UVB诱导下早衰人皮肤成纤维细胞中miR-34c及SIRT1表达的研究

Expression of senescence-associated miR-34c and SIRT1 in premature senescence of human skin fibroblasts induced by ultraviolet B

目的:运用反复亚毒性剂量紫外线B(ultraviolet B,UVB)辐射人皮肤成纤维细胞,构建应激诱导早衰(stress-inducedpremature senescence,SIPS)模型,观察衰老相关生物学标志、衰老相关基因信号分子p53、p21、p16、沉默信息调节因子2相关酶1(sirtuin1,SIRT1)以及miR-34c的表达情况。方法:体外培养人皮肤成纤维细胞,予连续5次、每次剂量10 mJ/cm2的UVB辐射,细胞衰老β-半乳糖苷酶(SA-β-Gal)染色检测衰老细胞,流式细胞术检测细胞周期阻滞情况,实时荧光定量PCR检测miR-34c及衰老相关基因p53、p21、p16、SIRT1 mRNA表达水平变化,Western blot检测p53、p21、p16及SIRT1蛋白水平表达。结果:与对照组相比,SIPS组细胞SA-β-Gal染色呈强阳性[对照组(8.13±1.92)%,SIPS组(94.72±2.08)%,P<0.05];多数细胞阻滞于G1期[对照组(52.96±1.76)%,SIPS组(77.31±2.05)%,P<0.05];另外,实时荧光定量PCR显示miR-34c mRNA的表达升高(miR-34c-3p、miR-34c-5p分别为对照组0.93±0.09、1.03±0.03,SIPS组2.08±0.19、12.28±1.64,P<0.05);衰老相关基因p53、p21、p16、SIRT1 mRNA的表达亦升高(对照组分别为0.74±0.23、1.06±0.23、0.86±0.13、0.74±0.25;SIPS组分别为1.84±0.55、20.25±2.34、16.7±3.94、2.15±0.22,P<0.05);Western bolt表明p53、p21、p16及SIRT1蛋白水平表达明显升高,差异有统计学意义(P<0.05)。结论:miR-34c在反复多次亚毒性剂量UVB辐射人皮肤成纤维细胞后表达升高,对p53起正反馈调节作用,而SIRT1并未受miR-34c的抑制。

Objective： To build the stress-induced premature senescence（SIPS） model with fibroblasts after repeated exposures to sub-toxic doses of ultraviolet B（UVB）,and observe the expressions of senescence-associated biomarkers,senescence-associated signals including p53,p16,p21,sirtuin 1（SIRT1）,as well as senescence-associated miRNAs such as miR-34c.Methods： Skin fibroblasts were repeatedly exposed to UVB irradiation at a dose of 10 mJ/cm2 for 5 times.After 5 days of treatment,SA-β-Gal staining was performed to evaluate the senescence state,flow cytometry to analyze cell cycle,and real-time quantitative PCR to determine mRNA expression of senescence-associated signals including p53,p16,p21,SIRT1 and miR-34c.Western blot was performed to detect the expression of p53,p21,p16 and SIRT1.Results： Compared with the control group,strong positive SA-β-Gal was observed in SIPS group,（94.72 ± 2.08）% in SIPS group compared with（8.13 ± 1.92）% in control group（P 0.05）.Most cells were arrested in G1 phase,（52.96 ± 1.76）% in control group compared with（77.31 ± 2.05）% in SIPS group（P 0.05）.Meanwhile,the expression of senescence-associated signals including p53,p16,p21,SIRT1 significantly increased after UVB exposure（0.74 ± 0.23,1.06 ± 0.23.0.86 ± 0.13,0.74 ± 0.25 in control group while 1.84 ± 0.55,20.25 ± 2.34,16.7 ± 3.94,2.15 ± 0.22 in SIPS group,P 0.05）,so as to that of senescence-associated miRNAs such as miR-34c（0.93 ± 0.09 for miR-34c-3p and 1.03 ± 0.03 for miR-34c-5p in control group while 2.08 ± 0.19 for miR-34c-3p and 12.28 ± 1.64 for miR-34c-5p in SIPS group,P 0.05）.Western blot showed that p53,p21,p16 and SIRT1 protein expression significantly increased in SIPS group compared with those in control group,and the difference was statistically significant（P 0.05）.Conclusion： The expression of miR-34c increases after repeated exposures to sub-toxic doses of UVB in skin fibroblasts,and it works as a positive feedback regulation to P53 while not inhibiting SIRT1.