摘要
目的探讨聚腺苷二磷酸-核糖聚合酶1(PARP-1)和p16/视网膜母细胞瘤(Rb)信号通路在氢醌(HQ)诱导的TK6细胞中的表达及其相关调控机制。方法按照2×2析因设计模型,将TK6细胞按HQ染毒水平分为磷酸缓冲盐溶液(PBS)-TK6组和HQ-TK6组,再按照多柔比星(DOX)干预水平分为无DOX干预组和DOX干预组,共4组。PBS-TK6组细胞以PBS处理,HQ-TK6组细胞予终浓度为20.0μmol/L的HQ染毒处理;无DOX干预组细胞加入体积分数为0.05%的二甲基亚砜,DOX干预组细胞加入终浓度为0.5μmol/L的DOX。采用流式细胞术检测细胞周期分布情况,蛋白免疫印迹法检测p16/Rb信号通路p16、细胞周期蛋白D1(cyclinD1)、多功能蛋白E2转录因子1(E2F1)、Rb、p-Rb蛋白表达水平,免疫荧光和免疫共沉淀法检测p16核糖化水平。结果主效应分析结果显示,与PBS-TK6组比较,HQ-TK6组细胞G0/G1期细胞周期分布百分比和p16蛋白相对表达水平均下调(P值均<0.05),S期细胞周期分布百分比和cyclinD1、p-Rb蛋白相对表达水平均上升(P值均<0.05);与无DOX干预组比较,DOX干预组细胞在G0/G1期、G2/M期的细胞周期分布百分比和p16蛋白相对表达水平均上升(P值均<0.05),在S期细胞百分比和cyclinD1、p-Rb蛋白相对表达水平均下调(P值均<0.05)。交互效应分析结果显示,与PBS-TK6细胞无DOX干预组比较,PBS-TK6细胞DOX干预组中Rb和E2F1蛋白相对表达水平均下调(P值均<0.05),HQ-TK6细胞无DOX干预亚组的Rb蛋白相对表达水平均下调(P<0.05),E2F1蛋白相对表达水平上调(P<0.05);与PBS-TK6细胞DOX干预组比较,HQ-TK6细胞DOX干预组的Rb蛋白相对表达水平下调(P<0.05),E2F1蛋白相对表达水平上调(P<0.05);与HQ-TK6细胞无DOX干预组比较,HQ-TK6细胞DOX干预组的Rb蛋白相对表达水平上调(P<0.05),E2F1蛋白相对表达水平下调(P<0.05)。结论PARP-1可能通过调控p16/Rb信号通路参与TK6细胞的周期调控。
ObjectiveTo investigate the expression of polyadenosine diphospho-ribose polymerase 1(PARP-1)and p16/retinoblastoma(Rb)protein in hydroquinone(HQ)-induced TK6 cells and their regulatory mechanisms.MethodsAccording to the 2×2 factorial design model,TK6 cells were divided into PBS-TK6 group and HQ-TK6 group based on HQ exposure,and then sub-divided into non-DOX intervention subgroup and DOX intervention subgroup based on DOX intervention,a total of four groups.The PBS-TK6 group was treated with phosphate buffer saline,and the HQ-TK6 group was treated with HQ at a final concentration of 20.0μmol/L.The non-DOX intervention subgroup was added with 0.05%dimethyl sulfoxide;and the DOX intervention subgroup was added with PARP-1 agonist DOX at a final concentration of 0.5μmol/L.The distribution of cell cycle was detected by flow cytometry.The protein expression of p16/Rb,cyclin D1(cyclinD1),multifunctional protein E2 transcription factor 1(E2F1),Rb,and p-Rb were detected by Western blot,and the level of p16 ribosylation was detected by immunofluorescence and immunoprecipitation.ResultsCompared with the PBS-TK6 group,the cell cycle distribution percentage in G0/G1 phase and the relative expression levels of p16 proteins were decreased in the cells of the HQ-TK6 group(all P<0.05).The cell cycle distribution percentage in S phase and the relative expression levels of cyclinD1 and p-Rb proteins were up-regulated(all P<0.05).Compared with the non-DOX intervention group,the cell cycle distribution percentage in G0/G1and G2/M phases and the relative expression level of p16 protein increased in the DOX intervention group(all P<0.05).The percentage of cells in S phase and the relative expression levels of cyclinD1 and p-Rb proteins were down-regulated(all P<0.05).The results of interaction effect analysis showed that compared with the non-DOX PBS-TK6 cells,the relative expression levels of Rb and E2F1 protein in the DOX PBS-TK6 cells intervention group were down-regulated(all P<0.05).The relative expression level of Rb protein in non-DOX HQ-TK6 cell group was down-regulated(P<0.05),and the relative expression of E2F1protein was up-regulated(P<0.05).Compared with DOX PBS-TK6 cell group,the relative expression level of Rb protein in DOX HQ-TK6 cell group was down-regulated and that of E2F1 protein was up-regulated(all P<0.05).Compared with the non-DOX HQ-TK6 cell group,the relative expression level of Rb protein in the DOX HQ-TK6 cell group was up-regulated and that of E2F1protein was down-regulated(all P<0.05).ConclusionPARP-1 participates in cell cycle regulation by regulating the p16/Rb signaling pathway in TK6 cells.
作者
邱伟峰
陈林
崔哲明
杨慧
罗皓
李华文
QIU Wei-feng;CHEN Lin;CUI Zhe-ming;YANG Hui;LUO Hao;LI Hua-wen(School of Public Health,Guangdong Medical University&Dongguan Key Laboratory of Environmental Medicine,Dongguan,Guangdong523808.China)
出处
《中国职业医学》
CAS
北大核心
2022年第2期126-132,共7页
China Occupational Medicine
基金
国家自然科学基金青年项目(81803278,82073582)
广东省自然科学基金面上项目(2018A030313580)
广东省医学科学技术研究基金项目(A2018197,A2019404)
广东医科大学学科建设项目(4SG22003G)。
关键词
氢醌
细胞周期
聚腺苷二磷酸-核糖聚合酶1
P16
恶性转化
Hydroquinone
Cell cycle
Polyadenosine diphospho-ribose polymerase 1
p16
Malignant transformation
