PKD1基因对主动脉平滑肌细胞自噬的影响

目的阐明多囊肾病基因1(PKD1)对小鼠主动脉平滑肌细胞自噬的影响。方法构建PKD1基因的shRNA慢病毒干扰及PKD1基因促进剂ETS-1慢病毒过表达载体,转染小鼠主动脉平滑肌细胞,采用实时荧光定量核酸扩增(qPCR)检验干扰及过表达效率。同时通过qPCR及Western blotting检测对照组、shPKD1组及ETS-1组小鼠主动脉平滑肌细胞自噬标志分子Atg5、Beclin1和LC3的表达水平。结果与正常对照组相比,shPKD1组PKD1 mRNA表达量下降(P<0.05),而ETS-1组ETS-1 mRNA及PKD1 mRNA表达量明显升高(P<0.05)。与对照组相比,shPKD1组自噬标志物Atg5和Beclin1 mRNA表达水平明显降低(P<0.05),ETS-1组Atg5和Beclin1 mRNA表达水平明显升高(P<0.001)。与对照组相比,shPKD1组Atg5、Beclin1和LC3蛋白表达水平明显降低(P<0.05),ETS-1组Atg5、Beclin1和LC3蛋白表达水平明显升高(P<0.05)。结论PKD1基因的表达能够影响小鼠主动脉平滑肌细胞自噬,即PKD1基因表达下调可抑制主动脉平滑肌细胞自噬,而PKD1基因过表达能够促进小鼠主动脉平滑肌细胞自噬。

The effect of PKD1 gene on autophagy in aortic smooth muscle cells

Objective To explore the effects of PKD1 gene on mouse aortic smooth muscle(MOVAS)cells autophagy.Methods The shRNA and over-expression lentiviral vectors for the target gene of PKD1 were constructed.MOVAS cells were infected by a number of successful packaging shRNA(PKD1 knockdown)or ETS-1(PKD1 overexpressing)lentiviral vectors,and qPCR was used to test interference and over-expressing effects.Then qPCR and Western blotting were used to detect the expression levels of autophagy markers including Atg5,Beclin1 and LC3 in control group,shPKD1 group and ETS-1 group.Results Compared with the control group,PKD1 mRNA level was decreased in the shPKD1 group(P<0.05);ETS-1 and PKD1 mRNA levels were increased in the ETS-1 group(P<0.05).In contrast with the control group,the mRNA levels of autophagy markers including Atg5(P<0.05)and Beclin1(P<0.01)were obviously decreased in the shPKD1 group,but they were obviously increased in the ETS-1 group(P<0.001).Protein levels of Atg5,Beclin1 and LC3 were significantly decreased in the shPKD1 group(P<0.05),but they were increased obviously in the ETS-1 group(P<0.05)in contrast with the control group.Conclusion PKD1 gene is involved in MOVAS cells autophagy,low expression of PKD1 gene can inhibit autophagy and high expression of PKD1 promotes autophagy in vascular smooth muscle cells.