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

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和蛋白相对表达量均升高(P<0.001)。与8 Gy+NC-sh组比较,8 Gy+MLL3-sh组SiHaR细胞的细胞活力降低,γ-H2AX的蛋白相对表达量和γ-H2AX foci数目升高,ATM、ATR、BRCA1和RAD50的mRNA水平降低(P<0.05)。结论:宫颈癌细胞MLL3的表达下调促进了其生长和转移,但降低DNA损伤修复能力,提高宫颈癌细胞的放射敏感性。

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-2 protein increased,the number of invasive cells and the relative expression of MMP2 and MMP9 protein of SiHa cells increased in MLL3-sh group(P<0.05).Compared with NC-OE group,the relative expression of MLL3 mRNA and protein increased,the cell viability decreased,the apoptosis rate,the relative expression of Bax and cleaved caspase3 protein increased,the relative expression of Bcl-2 protein decreased,the number of invasive cells and the relative expression of MMP2 and MMP9 protein of SiHa cells decreased in MLL3-OE group(P<0.05).Compared with SiHa cells,the relative expression of MLL3 mRNA and protein in SiHaR cells increased(t=18.476,P<0.001;t=29.126,P<0.001).Compared with 8Gy+NC-sh group,the cell viability decreased,the relative expression ofγ-H2AX protein and the number ofγ-H2AX foci increased,and the levels of ATM,ATR,BRCA1 and RAD50 mRNA of SiHaR cells decreased in 8Gy+MLL3-sh group(P<0.05).Conclusion:The downregulation of MLL3 expression promotes the growth and metastasis of cervical cancer cells,but reduces the ability of DNA damage repair and improves the radiosensitivity of cervical cancer cells.