基于高通量RNA测序技术的肝癌特异lncRNA表达谱及调控网络研究

Recently, accumulating articles have reported that long non-coding RNAs (lncRNAs) are aberrantly expressed in many cancers, including hepatocellular carcinoma (HCC). And lncRNA expression profiles are correlated with tumorigenesis, and progression. In this study, by utilizing rRNA-depletion strategy, we are applying next-generation RNA sequencing technology to profile the whole transcriptome of HCC tissue samples in search of HCC specific lncRNAs. By association analysis with clinical data, we could establish lncRNA prediction model related with HCC diagnosis and prognosis. Also, by combining RNA-RNA interaction prediction algorithm and statistical analysis of the correlation between differentially expressed lncRNA profile and gene expression pattern, we sought to build the HCC specific regulatory network based on the interaction between lncRNAs and coding genes. Finally, we will carry out some initial exploration on the biological functions of some candidate novel lncRNAs, including cell cycles, proliferation, migration and invasion, to evaluate the functional role of lncRNAs in the development and progress of HCC. This research project is technically feasible, and will systematically and comprehensively reveal the HCC specific lncRNA profiles from another point of view. And by establishing of lncRNA-coding gene interaction regulatory network and explorating on lncRNA biological functions in HCC, this study provides the deeper understanding of molecular mechanisms between lncRNAs and coding genes, as well as an alternative strategy for the early-stage diagnosis, treatment and prognosis of HCC patients.

最近研究显示：长链非编码RNA（lncRNA）在包括肝癌在内的多种肿瘤中异常表达，且与肿瘤的发生、发展密切相关。本课题拟利用第二代高通量测序技术，采用去rRNA的纯化策略，对肝细胞肝癌组织进行全转录组RNA测序，以获得肝癌特异lncRNA表达谱；同时，结合相关临床资料，建立与肝癌诊断及预后相关联的lncRNA预测模型；其次，对比分析差异lncRNA与编码基因表达谱，构建lncRNA-基因互作网络调控图；最后，筛选出肝癌特异性lncRNA，检测其对肝癌细胞细胞周期、增殖能力、侵袭转移能力的作用，以明确lncRNA在肝癌发生发展进展中的作用。该研究的开展，将从另一个层面更系统全面地揭示肝细胞肝癌特异的lncRNA表达谱，并通过lncRNA-基因表达互作网络调控图及lncRNA的生物学功能研究，更深入揭示lncRNA与编码基因的分子作用机制，为肝癌的早期诊断、治疗及预后分析提供新的策略。

肝细胞肝癌是全世界范围内最普遍且死亡率最高的肿瘤之一，对其全转录组水平的研究，尤其是长链非编码RNA的研究报道仍较少。通过对肝癌及癌旁组织样本进行第二代高通量测序，我们最终获得了12对肝癌组织全转录组表达谱，分别检测到了439及214特异表达基因和lncRNA，并在21对肝癌及癌旁组织中进行了Real-time PCR验证。经关联分析临床特征，我们发现了一系列lncRNA，如：NONHSAT003823，NONHSAT056213，NONHSAT015386，NONHSAT122051等，与包括肿瘤大小、肿瘤分化、门静脉癌栓、血清及组织甲胎蛋白水平等显著相关。尤其是NONHSAT122051，其表达水平与血清甲胎蛋白水平呈线性相关。此外，通过对差异表达lncRNA及基因的共表达网络分析，我们发现肝癌特异lncRNA可能涉及代谢、细胞周期、化学致癌、补体与凝血级联相关信号通路。最后，通过在肝癌细胞系中沉默内源性lncRNA的表达，我们对筛选出的肝癌特异性lncRNA的功能做了初步探索。尽管没有检测到lncRNA对细胞增殖的影响，我们发现沉默内源性NONSHAT122051与NONHSAT003826的表达水平能显著削弱肝癌细胞系的运动能力。这些发现不仅在全转录组水平增加了对肝癌特异的lncRNA的理解，更为后续对肝癌的诊断及治疗预后提供了切实可靠的靶标。