摘要
目的:探讨核受体亚家族2F组成员2(NR2F2)对人卵巢癌SKOV3细胞生物学行为的影响,并阐明其分子机制,为卵巢癌的临床治疗提供新的思路。方法:采用基因表达谱交互式分析(GEPIA)数据库分析卵巢组织中NR2F2基因表达水平,并分析其与卵巢癌患者临床预后的相关性。将人卵巢癌SKOV3细胞分为对照组和NR2F2过表达组(NR2F2 OE组),待细胞密度达到70%时,分别转染mCherry对照病毒和NR2F2 OE过表达病毒,48 h后通过嘌呤霉素(puro)筛选稳定转染的SKOV3细胞系。采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测2组细胞转染效率,RT-qPCR法检测2组细胞中NR2F2和性别决定区Y-box 2(SOX2)mRNA表达水平,Western blotting法检测2组细胞中NR2F2、SOX2、三磷酸腺苷结合转运蛋白G超家族成员2(ABCG2)和程序性细胞死亡配体1(PD-L1)蛋白表达水平。CCK-8法检测2组细胞增殖活性,细胞划痕实验检测2组细胞迁移率,Transwell小室实验检测2组穿膜细胞数,成球实验检测2组细胞成球数,外周血单核细胞(PBMCs)杀伤肿瘤细胞活性实验检测2组存活肿瘤细胞的相对密度,CCK-8法检测紫杉醇(PTX)和卡铂(CBP)对2组细胞的半数抑制浓度(IC_(50))。结果:与正常卵巢组织比较,卵巢肿瘤组织中NR2F2 mRNA表达水平降低(P<0.05),并随卵巢肿瘤临床病理分级提高而降低;NR2F2 mRNA表达水平较高的患者临床预后较好。成功构建过表达NR2F2的SKOV3细胞,与对照组比较,NR2F2 OE组细胞中NR2F2 mRNA和蛋白表达水平均明显升高(P<0.001);CCK-8实验,与对照组比较,不同时间点(1、2、3和4 d)NR2F2 OE组细胞增殖活性均降低(P<0.05或P<0.01);细胞划痕实验,与对照组比较,NR2F2 OE组细胞迁移率降低(P<0.001);Transwell小室实验,与对照组比较,NR2F2 OE组穿膜细胞数减少(P<0.01);与对照组比较,NR2F2 OE组细胞成球数减少(P<0.05),SKOV3细胞中SOX2 mRNA(P<0.01)和蛋白(P<0.001)表达水平均降低;与对照组比较,NR2F2 OE组存活肿瘤细胞的相对密度降低,但差异无统计学意义(P>0.05),PD-L1蛋白表达水平降低(P<0.05);与对照组比较,NR2F2 OE组细胞增殖活性减弱(P<0.05),对PTX和CBP的药物敏感性增强,PTX的IC_(50)明显减小,CBP的IC_(50)因其药物浓度过高未能计算;ABCG2蛋白表达水平降低(P<0.05)。结论:NR2F2过表达可抑制人卵巢癌SKOV3细胞的增殖、迁移和侵袭能力,降低SOX2、PD-L1和ABCG2蛋白表达水平,抑制人卵巢癌SKOV3细胞的干性和免疫逃逸能力,增强人卵巢癌SKOV3细胞对PTX和CBP的敏感性。
Objective:To investigate the effect of nuclear receptor subfamily 2 group F member 2(NR2F2)on the biological behaviors of human ovarian cancer SKOV3 cells,and to clarify its molecular mechauism and provide the new idea for treatment of ovarian cancer.Methods:Gene Expression Profiling Interactive Analysis(GEPIA)Database analyse the expression level of NR2F2 gene in ovarian tissue,and analyse its correlation with clinical prognosis of ovarian cancer patients.The human ovarian cancer SKOV3 cells were divided into control group and NR2F2 over-expression(NR2F2 OE)group,which were transfected with mCherry control virus and NR2F2 OE over-expression virus,respectively,when the cell deusity reached 70%,and the stable transfection SKOV3 cell lines were screened with puromycin(puro)48 h lafter.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the transfection efficiencies of the cells;RT-qPCR method was used to detect the expression levels of NR2F2 and sex-determining region Y-box 2(SOX2)mRNA in the cells in two groups;Western blotting method was used to detect the expression levels of NR2F2,ATP-binding cassette superfamily G member 2(ABCG2),and programmed cell death 1-ligand 1(PD-L1)protcins in the cells in two groups.CCK-8 assay was used to detect the proliferation activities of the cells in two groups;Wound assay was used to detect the migration rates of the cells in two groups;Transwell chamber assay was used to detect the number of transmembrane cells;Spheroidization assay was used to detect the numbers of spheroids in the cells;peripheral blood mononuclear cells(PBMCs)-mediated tumor cell killing assay was used to detect the relative densities of surviving tumor cells;CCK-8 assay was used to detect the half maximal inhibitory concentration(IC_(50))of paclitaxel(PTX)and carboplatin(CBP).Results:Compared with normal ovarian tissue,the expression level of NR2F2 gene in ovarian tumor tissue was decreased(P<0.05),and decreased with the improvement of clinical pathological grading of ovarian tumor.The patients with higher expression level of NR2F2 gene had better clincal prognosis.The SKOV3 cells with NR2F2 over-expresson were successfully constructed,and the expression levels of NR2F2 mRNA and protein in the cells in NR2F2 OE group were increased compared with control group(P<0.001).The CCK-8 assay results showed that compared with control group,the proliferation activities of the cells in NR2F2 OE group were decreased at different time points(1,2,3,and 4 d)(P<0.05 or P<0.01).The cell wound assay results showed that compared with control group,the migration rate of the cells in NR2F2 OE group was decreased(P<0.001).The Transwell assay results showed that compared with control group,the number of transmembrane cells in NR2F2 OE group was decreased(P<0.01).Compared with control group,the number of the spheroids in NR2F2 OE group was decreased(P<0.05),and the expression levels of SOX2 mRNA(P<0.01)and protein(P<0.001)were increased.Compared with control group,the relative density of surviving tumor cells in NR2F2 OE group was decreased,but the difference was not significant(P<0.05),and the expression level of PD-L1 protein was decreased(P<0.05).Compared with control group,the proliferation activities of cells in NR2F2 OE group were decreased(P<0.05),and the drug sensitivities of the cells to PTX and CBP were enhanced(P<0.05);the IC_(50)of PTX was significantly reduced,while the IC50 of CBP could not be calculated due to excessively high drug concentration;the expression level of ABCG2 protein was decreased(P<0.05).Conclusion:The overexpression of NR2F2 may inhibit the proliferation,migration,and invasion of the human ovarian cancer SKOV3 cells,decrease the expression levels of SOX2,PD-L1 and ABCG2 proteins,suppress the stemness and immune evasion ability of the SKOV3 cells,and enhance the sensitivities of the SKOV3 cells to PTX and CBP.
作者
张硕
夏云秀
陈微微
董洪亮
崔冰洁
刘翠兰
刘志强
王飞
杜静
ZHANG Shuo;XIA Yunxiu;CHEN Weiwei;DONG Hongliang;CUI Bingjie;LIU Cuilan;LIU Zhiqiang;WANG Fei;DU Jing(Department of Gynecology,Affiliated Hospital,Binzhou Medical University,Binzhou 256603,China;Medical Research Center,Affiliated Hospital,Binzhou Medical University,Binzhou 256603,China;Medical Integration and Practice Center,Shandong University,Jinan 250100,China)
出处
《吉林大学学报(医学版)》
北大核心
2025年第1期58-67,共10页
Journal of Jilin University:Medicine Edition
基金
国家自然科学基金项目(31900441,82373097)
中国博士后自然科学基金项目(2023M742089)
山东省科技厅自然科学基金项目(ZR2019MC026,ZR2023QH080,ZR2022MH305,ZR2022QH192)
山东省卫健委齐鲁卫生与健康杰出青年人才项目(2020)
山东省科技厅泰山学者青年基金项目(2021)。
关键词
核受体亚家族2F组成员2
卵巢肿瘤
肿瘤干性
免疫逃逸
耐药
Nuclear receptor subfamily 2 group F member 2
Ovarian neoplasm
Tumor stemness
Immune escape
Drug resistance
