缺氧诱导的UCHL5增强宫颈癌Hela细胞辐射抗性

Hypoxia-induced UCHL5 expression enhanced radioresistance of Hela cells in cervical cancer

目的研究缺氧诱导的泛素羧基末端水解酶L5(UCHL5)在调节宫颈癌Hela细胞辐射敏感性中的作用。方法以宫颈癌Hela细胞为研究对象,1%O_(2)条件下培养观察UCHL5表达水平的变化。采用Western blot和实时定量聚合酶链反应(PCR)检测慢病毒载体感染Hela细胞后稳定调变UCHL5的效率,转录本分为空白对照组、过表达对照组、过表达UCHL5组转录本1~4和沉默对照组、沉默UCHL5组转录本1~2。将实验所用的细胞分为过表达对照组、过表达UCHL5组、沉默对照组和沉默UCHL5组。采用流式细胞术检测8 Gyγ射线照射48 h后细胞的凋亡率;采用细胞克隆形成实验检测0、2、4、6 Gyγ射线单次照射培养2周后4组细胞的克隆形成率;采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)实验检测4组细胞培养1周后的增殖率以及联合0、2、4、6、8、10 Gyγ射线照射后的增殖率。使用基因表达谱数据动态分析癌症基因组图谱数据库宫颈癌组织和正常组织中抗缺氧诱导因子1α(HIF-1α)与UCHL5表达的相关性。采用双荧光素酶报告基因实验观察HIF-1α对UCHL5的激活作用。组间比较采用单样本t检验,采用Pearson检验进行相关性分析。结果缺氧可诱导宫颈癌Hela细胞UCHL5的表达。Western blot和实时定量PCR结果显示,感染后的Hela细胞可显著上调或下调UCHL5的表达,其中,过表达UCHL5组转录本2和沉默UCHL5组转录本2的表达均较高,所以选择此2种转录本进行后续实验。克隆形成实验结果显示,与接受相同剂量照射的过表达对照组相比,上调UCHL5增加了Hela细胞的克隆形成率,在0、2、4、6 Gy剂量照射后克隆形成率的差异均有统计学意义(t=14.16、19.22、8.76、6.79,均P<0.05)。流式细胞术结果显示,与沉默对照组相比,沉默UCHL5促进了Hela细胞的凋亡(t=10.29,P<0.05),增加了γ射线诱导的细胞凋亡率(t=52.01,P<0.05)。MTT实验结果显示,与过表达对照组相比,上调UCHL5可升高宫颈癌Hela细胞的增殖率,增殖率在第3天时差异有统计学意义(t=3.905,P<0.05);与过表达对照组相比,等剂量照射UCHL5上调组升高了受照细胞的增殖率,在剂量为6、8、10 Gy时,细胞的增殖率差异均有统计学意义(t=3.40、4.06、3.68,均P<0.05)。宫颈癌组织中HIF-1α表达水平和UCHL5表达水平呈正相关(R=0.31,P<0.01),双荧光素酶报告基因结果显示HIF-1α结合并激活UCHL5启动子的活性,其活性增加了2.5倍(t=30.47,P<0.05)。结论缺氧条件下,宫颈癌Hela细胞中UHCL5的诱导表达降低了细胞的辐射敏感性,其潜在的机制可能与HIF-1α转录激活UCHL5的表达有关。

Objective To investigate the role of hypoxia-induced ubiquitin carboxyl-terminal hydrolase isozyme L5(UCHL5)in the regulation of radiosensitivity of Hela cells in cervical cancer.Methods UCHL5 levels were detected in Hela cells cultured under 1%O_(2) condition.Western blot and qRT-PCR analyses verified the efficiency of lentiviral vector infection on stable UCHL5 modulation in HeLa cells,including oe-vector,oe-UCHL5-1,oe-UCHL5-2,oe-UCHL5-3,oe-UCHL5-4 and sh-vector,sh-UCHL5-1 and sh-UCHL5-2.The cells used in the experiment were divided into the following groups:oe-vector,oe-UCHL5,sh-vector and sh-UCHL5.Cell colony-formation rate and radiosensitivity were detected by colony-formation assay combined with single-dose(0,2,4 and 6 Gy)γ-ray irradiation after culturing for 2 weeks.The effect of radiation on cell apoptosis was determined by flow cytometry after 48 h of 8 Gyγ-ray irradiation.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect the cell viability of down-or up-regulated UCHL5 cells before and after 0,2,4,6,8 and 10 Gy ofγ-ray irradiation.Gene expression profiling interactive analysis was used to analyze the correlated expression between UCHL5 and hypoxia inducible factor-1α(HIF-1α).The transcriptional activation effect of UCHL5 by HIF-1αwas detected using dual-luciferase reporter-gene assay.Differences between groups were compared by single-sample t test,and Pearson test was used for correlation analysis.Results UCHL5 was significantly induced by hypoxia after culturing at different time points.Hela cell lines with stably overexpressed and silenced UCHL5 were successfully constructed,with oe-UCHL5-2 and sh-UCHL5-2 having the highest regulatory efficiency.These two groups were selected for subsequent experiments.Compared with the control group that received the same dose of irradiation,significant differences existed at doses of 0,2,4 and 6 Gy(t=14.16,19.22,8.76,6.79,all P<0.05),respectively.Knock-down of UCHL5 promoted apoptosis(t=10.29,P<0.05)and radiation-induced apoptosis(t=52.01,P<0.05).UCHL5 up-regulation promoted cell proliferation,and the proliferation rate was statistically significant on the third day(t=3.905,P<0.05).Furthermore,UCHL5 strengthened the viability(t=3.40,4.06,3.68,all P<0.05)of irradiated Hela cells,with significant differences at doses of 6,8 and 10 Gy(t=3.40,4.06,3.68,all P<0.05),respectively.The expression levels of HIF-1αand UCHL5 in cervical cancer tissues were positively correlated(R=0.31,P<0.01).Additionally,HIF-1αwas a potential transcriptional activator of UCHL5 in Hela cells,and its activity increased 2.5 times(t=30.47,P<0.05).Conclusions The induced expression of UHCL5 in cervical cancer Hela cells under hypoxia condition can reduce the radiation sensitivity of cells.The underlying mechanism may be related to the HIF-1αtranscriptional activation of UCHL5 expression.