泛素连接酶MuRF2抑制心肌细胞线粒体自噬  

Ubiquitin Ligase MuRF2 Inhibits Mitochondrial Autophagy in Cardiomyocytes

作　　者：孙宾 马慧艳 严宁[2] 田进海[3] 何军 SUN Bin;MA Huiyan;YAN Ning;TIAN Jinhai;HE Jun(Ningxia Medical University,Yinchuan750004,China;Department of Cardiovascular Medicine,Hospital of Ningxia Medical University,Yinchuan750004,China;Sub-center of Biochip Research Center,the General Hospital of Ningxia Medical University,Yinchuan750004,China)

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学总医学院心血管内科,银川750004 [3]宁夏医科大学总医院生物芯片中心,银川750004

出　　处：《宁夏医科大学学报》2020年第3期230-236,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81660045)。

摘　　要：目的观察泛素连接酶肌肉环指状蛋白2(muscle ring finger protein2,MuRF2)在HL-1心肌细胞自噬中的作用。方法采用慢病毒转染技术上调和下调HL-1心肌细胞中MuRF2的表达。实验分为阴性对照组、阳性对照组(空载病毒对照)、雷帕霉素处理组、MuRF2过表达组和MuRF2敲减组。用雷帕霉素处理细胞,蛋白免疫印迹法(Western blot)检测自噬蛋白LC3和BNIP3的表达。透射电镜观察HL-1心肌细胞超微结构。结果(1)Western blot验证,MuRF2过表达组中MuRF2蛋白表达水平均高于阴性对照组、阳性对照组、雷帕霉素处理组(P均<0.05),MuRF2敲减组中MuRF2蛋白表达水平均低于阴性对照组、阳性对照组、雷帕霉素处理组(P均<0.05)。(2)Western blot结果表明,雷帕霉素处理组LC3蛋白Ⅱ/Ⅰ比值和BNIP3蛋白表达均高于阴性对照组(P均<0.05),MuRF2过表达组LC3蛋白Ⅱ/Ⅰ比值和BNIP3蛋白表达均低于雷帕霉素处理组(P均<0.05)。与阴性对照组相比,MuRF2敲减组的LC3蛋白Ⅱ/Ⅰ比值和BNIP3蛋白表达均升高(P均<0.05),雷帕霉素处理组和MuRF2敲减组在LC蛋白Ⅱ/Ⅰ比值和BNIP3蛋白表达上差异均无统计学意义(P均>0.05)。透射电镜显示,雷帕霉素处理组线粒体分裂、碎片化,自噬体和自噬溶酶体散在胞质内。MuRF2过表达组线粒体处于融合状态,自噬体明显减少。结论MuRF2可以抑制心肌细胞线粒体自噬。Objective To assess the role of E3 ubiquitin ligase muscle ring finger protein 2(MuRF2)in cardiomyocyte autophagy.Methods By using the means of lentiviral transfection technique,we upregulated and down-regulated the expression of MuRF2 in HL-1 cardiomyocytes.Cells were grouped as negative control,positive control,Rapamycin treatment group,MuRF2 overexpression and MuRF2 knockdown group.The cells were treated with Rapamycin and the expressions of autophagy proteins LC3 and BNIP3 were detected by Western blot.The ultrastructure of HL-1 cardiomyocytes was observed by transmission electron microscopy(TEM).Results(1)Western blot validation showed that the level of MuRF2 protein in the MuRF2 overexpression group was higher than that in the negative control group,positive control group and the Rapamycin treatment group(P<0.05).The expression level of MuRF2 protein in the MuRF2 knockdown group was significantly lower than that in the negative control group,positive control group and Rapamycin treatment group(P<0.05).(2)LC3Ⅱ/Ⅰand BNIP3 protein expressions in Rapamycin treatment group significantly increased compared with the negative control group(P<0.05).In MuRF2 overexpression group,LC3Ⅱ/Ⅰand BNIP3 protein levels were lower than that in rapamycin treatment group(P<0.05).Compared with the negative control group,LC3Ⅱ/Ⅰand BNIP3 protein expression levels in the MuRF2 knockdown group were higher(P<0.05).There was no difference between Rapamycin treatment group and MuRF2 knockdown in LC3Ⅱ/Ⅰand BNIP3 protein levels(P>0.05).TEM analysis showed that in the Rapamycin treatment group,mitochondria were being fission and fragmented,autophagosomes and autophagolysosomes scattered in the cytoplasm.In MuRF2 overexpression group,mitochondria were much less in fusion status and autophagosomes.Conclusion MuRF2 inhibits mitochondrial autophagy in HL-1 cardiomyocytes.

关 键 词：肌肉环指状蛋白2 心肌细胞HL-1 线粒体 自噬 雷帕霉素 

分 类 号：R541.6[医药卫生—心血管疾病]