卵巢上皮性癌转移的关键基因筛选与功能分析

Screening and functional analysis of key genes in ovarian epithelial carcinoma metastasis

目的:筛选卵巢上皮性癌(卵巢癌)转移的关键基因,并基于卵巢癌细胞株SKOV3,探索其对卵巢癌细胞迁移侵袭的影响。方法:基于癌症基因组图谱(TCGA)数据库、基因表达综合(GEO)数据库及人类基因数据库(GeneCards),通过limma差异表达分析及Kaplan-Meier生存分析挖掘在卵巢癌中高表达且与患者不良预后相关的迁移侵袭相关基因,并通过基因本体(GO)功能富集分析预测基因参与的生物学过程。利用PXN敲减慢病毒感染SKOV3细胞后,通过细胞划痕实验和Transwell迁移侵袭实验探讨PXN敲减对卵巢癌细胞迁移和侵袭能力的影响。利用癌症药物敏感性基因组学(GDSC)数据库对样本的化疗反应进行预测,评估PXN基因的卵巢癌细胞化疗敏感性的影响。结果:共筛选到36个迁移侵袭相关基因在卵巢癌组织中高表达,其中PXN基因高表达与卵巢癌患者不良预后相关(P<0.05)。PXN的功能与组蛋白修饰、染色质共价修饰、赖氨酸肽基修饰、蛋白质去泛素化、通过去除小蛋白进行蛋白质修饰、组蛋白去泛素化以及组蛋白H3-K4三甲基化等相关。荧光显微镜、Western blot结合RT-qPCR结果显示,在SKOV3细胞中成功敲减PXN基因。细胞功能学实验表明,PXN敲减组相对于空白对照组和阴性对照组,划痕愈合面积更小,穿膜细胞数更少。PXN高表达组中的顺铂及紫杉醇IC 50明显高于PXN低表达组(P<0.001),而其吉西他滨及多柔比星的IC 50明显低于PXN低表达组(P<0.0001)。肿瘤细胞中紫杉醇的IC 50明显低于正常卵巢细胞(P<0.0001),其西他滨及多柔比星的IC 50值高于正常卵巢细胞(P<0.0001)。结论:迁移侵袭相关基因PXN在卵巢癌中高表达,且与患者不良预后相关。PXN表达水平降低能抑制卵巢癌细胞SKOV3的迁移和侵袭。PXN基因表达水平与卵巢癌细胞对化疗药物的敏感性相关。

Objective:To screen key genes for ovarian epithelial carcinoma(ovarian cancer)metastasis and to explore their effects on the migration and invasion of ovarian cancer cells based on ovarian cancer cell line SKOV3.Methods:By using limma differential expression analysis and Kaplan-Meier survival analysis,we mined migratory invasion-related genes that were highly expressed in ovarian cancer and associated with poor patient prognosis based on The Cancer Genome Atlas(TCGA)database,Gene Expression Omnibus(GEO)database,and human gene databases(GeneCards).The biological function of genes was predicted using a Gene Ontology(GO)functional enrichment study.Following PXN knockdown lentivirus infection of SKOV3 cells,the impact of PXN knockdown on the migration and invasion capacity of ovarian cancer cells was examined using the cell scratch test and the Transwell migration invasion assay.Analysis of the effect of the PXN gene on the chemosensitivity of ovarian cancer cells utilizing the Genomics of Drug Sensitivity in Cancer(GDSC)database to predict the chemotherapy response in samples.Results:A total of 36 migration invasion-related genes were screened for high expression in ovarian cancer tissues,among which,high expression of PXN gene was associated with poor prognosis in ovarian cancer patients(P<0.05).The function of PXN was associated with histone modification,covalent chromatin modification,peptidyl-lysine modification,protein deubiquitination,protein modification by small protein removal,histone deubiquitination,and histone H3-K4 trimethylation.The PXN gene in SKOV3 cells was successfully knocked down as evidenced by results from fluorescence microscopy,Western blot analysis,and RT-qPCR.Comparative to the blank control and negative control groups,cell functional experiments revealed that the PXN knockdown group had a smaller scratch healing area and fewer cells crossing the vesicle membrane.While the IC 50 of its gemcitabine and doxorubicin was significantly lower than that of the PXN low expression group(P<0.0001),the IC 50 of cisplatin and paclitaxel in the PXN high expression group was significantly greater than that in the PXN low expression group(P<0.001).The IC 50 values of both gemcitabine and doxorubicin were greater than those of normal ovarian cells(P<0.0001),and the IC 50 of paclitaxel was significantly lower in tumor cells than in normal ovarian cells(P<0.0001).Conclusion:The migration invasion-related gene PXN is highly expressed in ovarian cancer and is associated with poor patient prognosis.The reduction of PXN expression level could inhibit the migration and invasion of ovarian cancer cells SKOV3.The expression level of PXN gene correlated with the sensitivity of ovarian cancer cells to chemotherapeutic drugs.