RNA干扰p53基因对中波紫外线诱导的早衰和光致癌相关基因表达的影响

Influence of RNA interference in p53 gene on the expressions of genes involved in ultraviolet B-induced premature senescence and photocarcinogenesis in human skin fibroblasts

目的探讨RNA干扰p53基因对中波紫外线（UVB）诱导的人皮肤成纤维细胞（HSF）早衰和光致癌相关基因表达的影响。方法利用已构建成功的RNA干扰抑制p53表达的HSF细胞系，加以反复多次亚毒性剂量UVB照射，衰老相关的B半乳糖苷酶（SAβ-gal）染色法检测细胞衰老，定制实时定量PCR芯片，检测多种光致癌发生相关基因的表达，包括：参与细胞衰老通路的p53、p21、p19、p16、pRb，衰老相关基因纤维结合素（FN）、骨结合素（ON）、平滑肌22（SM22），p53依赖的细胞凋亡相关基因hax和bcl-2，UVB诱导的癌基因缺氧诱导因子1a（HIF-1a）、血管内皮生长因子（VEGF）以及负性调控p53的人双微球蛋白2基因（hdm2）。使用SPSS10．0软件对各组间数据进行配对t检验。结果抑制p53表达的HSF经UVB照射后SA β-gal活性（19．70％±0．85％）较照射前（12．77％±0．81％）有所增加（t=6．45，P〈0．05），但显著低于正常HSF经UVB照射后（50．48％±5．30％，t=7．86，P〈0．05），与正常HSF组（18．50％±0．45％）差异无统计学意义（t=2．57，P〉0．05）。基因检测结果表明，抑制p53表达可抑制衰老基因p21、p19、FN、ON、SM22等的表达，但p16通路并不受之影响，在UVB作用下，p16、pRb的表达显著增加，分别上调。抑制p53的表达可使抑凋亡基因bcl-2上调及促凋亡基因bax下调，癌基因HIF-1a、VEGF、hdm2表达明显增加。UVB照射对这些基因表达改变无明显影响。结论抑制p53表达能够延缓UVB诱导的早衰。UVB诱导的早衰具有p53依赖的相关肿瘤抑制作用。

Objective To evaluate theeffect of RNA interference in p53 gene on the expressions of genes involved in ultraviolet B （UVB）-induced premature senescence and photocarcinogenesis in human skin fibroblasts （HSFs）. Methods A previously established HSF cell clone with repressed expression of p53, which was named as HSF-p53, was cultured and irradiated with a subcytotoxic dose （ 10 mJ/cm2） of UVB once a day for five consecutive days. The HSFs with normal expression of p53 served as the control. Subsequently, [3-galactosidase （SA-β-gal）- staining was performed to estimate the degree of senescence, quantitative real-time PCR array was performed to determine the mRNA expressions of photocarcinogenesis- and senescence-associated genes, including p53, p21, p19, p16, pRb, fibronectin, osteonectin, smooth muscle 22 （SM22）, bax, bcl-2, hypoxia-inducible factor-1 a （HIF-1a）, vascular endothelial growth factor（VEGF）, and human double minute-2 （hdm2）. Statistical analysis was carried out by Student＇s t test using the software SPSS 10.0. Results The percentage of SA-β-gal-positive cells in irradiated HSF-p53 was 19.70% ± 0.85%, significantly higher than that in unirradiated HSF-p53 （ 12.77% ± 0.81%, t = 6.45, P 〈 0.05）, but lower than that in irradiated control HSFs （50.48% ± 5.30%, t = 7.86, P 〈 0.05）, and similar to that in unirradiated control HSFs （18.50% ± 0.45%, t = 2.57, P 〉 0.05）. Compared with the control HSFs, the HSF-p53 showed decreased expressions of p21, p19, fibronectin, osteonectin, SM22 and bax genes （all P 〈 0.05）, but increased expressions of bcl-2, HIF-1a , VEGF and hdm2 genes （all P 〈 0.05）, and a similar expression of p16 gene （P 〉 0.05）; the repeated UVB radiation significantly promoted the expressions of p16 and pRb genes （both P 〈 0.05）, but had no obvious effect on the expressions of the other genes in HSF-p53 compared with unirradiated HSF- p53 （all P 〉 0.05）. Conclusions The inhibition of p53 expression may decelerate the UVB-induced premature senescence in HSFs, which may be involved in the p53-dependent tumor suppression.