MLL3基因对宫颈癌细胞放射敏感性和DNA损伤修复能力的影响  

Effects of MLL3 Gene on Radiosensitivity and DNA Damage Repair of Cervical Cancer Cells

作　　者：李佩[1] 唐阳芳[2] 高雪[3] 程柳 刘玉 马素叶 张玮 王金声 康媛[1] LI Pei;TANG Yang-fang;GAO Xue;CHENG Liu;LIU Yu;MA Su-ye;ZHANG Wei;WANG Jin-sheng;KANG Yuan(Department of Obstetrics and Gynecology,The Second People's Hospital of Shaanxi Province,Xi'an,Shaanxi,710005,China;Department of Gynecology,The First Affiliated Hospital of Xi'an Medical University,Xi'an,Shaanxi,710005,China;Department of Obstetrics and Gynecology,Xi'an Fourth Hospital,Xi'an,Shaanxi,710004,China;Department of Gynecology,Xi'an New Chang'An Maternity Hospital,Xi'an,Shaanxi,710001,China)

机构地区：[1]陕西省第二人民医院妇产科,陕西西安710005 [2]西安医学院第一附属医院妇科,陕西西安710005 [3]西安市第四医院妇产科,陕西西安710004 [4]西安新长安妇产医院妇科,陕西西安710001

出　　处：《现代生物医学进展》2024年第1期46-53,135,共9页Progress in Modern Biomedicine

基　　金：陕西省重点研发计划项目(2022SF-553)。

摘　　要：目的:探究髓系/淋巴系或混合谱系白血病3基因(myeloid/lymphoid or mixed-lineage leukemia 3,MLL3)对宫颈癌细胞生长、转移、放射敏感性的影响。方法:选择60例宫颈鳞癌患者的癌组织及配对癌旁组织,采用实时定量聚合酶链式反应(qRT-PCR)检测检测MLL3 mRNA水平。体外培养SiHa细胞,将其分为以下5组:Control组(不转染)、NC-sh组(转染阴性对照shRNA慢病毒)、MLL3-sh组(转染MLL3 shRNA慢病毒)、NC-OE组(转染阴性对照过表达慢病毒)和MLL3-OE组(转染MLL3过表达慢病毒)。进一步采用2300EX直线加速器9 MeVβ射线照射细胞建立放射抵抗SiHa细胞(SiHaR),然后将其分为:NC-sh组、MLL3-sh组、8 Gy+NC-sh组和8 Gy+MLL3-sh组。NC-sh组和MLL3-sh组细胞不照射,8 Gy+NC-sh组和8 Gy+MLL3-sh组细胞用9 MeVβ射线照射8 Gy。采用MTT法检测细胞增殖情况;Annexin V-FITC/PI双染色法检测细胞凋亡;Transwell检测细胞侵袭能力;qRT-PCR检测MLL3、共济失调毛细血管扩张征突变基因(ATM)、ATM-Rad3相关基因(ATR)、乳腺癌易感基因(BRCA1)和RAD50双链断裂修复蛋白(RAD50)的mRNA水平;Western blot检测MLL3、Bcl-2相关X蛋白基因(Bax)、B细胞淋巴瘤/白血病-2基因(Bcl-2)、cleaved caspase 3、基质金属蛋白酶(MMP)2、MMP9和γ-H2AX的表达;免疫荧光染色检测γ-H2AX的表达。结果:与癌旁组织相比,宫颈鳞癌组织中的MLL3 mRNA水平显著降低(P<0.001)。与NC-sh组比较,MLL3-sh组SiHa细胞的MLL3 mRNA和蛋白相对表达量降低,细胞活力升高,细胞凋亡率、Bax和cleaved caspase 3蛋白相对表达量降低,Bcl-2蛋白相对表达量升高,侵袭细胞数量、MMP2和MMP9蛋白相对表达量升高(P<0.05)。与NC-OE组比较,MLL3-OE组SiHa细胞的MLL3 mRNA和蛋白相对表达量升高,细胞活力降低,细胞凋亡率、Bax和cleaved caspase 3蛋白相对表达量升高,Bcl-2蛋白相对表达量降低,侵袭细胞数量、MMP2和MMP9蛋白相对表达量降低(P<0.05)。与SiHa细胞相比,SiHaR细胞中的MLL3 mRNA�Objective:To explore the effects of myeloid/lymphoid or mixed-lineage leukemia 3(MLL3)gene on the growth,metastasis,radiosensitivity and DNA damage repair of cervical cancer cells.Methods:Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the level of MLL3 mRNA in 60 patients with cervical squamous cell carcinoma and their adjacent tissues.SiHa cells were cultured in vitro and divided into the following 5 groups:Control group(no transfection),NC-sh group(transfection of negative control shRNA lentivirus),MLL3-sh group(transfection of MLL3shRNA lentivirus),NC-OE group(transfection of negative control overexpression lentivirus)and MLL3-OE group(transfection of MLL3 overexpression lentivirus).Radio-resistant SiHa cells(Si-HaR)were further established by using 2300EX linear accelerator 9 MeVβirradiation,and then divided into NC-sh group,MLL3-sh group,8Gy+NC-sh group and 8Gy+MLL3-sh group.The cells in NC-sh group and MLL3-sh group were not irradiated,while the cells in 8Gy+NC-sh group and 8Gy+MLL3-sh group were irradiated with 9 MeVβrays for 8 Gy.Cell proliferation was detected by MTT method.Apoptosis was detected by AnnexinV-FITC/PI double staining.Cell invasion was detected by Transwell.The levels of MLL3,ataxia-telangiectasia mutated gene(ATM),ATM-Rad3-Related(ATR),breast cancer susceptibility gene 1(BRCA1)and RAD50 double strand break repair protein(RAD50)mRNA were detected by qRT-PCR.The expression of MLL3,B-cellymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cleaved caspase3,matrix metalloproteinase(MMP)2,MMP9 andγ-H2AX was detected by Western blot.The expression level ofγ-H2AX was detected by immunofluorescence staining.Results:Compared with the adjacent tissues,the level of MLL3 mRNA in cervical squamous cell carcinoma decreased significantly(P<0.001).Compared with NC-sh group,the relative expression of MLL3 mRNA and protein decreased,the cell viability increased,the apoptosis rate,the relative expression of Bax and cleaved caspase3 protein decreased,the relative expression of Bcl

关 键 词：宫颈癌 髓系/淋巴系或混合谱系白血病3 生长 转移 放射敏感性 DNA损伤修复 

分 类 号：R-33[医药卫生] R737.33