长链非编码RNA-ASLNC02233在肝癌中的功能及机制研究

A handful of studies have implicated altered lncRNA levels can result in aberrant expression of downstream gene products that may contribute to tumorigenesis. However, the role of lncRNAs to hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remain largely unknown, the molecular mechanisms of their functions are still lacking. In this study, we have identi?ed differentially expressed lncRNAs between HBV-related HCC and paired peritumoral tissues by lncRNA /mRNA microarray. We have built coding-noncoding gene co-expression network to identify interactions among lncRNAs in cancer tissues. Furthermore, we have find the corresponding mRNAs and developed the GO and pathway annotation in order to predict the function of lncRNAs. To validate microarray analysis ?ndings, we randomly selected lncRNAs among the differential lncRNAs and analyzed their expression using qRT-PCR in 14 pairs of HCC and corresponding nontumor liver tissues, as well as 8 HCC cell lines. The results confirmed that the LncRNA-ASLNC02233 was down-regulated in HCC tissues and cell lines with varying degrees. To investigate the roles of the ASLNC02233, we knockdown the ASLNC02233 expression by siRNA in Huh7 and SMMC-7721 cells, transwell assays indicated that the migration and invasion ability were significantly increased. This suggested that the ASLNC02233 played a metastasis inhibition role in HCC. Based on the above results, we will further study the possible regulation genes of the ASLNC02233 and then explore the molecular mechanism of the ASLNC02233 in HCC. In addtion, we will examine the expression levels of the ASLNC02233 in a large sample of liver tissues to determine the correlation between ASLNC02233 expression and metastasis, prognosis, recurrence and survival of HCC patients. Taken together, our results will identify the contribution of ASLNC02233 in hepatocarcinogenesis, and determine whether deregulation of the ASLNC02233 could be used as prognostic biomarkers of HCC.

肿瘤细胞中长链非编码RNA表达水平变化能影响下游基因表达从而引发肿瘤发生，目前肝癌中LncRNA的功能鲜有研究，作用机制也尚不清楚。本研究结合LncRNA/mRNA表达谱芯片和生物信息学分析整合筛选出肝癌中差异表达的LncRNA并构建共表达网络，从已知功能的mRNA推测LncRNA的作用。此外，我们验证了LncRNA-ASLNC02233在14对肝癌组织及8株肝癌细胞系中均有不同程度的表达下调，对ASLNC02233进行功能研究时发现干扰ASLNC02233，肝癌细胞的迁移、侵袭能力均显著增加。基于上述基础，我们将进一步研究ASLNC02233在肝癌中发挥功能的分子机制，寻找ASLNC02233的可能作用蛋白，探索其调控机制；本项目还将在大样本肝癌组织中检测ASLNC02233的表达水平，结合临床资料分析其与肝癌转移、预后、生存期等临床指标的相关性，为肝癌诊断提供参考。

为了研究肝癌中lncRNA的功能和机制，本项目用lncRNA表达谱芯片检测了12对肝癌及癌旁组织，发现713个在肝癌组织中异常表达的lncRNA。其中miR503的宿主基因miR503HG（ASLNC02233）在肝癌中显著下调。Kaplan-Meier 分析发现，miR503HG的下调表达与肝癌病人的复发（time to tumor recurrence ，TTR，p=0.043）和总生存期（overall survival ,OS,p=0.045） 相关，miR503HG表达高的病人复发率低且生存期较好.Cox multivariate分析发现，miR503HG的表达量可以作为复发和生存的一个独立的因素（OS，HR: 0.37; 95% CI: 0.16-0.84; P=0.018；TTR，HR: 0.48; 95% CI: 0.24-0.98; P=0.045）。功能实验研究发现，miR503HG过表达能够在细胞和裸鼠体内显著抑制肝癌细胞的迁移和转移。进一步分子机制研究发现，过表达miR503HG能够与IκBα竞争性的结合HNRNPA1，从而阻止IκBα与HNRNPA1结合，使得IκBα不能发生降解得到累积，进而抑制NF-κB 的转录活性及其下游一些与转移相关基因的表达，从而抑制肝癌的转移。此外，本项目还发现，miR503HG与miR503对肝癌细胞迁移的抑制作用具有协同效应。总结：本项目阐明了miR503HG在肝癌病人的复发危险和生存预测方面的重要意义，探索了miR503HG通过抑制NF-κB 信号通路抑制肝癌的转移，这为肝癌转移的分子机制提供了思路。