缺氧相关长链非编码RNA参与调控肝癌发生发展及化疗耐药的机制研究

Even through improved interventional and surgical treatment, the 5-year survival rate of Hepatocellular carcinoma (HCC) is less than 15%. The major reason is cancer recurrence and metastasis. Hypoxia is a common phenomenon in many solid tumors，promoting the proliferation, migration and invasion of tumor cells. Our previous studies have found that Hypoxia promoted Epithelial-mesenchymal transition (EMT) , invasion and metastasis of hepatic cancer cells by increasing the expression of HIF1ɑ. In addition, hypoxia induced drug resistance of cancer cells to sorafenib. In this study, human LncRNA microarray is used to detect and identify lncRNAs that are increased or reduced significantly by hypoxia in HCC cells. qRT-PCR is then used to verify the expression of these lncRNAs in HCC cell lines and tissues. SPSS 15.0 software was employed to analyze the correlation of lncRNAs expression and clinical factors of HCC patients. 5’RACE and 3’RACE was employed to gain the full-length sequence of lncRNA that will be cloned into expression vector to establish lncRNA stably expressed cell lines. Cell proliferation assay, flow cytometry analysis, colony formation and xenograft mice model will be applied to analyze the effect of lncRNA on HCC tumor growth. CHIRP combined with Mass Spectrometry and Sequencing are used to identify the proteins and DNAs that binds to lncRNA. Furthermore, MTT is used to detect the sensitivity of lncRNA unexpressed and stably expressed HCC cells to sorafenib.

我们的前期研究发现，肝癌细胞缺氧条件下发生上皮细胞间质化，且侵袭转移能力增强、导致索拉菲尼耐药。本研究将应用LncRNA芯片等技术获得肝癌细胞缺氧诱导后差异表达的长链非编码RNA（lncRNAs），并进一步利用qRT-PCR方法检测并验证lncRNAs在肝癌细胞系和组织中的表达，明确其与缺氧诱导因子HIF1ɑ表达的相关性；通过临床病例资料分析明确lncRNA与患者分期、分化、淋巴结转移、化疗敏感性、预后等的相关性。克隆、鉴定lncRNA全长并构建稳定表达的肝癌细胞系，通过克隆形成、增殖实验、凋亡、细胞周期检测、裸鼠成瘤等实验探索lncRNA在肝癌中的功能。利用CHIRP结合质谱分析和测序分析等技术明确与lncRNA相互作用的蛋白和DNA，探讨其所涉及的重要信号通路和作用机制。通过MTT实验探讨lncRNA表达缺失和稳定表达的肝癌细胞对索拉菲尼的敏感性，明确lncRNA是否参与索拉菲尼耐药机

研究已证实肿瘤组织缺氧，缺氧诱导因子HIF-1在肿瘤组织中表达较正常组织明显升高，进一步导致上皮-间充质转化和促进肿瘤的侵袭和转移。本项目寻找缺氧相关的LncRNA，探索其影响肝细细胞癌的功能机制。通过microarray获得缺氧相关表达差异的mRNA和ncRNA，利用生物信息学分析总结了肝细胞癌转录谱特点。筛选出差异最显著lncRNA, 构建稳转细胞株，通过细胞功能试验来研究lncRNA对细胞功能的影响。数据库预测lncRNA靶基因靶位点，转染mimic后验证靶基因和靶位点。裸鼠皮下成瘤实验在体内进一步验证lncRNA对肿瘤生长和靶基因表达的影响。通过研究我们发现了727个缺氧相关的差异表达的mRNA和203个差异表达非编码的RNA(ncRNA)，其中差异表达的lncRNA共78个，癌组织中下调表达的47个，上调表达的31个。linc01903在肝细胞癌组织中的表达显著低于对应的癌旁组织，ROC曲线下面积为0.754，具有成为潜在的诊断标志物的价值，linc01093表达越低，肿瘤组织病理分级越低，患者预后越差。Linc00193在多个肝癌细胞株中表达量低，慢病毒感染构建稳定过表达linc01093的细胞株显示过表达linc01093后显著抑制了肝细胞的增殖、侵袭和迁移，抑制了细胞由G1向S期的转变，促进了细胞的凋亡。数据库预测linc01093潜在结合位点miR-3916和靶基因EBF1。利用QPCR和WB验证发现，linc01093过表达后miR-3916显著下调表达，EBF1显著上调表达。通过转染miR-3916 mimic后，linc01093和EBF1的表达均下降。构建裸鼠皮下移植瘤模型，结果显示过表达linc01093后能够显著抑制裸鼠皮下瘤的生长，并且EBF1表达显著上调，提示linc01093能够促进EBF1的表达，抑制肿瘤的生长。肝细胞癌转录谱生信分析为我们研究缺氧相关的肝细胞癌提供了分子生物层面的重要信息。linc01093具有诊断价值和潜在的靶向药物的临床意义。