LncRNAs调控卵巢癌细胞卡铂耐药的机制和功能研究  

Study on the mechanisms and functions of lncRNAs in regulating carboplatin resistance in ovarian cancer cells

作　　者：洪旖 陈翠兰 尹富强 刘夏[1,4] HONG Yi;CHEN Cuilan;YIN Fuqiang;LIU Xia(Key Laboratory of Longevity and Aging-Related Disease of Chinese Ministry of Education,Center for Translational Medicine,School of Basic Medical Sciences,Institute of Neuroscience and Guangxi Key Laboratory of Brain Science,Key Laboratory of Human Development and Disease Research,Education Department of Guangxi Zhuang Autonomous Region,Guangxi Medical University,Nanning 530021,China;Life Sciences Institute,Guangxi Medical University,Nanning 530021,China;Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumour,Ministry of Education,Guangxi Medical University,Nanning 530021,China;Collaborative Innovation Centre of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry,Guangxi Key Laboratory of Regenerative Medicine,Nanning 530021,China)

机构地区：[1]广西医科大学基础医学院,转化医学研究中心“长寿与老年相关疾病”教育部重点实验室,神经科学研究所“广西脑科学研究”重点实验室,广西高校人体发育与疾病研究重点实验室,南宁530021 [2]广西医科大学生命科学研究院,南宁530021 [3]广西医科大学区域性高发肿瘤早期防治研究教育部重点实验室,南宁530021 [4]广西医科大学再生医学与医用生物资源开发应用省部共建协同创新中心广西再生医学重点实验室,南宁530021

出　　处：《广西医科大学学报》2025年第1期74-86,共13页Journal of Guangxi Medical University

基　　金：国家自然科学基金项目(No.82260721;No.81903644);广西自然科学基金重点项目(No.2024GXNSFDA010045);广西医科大学高水平创新团队及杏湖学者计划项目(No.24/02304001018X)。

摘　　要：目的:基于表达谱芯片筛选卵巢癌卡铂耐药中的差异长链非编码RNA(lncRNAs)并进行功能富集分析,从整体上了解lncRNA响应卵巢癌铂类耐药的信号途径;筛选卡铂耐药调控关键lncRNAs并进行相关功能研究,初步探究分子机制。方法:表达谱芯片筛选两对卵巢癌卡铂耐药和敏感细胞(HeyA8-CBP/HeyA8及SKOV3-CBP/SKOV3)中差异表达lncRNAs;基因本体(GO)和京都基因与基因组百科全书(KEGG)对差异表达的lncRNAs进行功能注释和通路富集;实时荧光定量逆转录聚合酶链反应(RT-qPCR)检测lncRNAs的表达,蛋白免疫印迹法(western blotting)检测蛋白表达。慢病毒转染构建lncRNA DAPK1-IT1过表达细胞,克隆形成实验检测细胞增殖能力,流式细胞术检测细胞周期变化。结果:通过表达谱芯片分析,筛选出在两株耐药细胞中稳定差异表达lncRNAs 161个,其中表达上调lncRNAs 107个,表达下调lncRNAs 54个。KEGG通路富集结果表明,差异表达的lncRNAs显著富集于p53信号通路、溶酶体、粘着斑等经典耐药途径以及PPAR免疫相关信号通路。基于表达谱芯片和RT-qPCR验证,筛选出在两株卡铂耐药细胞中表达均显著下调4倍以上的lncRNA DAPK1-IT1进行功能验证。在卡铂处理下,过表达lncRNA DAPK1-IT1的卵巢癌卡铂耐药细胞SKOV3-CBP的克隆形成能力显著降低,并导致细胞周期阻滞于G2/M期;western blotting结果表明,lncRNA DAPK1-IT1显著影响细胞周期和细胞自噬相关蛋白的表达。结论:lncRNA DAPK1-IT1在卵巢癌卡铂耐药细胞中的表达稳定下调,其表达抑制卡铂耐药细胞克隆形成,阻滞细胞周期,影响细胞自噬,是卵巢癌卡铂耐药调控的潜在关键因子,而p53信号通路、溶酶体、PPAR信号通路和IL-18可能是lncRNAs调控卡铂耐药的潜在途径,研究结果为进一步理解卵巢癌耐药调控提供了新的思路。Objective:To screen for differentially expressed long non-coding RNAs(lncRNAs)in carboplatinresistant ovarian cancer(OC)based on expression profiling microarrays and conduct functional enrichment analysis to comprehensively understand the signaling pathways of lncRNAs in response to carboplatin resistance in OC.Additionally,to screen for key lncRNAs regulating carboplatin resistance and explore their molecular mechanisms through functional studies.Methods:Differential expressed lncRNAs were screened using expression profiling microarrays in two pairs of carboplatin-resistant and sensitive OC cells(HeyA8-CBP/HeyA8 and SKOV3-CBP/SKOV3).Functional annotation and pathway enrichment of differentially expressed lncRNAs were performed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was used to detect lncRNA expression,and western blotting was employed to assess protein expression.Lentiviral transfection was utilized to construct lncRNA DAPK1-IT1 over-expressing cells,colony formation assays were conducted to examine cell proliferation,and flow cytometry was used to analyze cell cycle changes.Results:Through expression profiling microarray analysis,161 stably differentially expressed lncRNAs were identified in two resistant cell lines,including 107 upregulated and 54 down-regulated lncRNAs.KEGG pathway enrichment revealed that the differentially expressed lncRNAs were significantly enriched in classical drug resistance pathways such as the p53 signaling pathway,lysosome,focal adhesion,and immune-related signaling pathways like PPAR.Based on expression profiling microarrays and RT-qPCR validation,lncRNA DAPK1-IT1,which was significantly down-regulated by more than 4-fold in both two carboplatin-resistant cell lines,was selected for functional verification.The results showed that under carboplatin treatment,the colony-forming ability of the carboplatin-resistant OC cells SKOV3-CBP overexpressing lncRNA DAPK1-IT1 was significantly

关 键 词：卵巢癌 卡铂耐药 长链非编码RNA DAPK1-IT1 

分 类 号：R737.31[医药卫生—肿瘤]