长链非编码RNA-ASLNC00850在HBV相关原发性肝癌发生过程中的功能与机制研究

Hepatocellular carcinoma is the commonest malignant tumor of liver and the complicated mechanisms under tumorgenesis remains unclear, restricting the improvement of diagosis and treatment of HCC. Long non-coding RNA could function as scaffolds for chromatin-modifying complexes and regulate downstream molecules on transcriptional or post-transcriptional levels, but roles of lncRNA in tumorgenesis of HCC was not studied. We previously measured the system expression and screened the lncRNAs associated with the tumorgenesis of HCC.Bioinformatic analysis reveal that the lncRNA-ASLNC00850, might be central in the regulatory network during the tumorgenesis. Our preliminary experiments revealed that ASLNC00850 was upregulated in tumor initial cells and ASLNC00850 knock-down would significantly downregulate markers of stemness and upregulate hepatocyto markers. Besides, ASLNC00850 might be regulated by transcription factor-NF-kB. In this project, the expression of ASLNC00850 will be measured in multicentre,large cohort of clinical samples, in vitro and in vivo experiments will be applied to investigate the role of ASLNC00850 in tumorgenesis, and the key factors mediating the function of ASLNC00850 were screened by high-throughput interactome analysis and validated by pull down experiments. For HCC is classical malignant tumor drove by chronic inflammation and ASLNC00850 might be regulated by NF-kB，the role of inflammation in regulation of ASLNC00850 and subsequently tumorgenesis will be tested. Additionally, the homologous conserved gene of ASLNC00850 in mice will be validated , transgenic mice will be constructed to study the critical effect in tumorgenesis promotion and the potential value in therapy. Collectively, our study will systematically screen the lncRNAs associated with tumorgenesis of HCC and thoroughly classify the central role of ASLNC00850 in the process of chronic inflammation transforming to HCC.

长链非编码RNA（lncRNA）可在染色质构象、基因转录及转录后等水平发挥重要作用，但lncRNA在肝癌发生中的作用少有文献报道。课题组前期利用芯片检测正常肝、慢性肝炎、肝硬化及肝癌等组织中lncRNA的表达，筛选与肝癌发生相关的lncRNA，并锚定关键lcnRNA-ASLNC00850。预实验发现ASLNC00850可能受NF-kB的转录调控并在肿瘤起始细胞中上调表达，干扰ASLNC00850可显著下调肿瘤细胞干性标志分子及上调肝细胞标志分子的表达。据此，本项目拟在临床样本中观察ASLNC00850与肿瘤发生的相关性及预后预测价值；探讨ASLNC00850在肝癌发生中的作用及明确ASLNC0085促进肝癌发生的分子机制；阐明ASLNC00850受炎症通路调控的机制；确认ASLNC00850的小鼠同源基因，观察同源基因的功能及在体内干预同源基因对肝癌发生的影响，为临床应用提供理论依据。

原发性肝细胞癌（Hepatocellular Carcinoma, HCC）是最常见的肝脏恶性肿瘤，现行的治疗手段疗效有限，所以深入探讨原发性肝癌发生的分子机制，寻找理想的早期诊断标志物以及治疗靶点成为突破瓶颈，是提高HCC诊治效果的关键。长链非编码RNA（Long noncoding RNA, LncRNA）是一类长度大于200nt，不具有编码蛋白能力的RNA，近年来大量证据证实长链非编码RNA可作为广谱调控因子，能在染色体修饰、转录、转录后等多水平发挥重要的生物学作用。我们利用高通量手段检测肝炎/肝硬化/肿瘤组织中差异表达的LncRNA，并在体内外水平及临床水平探讨其上游调控机制及生物学功能。我们发现受炎症相关转录因子NF-kB和STAT3激活的长链非编码RNA-DANCR在肝癌组织中和干性特征明显的细胞中高表达，其高表达预示着肝癌病人较差的无瘤生存率和总体生产率。过表达DANCR能上调肿瘤起始细胞相关基因的表达水平，扩增肿瘤起始细胞的数量并能在体外和体内促进肿瘤的形成。抑制DANCR则能显著抑制肿瘤起始细胞的扩增并抑制肿瘤的形成。干扰小鼠皮下荷瘤组织中的DANCR可显著降低肿瘤的生长速度并延长小鼠的生存时间。进一步分子机制研究发现DANCR可结合CTNNB1的转录本，并抑制miR199a等对CTNNB1 mRNA的负调控，从而促进细胞内b-catenin的聚集。在稳定过表达DANCR的肿瘤细胞中干扰CTNNB1可显著抵消DANCR过表达的生物学效应。总之，炎症因子诱导表达的DANCR能扩增肿瘤起始细胞，可作为肝癌诊断和治疗潜在的靶基因，也提示长链非编码RNA在炎症促进肿瘤发生、发展过程中的重要作用，进一步揭示肝脏炎癌转化过程中的分子机制。