FADD基因对宫颈癌细胞凋亡和侵袭及NF-κB信号通路的影响  

Effects of FADD gene on apoptosis,invasion and NF-κB signal pathway of cervical cancer cells

作　　者：康媛[1] 唐阳芳[2] 高雪[3] 程柳 刘玉 马素叶 张玮 王金声 李佩[1] KANG Yuan;TANG Yangfang;GAO Xue;CHENG Liu;LIU Yu;MA Suye;ZHANG Wei;WANG Jinsheng;LI Pei(Department of Obstetrics and Gynecology,Second People’s Hospital of Shaanxi Province,Xi’an 710005,China;Department of Gynecology,First Affiliated Hospital of Xi’an Medical University;Department of Obstetrics and Gynecology,Xi’an Fourth Hospital;Department of Gynecology,Xi’an New Chang’an Maternity Hospital)

机构地区：[1]陕西省第二人民医院妇产科,西安710005 [2]西安医学院第一附属医院妇科 [3]西安市第四医院妇产科 [4]西安新长安妇产医院妇科

出　　处：《山西医科大学学报》2023年第9期1183-1191,共9页Journal of Shanxi Medical University

基　　金：陕西省重点研发计划项目(2022SF-553)。

摘　　要：目的探讨Fas相关死亡结构域(FADD)基因对宫颈癌细胞凋亡和侵袭及NF-κB信号通路的影响。方法qRT-PCR检测60例宫颈鳞癌患者的癌组织及配对癌旁组织,人宫颈鳞癌细胞系SiHa及人宫颈上皮永生化细胞系H8中FADD mRNA水平。将SiHa细胞分为对照组、NC-sh组、FADD-sh组、NC-OE组和FADD-OE组。对照组SiHa细胞正常培养,不进行转染;NC-sh组、FADD-sh组、NC-OE组和FADD-OE组SiHa细胞分别使用Lipofectamine 2000试剂转染shRNA慢病毒阴性对照(NC-sh)、FADD shRNA慢病毒(FADD-sh)、过表达慢病毒阴性对照(NC-OE)和FADD过表达慢病毒(FADD-OE)。通过MTT法和集落形成实验检测细胞增殖。通过AnnexinⅤ-FITC/PI双染色法检测细胞凋亡。通过Transwell检测细胞侵袭。通过qRT-PCR检测FADD mRNA水平。通过Western blot检测cleaved Caspase-3、cleaved Caspase-8、基质金属蛋白酶(MMP)2、MMP9、核因子(NF)-κB p65和p-NF-κB p65的表达水平。结果与配对癌旁组织相比,宫颈鳞癌组织中FADD mRNA水平降低(t=29.333,P<0.001)。与H8细胞相比,SiHa细胞中FADD mRNA水平降低(t=27.219,P<0.001)。与Ⅰ-Ⅱ期相比,Ⅲ-Ⅳ期患者癌组织中FADD mRNA水平降低(t=7.727,P<0.001)。与淋巴结未转移患者相比,淋巴结转移患者癌组织中FADD mRNA水平降低(t=7.618,P<0.001)。与NC-sh组比较,FADD-sh组细胞的FADD mRNA和蛋白相对表达量降低(P<0.05),相对细胞活力和集落数量升高(P<0.05),细胞凋亡率、cleaved Caspase-3和cleaved Caspase-8蛋白相对表达量降低(P<0.05),侵袭细胞数量、MMP2和MMP9蛋白相对表达量升高(P<0.05),NF-κB p65相对磷酸化水平升高(P<0.05)。与NC-OE组比较,FADD-OE组细胞的FADD mRNA和蛋白相对表达量升高(P<0.05),相对细胞活力和集落数量降低(P<0.05),细胞凋亡率、cleaved Caspase-3和cleaved Caspase-8蛋白相对表达量升高(P<0.05),侵袭细胞数量、MMP2和MMP9蛋白相对表达量降低(P<0.05),NF-κB p65相对磷酸化水平降低(P<0.05)。对照组、NC-sh组Objective To investigate the effect of Fas-associated death domain(FADD)gene on apoptosis,invasion of cervical cancer cells and NF-κB signal pathway.Methods The level of FADD mRNA was detected in 60 pairs of cervical squamous cell carcinoma and matched adjacent tissues,human cervical squamous cell line SiHa and human cervical epithelial immortalized cell line H8 by qRT-PCR.SiHa cells were divided into control group,NC-sh group,FADD-sh group,NC-OE group and FADD-OE group.SiHa cells in control group were cultured normally.SiHa cells in NC-sh group,FADD-sh group,NC-OE group and FADD-OE group were transfected with negative control shRNA lentivirus(NC-sh),FADD shRNA lentivirus(FADD-sh),negative control overexpression lentivirus(NC-OE)and FADD overexpression lentivirus(FADD-OE)via Lipofectamine2000 reagent,respectively.Cell proliferation was detected by MTT assay and colony formation test.Apoptosis was detected by AnnexinⅤ-FITC/PI double staining.Cell invasion was detected by Transwell.The level of FADD mRNA was detected by qRT-PCR.The expression levels of cleaved Caspase-3,cleaved Caspase-8,matrix metalloproteinase(MMP)2,MMP9,nuclear factor-κB(NF-κB)p65 and p-NF-κB p65 were detected by Western blot.Results Compared with matched adjacent tissues,the level of FADD mRNA in cervical squamous cell carcinoma was decreased(t=29.333,P<0.001).Compared with H8 cells,the level of FADD mRNA was decreased in SiHa cells(t=27.219,P<0.001).Compared with stageⅠ-Ⅱpatients,the level of FADD mRNA was decreased in cancer tissue of patients with stageⅢ-Ⅳ(t=7.727,P<0.001).Compared with patients without lymph node metastasis,the level of FADD mRNA was decreased in cancer tissues of patients with lymph node metastasis(t=7.618,P<0.001).Compared with NC-sh group,the relative expression levels of FADD mRNA and protein were decreased(P<0.05),the relative cell viability and the colony number were increased(P<0.05),the apoptosis rate,and the relative expression of cleaved Caspase-3 and cleaved Caspase-8 protein were decreased(P<0.05),

关 键 词：宫颈癌 Fas相关死亡结构域 凋亡 侵袭 核因子-ΚB 

分 类 号：R737.33[医药卫生—肿瘤]