI3 K(0.316±0.031, 0.204±0.021, 0.189±0.024, 0.095±0.086) and p-Akt(0.586±0.062, 0.306±0.032, 0.182±0.021, 0.071±0.008) decreased gradually in turn in miR-34 a agonist group, PI3 K channel block group,normal control group and hypoxia-reoxygenation group(P<0.05).The relative expression of Bcl-2 protein was lower and the relative expression of caspase-3 protein was higher in miR-34 aagonist group(0.385±0.042,0.526±0.061),PI3 Kchannel block group(0.381±0.045,0.544±0.059)and hypoxia-reoxygenation group(0.179±0.018,0.854±0.094)than that in normal control group(0.832±0.062,0.518±0.021)(P<0.05).The relative expression of Bcl-2 protein was lower and the relative expression of caspase-3 protein was higher in hypoxia-reoxygenation group than that in miR-34 aagonist group and PI3 Kchannel block group(P<0.05),and both of them two showed no significant differences between miR-34 aagonist group and PI3 Kchannel block group(P>0.05).Conclusion miR-34 acan suppress the apoptosis of H9 C2 cardiomyocytes induced by hypoxia and reoxygenation,probably by promoting PI3 K phosphorylation,activating PI3 K/Akt signaling pathway and inhibiting the expression of apoptosis proteins.