宫颈癌顺铂耐药相关基因筛选及KRT6A表达对顺铂耐药宫颈癌SiHa细胞生物学行为的影响  

Screening of cisplatin-resistant genes in cervical cancer and influence of KRT6A expression on the biological behavior of cisplatin-resistant cervical cancer SiHa cells

作　　者：夏娜娜 杨安银 余敏敏 XIA Na-na;YANG An-yin;YU Min-min(Department of Gynaecology,Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine,the Second Hospital of Nanjing,Nanjing,Jiangsu 210003,China)

机构地区：[1]南京中医药大学附属南京医院、南京市第二医院妇科,江苏南京210003

出　　处：《中华实用诊断与治疗杂志》2023年第3期252-260,共9页Journal of Chinese Practical Diagnosis and Therapy

基　　金：江苏省医学重点人才基金(ZDRCA2016072);南京中医药大学自然科学基金(XZR2020070)。

摘　　要：目的应用生物信息学方法筛选宫颈癌顺铂耐药相关的枢纽基因,探讨KRT6A表达对顺铂耐药宫颈癌SiHa细胞生物学行为的影响。方法(1)收集TCGA数据库306份宫颈癌标本的mRNA表达数据,基于GDSC数据库,应用R软件包“pRRophetic”预测宫颈癌样本顺铂化疗的敏感度,采用线性回归分析顺铂的半数抑制浓度(the half inhibitory concentration,IC_(50))。依据IC_(50)值将宫颈癌样本分为顺铂敏感组和耐药组,应用limma软件包进行差异表达mRNA分析,对其进行基因本体(gene ontology,GO)和京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,应用Cytoscape软件构建差异表达基因的蛋白互作网络,进行模块分析并筛选枢纽基因。选择KRT6A基因进行细胞实验。(2)取对数生长期SiHa和顺铂耐药SiHa(SiHa/DDP)细胞,采用实时荧光定量PCR法检测KRT6A mRNA相对表达量;分别应用0、0.625、1.25、2.5、5、10、20 mg/L顺铂处理48 h,采用CCK-8法检测2组细胞增殖率。(3)取对数生长期SiHa/DDP细胞分为转染组(转染siRNA-KRT6A)和对照组(转染siRNA-NC),转染48 h,2组采用实时荧光定量PCR法检测KRT6A mRNA相对表达量,采用Western blot法检测KRT6A蛋白相对表达量;然后采用CCK-8法检测应用0、0.625、1.25、2.5、5、10、20 mg/L顺铂处理48 h细胞增殖,计算顺铂的IC_(50)值。转染48 h取转染组和对照组细胞,应用5 mg/L(IC_(50)值)浓度的顺铂处理48 h,采用流式细胞术检测处理0、48 h时细胞凋亡。结果(1)顺铂敏感组与顺铂耐药组差异表达基因有190个,与顺铂敏感组比较,顺铂耐药组74个基因表达上调、116个基因表达下调。GO分析结果提示差异表达基因参与表皮发育、肽酶活性调节、角质形成细胞分化、蛋白质水解和水解酶活性的负调控等生物过程,其产物参与细胞间连接、顶端质膜、中间体、侧质膜等细胞组分,发挥丝氨酸型内肽酶抑制剂活性、氧化还原酶Objective To screen the cervical cancer cisplatin resistance-associated pivotal genes by bioinformatic methods and to investigate the influence of KRT6A expression on the biological behaviors of cisplatin-resistant cervical cancer SiHa cells.Methods(1)The mRNA expression data of 306 cervical cancer specimens were collected from the TCGA database,and the sensitivity to cisplatin chemotherapy in cervical cancer samples was predicted using the R package“pRRophetic”based on the GDSC database,and the half inhibitory concentration(IC_(50))of cisplatin was analyzed by linear regression.The cervical cancer samples were divided into cisplatin-sensitive and cisplatin-resistant groups based on IC_(50)values,and the differential expression mRNA analysis was performed using the limma package.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed for differential genes.The differential genes were used to construct their protein interaction networks by Cytoscape software to perform modular analysis and screen pivotal genes.The KRT6A gene was chosen for cell experiment.(2)The SiHa and SiHa/DDP cells in logarithmic growth phase were taken and the relative expression of KRT6A mRNA was detected by real-time fluorescence quantification PCR.After being treated with 0,0.625,1.25,2.5,5,10 and 20 mg/L cisplatin for 48 h,the proliferation rate of SiHa and SiHa/DDP cells was detected by CCK-8.(3)The SiHa/DDP cells in logarithmic growth phase were divided into transfected group(transfected with siRNA-KRT6A)and control group(transfected with siRNA-NC).The relative expressions of KRT6A mRNA and protein were detected after transfection for 48 h by real-time fluorescence quantitative PCR and Western blot,respectively.The proliferation rate was detected by CCK-8 after being treated with 0,0.625,1.25,2.5,5,10 and 20 mg/L cisplatin for 48 h,and the IC_(50)value of cisplatin was calculated.The cells in transfected and control groups were treated with 5 mg/L cisplatin(IC_(50))for 48 h,and the apopto

关 键 词：宫颈癌 差异表达基因 顺铂耐药 KRT6A基因 SIHA细胞 SiHa/DDP细胞 

分 类 号：R737.33[医药卫生—肿瘤]