UVB诱导下早衰人皮肤成纤维细胞中miR-34c及SIRT1表达的研究
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国家自然科学基金(30771946)


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

    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-induced premature 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。

    Abstract:

    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.

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郭娴菲,周炳荣,李 巍,黄秋红,骆 丹. UVB诱导下早衰人皮肤成纤维细胞中miR-34c及SIRT1表达的研究[J].南京医科大学学报(自然科学版),2011,(9):1289-1293

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  • 收稿日期:2011-04-06
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