miR-17对大鼠血管平滑肌细胞衰老的调控作用

miR-17 regulates senescence of rat vascular smooth muscle cells

目的:探讨微小RNA-17(miR-17)对大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)衰老的影响。方法:取SD大鼠胸主动脉中层,分离并原代培养VSMCs。取第4~6代VSMCs,将细胞分为6组:诱导衰老+miR-17过表达(A-miR-17)组,诱导衰老+miR-17敲减(A-anti-miR-17)组,诱导衰老对照(A-control)组,正常培养+miR-17过表达(N-miR-17)组,正常培养+miR-17敲减(N-anti-miR-17)组和正常培养对照(N-control)组。通过脂质体将miR-17-mimics和miR-17-inhibitor转入VSMCs,24 h后采用D-半乳糖诱导细胞衰老,在加入D-半乳糖后的第3天采用β-半乳糖苷酶染色方法鉴定细胞衰老;分别采用实时荧光定量PCR和免疫组化方法检测血管平滑肌细胞miR-17、p16和p21的表达。结果:(1)与A-control组相比,VSMCs内miR-17的表达在A-miR-17组显著升高(P<0.01),而在A-anti-miR-17组显著降低(P<0.01);(2)D-半乳糖诱导3天后,A-anti-miR-17组β-半乳糖苷酶阳性细胞显著高于A-miR-17组;(3)A-miR-17组VSMCs中p21蛋白和mRNA表达显著低于A-control组(P<0.01),而A-anti-miR-17组p21蛋白和mRNA表达显著高于A-miR-17组(P<0.01)。结论:miR-17能够抑制D-半乳糖诱导的大鼠VSMCs衰老,其机制与抑制p21过表达有关。

AIM:To investigate the effect of microRNA-17(miR-17)on the senescence of vascular smooth muscle cells(VSMCs)and the underlying mechanism.METHODS:The medial layer of the thoracic aorta was collected from the SD rats and isolated for primary culture.VSMCs were identified by immunofluorescence staining.The VSMCs were collected at the 4th^6th generations,and then the miR-17 mimics and miR-17 inhibitor were transfected into the VSMCs by liposome method.After 24 h,the cell senescence was induced by D-galactose.The VSMCs were divided into the following 6 groups:aging induction+miR-17 mimics(A-miR-17)group,aging induction+miR-17 inhibitor(A-anti-miR-17)group,A-control group,normal(N)+miR-17-mimics(N-miR-17)group,N-anti-miR-17 group,and N-control group.On day 3 after the addition of D-galactose,the senescence of VSMCs was observed withβ-galactosidase staining.The expression of miR-17,p16 and p21 was detected by RT-qPCR and immunohistochemistry.RESULTS:miR-17 expression in the VSMCs was significantly lower in A-control group than that in N-control group(P<0.01).Compared with A-control group,the expression of miR-17 in the VSMCs was significantly increased in A-miR-17 group(P<0.01),while that was significantly decreased in A-anti-miR-17 group(P<0.01).The number ofβ-galactosidase positive staining cells in A-anti-miR-17 group was significantly higher than that in A-miR-17 group(P<0.01).The expression of p21 at mRNA and protein levels in the VSMCs was significantly lower in A-miR-17 group than that in A-control group(P<0.01),and the expressions of p21 at mRNA and protein levels was significantly higher in A-anti-miR-17 group than that in A-miR-17 group(P<0.01).CONCLUSION:miR-17 inhibits rat VSMCs senescence induced by D-galactose,the underlying mechanism is associated with the inhibition of p21 expression.