长链非编码RNA-NCRNA00061正反馈调节c-Myc对肝癌生物学功能的影响及其机制研究

Myc is an oncogenic factor that is generally over-expressed in tumors: this can happen either indirectly through the activation of oncogenic pathways that stabilize Myc and/or augment c-myc transcription, or directly through amplification or translocation of c-myc gene. Long noncoding RNAs (lncRNAs) have been reported to be dysregulated in cancer and to be important in the regulation of carcinogenesis, thus suggesting that this class of molecules may be used as biomarkers in cancer. Our previous work showed that the expression of long noncoding RNA NCRNA00061 is significantly upregulated in HCC, NCRNA00061 knockdown inhibit HCC cell proliferation and clonogenicity. c-myc may transcriptional activates NCRNA00061 and promotes the HCC cell proliferation. NCRNA00061 knockdown inhibit c-myc mRNA level, vice versa, overexpression of NCRNA00061 can increase c-myc mRNA expression level. The NCRNA00061 and c-Myc expression levels were significantly correlated in hepatocellular carcinoma samples. Collectively, these data demonstrate that there is a positive feedback loop between c-myc and long noncoding RNA NCRNA00061.Therefore, this study will focus on to elucidate the mechanisms of a positive feedback loop between c-myc and long noncoding RNA NCRNA00061 regulates cell proliferation and tumor growth in hepatocellular carcinoma, to explore mechanism of NCRNA00061 promotes cell proliferation, invasion and migration ability in HCC. To identify the NCRNA00061 binding protein, microRNA and target genes, To clarify the regulating networks of between NCRNA00061, binding protein, microRNA and target genes. In order to provide a theoretical basis that NCRNA00061 as hepatocellular carcinoma (HCC) new biomarkers and therapeutic drug targets.

原癌基因c-myc和长链非编码RNA（lncRNA）在肝癌(HCC)的发生发展中起关键的作用。前期工作表明：lncRNA-NCRNA00061在HCC中的表达显著上升；RNA干扰NCRNA00061明显抑制HCC细胞的增殖及克隆形成能力；RNA干扰和过表达c-myc能显著抑制和促进NCRNA00061的表达，反之RNA干扰和过表达NCRNA00061后c-myc的mRNA表达量亦显著降低和升高；两者表达量呈显著正相关，由此推断NCRNA00061和c-myc之间存在正反馈调节作用。因此，本研究将重点研究c-myc和NCRNA00061正反馈调节的分子机制；阐明NCRNA00061对HCC细胞的增殖、侵袭和迁移能力的影响及其分子机制、以及c-myc在此过程中的作用；分析NCRNA00061与HCC临床分期及预后的相关性，为NCRNA00061作为HCC新型的诊断标志物和药物靶点提供理论依据。

肝癌（HCC）是我国最常见的恶性肿瘤之一，长链非编码RNA（lncRNA）与肿瘤发生发展密切相关。原癌基因c-Myc的异常激活在HCC的发生、进展中起关键的调控作用，其分子机制还为完全阐明。本项目通过前期工作推断NCRNA00061（SNHG7）和c-Myc之间存在正反馈调节作用。通过三年的研究得出如下结论：第一、利用体内裸鼠模型、体外细胞系以及临床样本的分析研究，表明SNHG7在HCC中表达量明显上升，且与患者预后差显著相关，抑制SNHG7后HCC细胞增殖、克隆形成能力减弱以及肿瘤的体积明显减小。第二、通过对细胞系和患者组织实验分析表明，SNHG7主要定位于细胞质中；通过RNA-pulldown和质谱实验未发现特异性结合蛋白，说明主要通过ceRNA的方式调控c-Myc等下游靶基因。第三、阐明了转录因子c-Myc转录激活SNHG7，SNHG7通过竞争性结合miR-33a-5p增强了c-Myc的稳定性，两者形成正反馈调节促进HCC细胞的生长的分子机制。第四、用RNA-seq对SNHG7的调控网络研究发现SNHG7与HCC细胞周期以及糖酵解信号通路相关。第五、首次阐明了细胞周期蛋白依赖性激酶CDK7在HCC中表达升高，并与HCC患者的预后差相关，CDK7作为一个原癌基因，对促进HCC细胞的生长发挥重要作用。第六、阐明了SNHG7通过竞争性结合miR-199a-3p，抑制了miR-199a-3p对CDK7 mRNA的降解，增加了CDK7 mRNA的稳定性，CDK7的积累促进了HCC细胞生长的分子机制。第七、阐明了SNHG7通过miR-122/PKM调控HCC细胞糖酵解信号通路的分子机制。这些研究结果表明，SNHG7在HCC细胞的生长过程中发挥着关键的作用，为SNHG7作为HCC诊断的新型标志物和治疗的药物靶点提供理论依据。