聚ADP-核糖聚合酶-1在喉鳞状细胞癌细胞DNA氧化损伤及凋亡中的作用  

The role of poly ADP-ribose polymerase-1 in oxidative DNA damage and apoptosis of laryngeal squamous cell carcinoma cells

作　　者：宿伟鹏[1] 赵化荣[1] 刘攀[1] 张洋[1] 王松[2] SU Wei-peng;ZHAO Hua-rong;LIU Pan;ZHANG Yang;WANG Song(Tumor Center,First Affiliated Hospital of Xinjiang Medical University,Urumqi Xinjiang 830011,China;Department of Otolaryngology,First Affiliated Hospital of Xinjiang Medical University,Urumqi Xinjiang 830011,China)

机构地区：[1]新疆医科大学第一附属医院肿瘤中心,新疆乌鲁木齐830011 [2]新疆医科大学第一附属医院耳鼻喉科,新疆乌鲁木齐830011

出　　处：《局解手术学杂志》2023年第8期666-672,共7页Journal of Regional Anatomy and Operative Surgery

基　　金：国家重点实验室开放课题(SKL-HIDCA-2021-6)。

摘　　要：目的探究聚ADP-核糖聚合酶-1(PARP1)在喉鳞状细胞癌(LSCC)中的表达及其对LSCC细胞DNA氧化损伤与凋亡的影响。方法收集40例患者的LSCC组织及癌旁组织样本,采用免疫组化染色和qRT-PCR检测LSCC组织与癌旁组织中PARP1的表达。采用不同浓度Menadione诱导LSCC细胞系Hep-2,MTT法检测不同浓度诱导下细胞增殖抑制率。将Hep-2细胞随机分为对照组(正常培养)、Men组(20μmol/L Menadione处理)、Men+PARP1抑制剂组(20μmol/L Menadione+10μmol/L PARP1抑制剂Olaparib共处理)。MTT法计算各组细胞增殖抑制率;流式细胞术检测各组细胞内活性氧(ROS)水平;细胞免疫荧光染色观察各组细胞DNA损伤标记分子γ-H2AX焦点生成情况;Western blot测定各组细胞γ-H2AX、DNA-PKcs、BRCA1、LIG4、Caspase-3及Caspase-9蛋白表达;Hoechst33258染色观察各组细胞凋亡情况。结果LSCC组织中PARP1阳性表达率及PARP1 mRNA表达均明显高于癌旁组织(P<0.01)。Hep-2细胞经不同浓度Menadione诱导后,细胞增殖抑制率均升高(P<0.05)。与对照组比较,Men组和Men+PARP1抑制剂组的细胞增殖抑制率升高,细胞ROS水平升高,γ-H2AX焦点形成明显增加,γ-H2AX蛋白表达上调,DNA-PKcs、BRCA1、LIG4蛋白表达均下调,细胞出现浓缩、碎裂的蓝色凋亡体,Caspase-3和Caspase-9蛋白表达上调,差异均有统计学意义(P<0.05),且Men+PARP1抑制剂组以上变化较Men组更明显(P<0.05)。结论PARP1在LSCC组织中高表达,抑制其表达能够进一步加重Menadione诱导的Hep-2细胞DNA氧化损伤,并促进细胞凋亡。Objective To investigate the expression of poly ADP-ribose polymerase-1(PARP1)in laryngeal squamous cell carcinoma(LSCC)and its effect on DNA oxidative damage and apoptosis of LSCC cells.Methods LSCC tissue and paracancerous tissue samples from 40 patients were collected.Immunohistochemical staining and qRT-PCR were used to detect the expression of PARP1 in LSCC tissue and paracancerous tissue.LSCC cell line Hep-2 was induced by different concentrations of Menadione,and the inhibition rate of cell proliferation was detected by MTT assay.The Hep-2 cells were randomly divided into the control group(normal culture),Men group(treated with 20μmol/L Menadione),Men+PARP1 inhibitor group(co-treated with 20μmol/L Menadione and 10μmol/L PARP-1 inhibitor Olaparib),the inhibition rate of cell proliferation was calculated by MTT assay,the intracellular reactive oxygen species(ROS)levels in each group was detected by flow cytometry,the focus formation of DNA damage markerγ-H2AX in each group was observed by immunofluorescence staining,the protein expression ofγ-H2AX,DNA-PKcs,BRCA1,LIG4,Caspase-3 and Caspase-9 in each group were determined by Western blot,the apoptosis in each group was observed by Hoechst33258 staining.Results The positive expression rate of PARP1 and the relative expression of PARP1 mRNA in LSCC tissues were significantly higher than those in paracancerous tissue(P<0.01).The proliferation inhibition rates of Hep-2 cells after induced by different concentration of Menadione were increased(P<0.05).Compared with the control group,the inhibition rates of cell proliferation in the Men group and the Men+PARP1 inhibitor group were increased,the levels of ROS were increased,the formations ofγ-H2AX foci were significantly increased,and the relative protein expression ofγ-H2AX protein were up-regulated,the relative protein expression of DNA-PKcs,BRCA1 and LIG4 were all down-regulated,the cells appeared condensed and fragmented blue apoptotic bodies appeared in cells,the relative protein expression of Caspase-3 a

关 键 词：喉鳞状细胞癌 聚ADP-核糖聚合酶-1 DNA氧化损伤 凋亡 

分 类 号：R739.8[医药卫生—肿瘤]