长链非编码RNA-uc002mbe.2介导的HDACi凋亡效应及其在肝癌中的作用

Aberrant expression of long non-coding RNAs (lncRNAs) plays an important role in hepatocellular carcinogenesis. However the mechanisms that account for aberrant lncRNA expressions are unknown. LncRNAs are may have the potential to be hepatocellular carcinoma (HCC) diagnostic and prognostic markers as well as therapy targets. My laboratory has already studied the global profile of lncRNA and coding gene expression affected by HDACi trichostatin (TSA) in HCC cells. Constructing network modeling was used to discover the interaction between lnRNAs and coding RNAs as well as the function of lncRNAs. The differentially expressed lncRNAs and coding gene RNAs were clustered into phenotypically relevant co-expression modules. The functions of coding RNAs were found in NCBI RefSeq. The functions of lncRNAs in co-expression modules were predicted through the associated coding RNAs. Using this method, we identified a few lncRNAs associated TSA-induced apoptosis of liver cancer cells. For example, TSA selectively induced lncRNA uc002mbe.2 in four studied HCC cell lines. Compared with normal human hepatocytes, uc002mbe.2 expression level was significantly lower in all four studied HCC cell lines. This finding was confirmed in human HCC and adjacent normal livers. Moreover, TSA-induced uc002mbe.2 expression was positively correlated with the apoptotic effect of TSA in HCC cells. In addition, knockdown the expression of uc002mbe.2 increased the proliferation of untreated Huh7 liver cancer cells and reduced TSA-induced apoptosis of Huh7 cells. Based on these novel findings, the tested hypothesis of this application is that the expression of lncRNA uc002mbe can be regulated by histone acetylation and that reduced expression of uc002mbe.2 accounts for liver carcinogenesis. In the proposed application, we will use different approaches such as chromation immunoprecipitation (ChIP) assay to understand how histone acetylation can alter the expression level of uc002mbe.2. Loss-and gain-of-function studies on uc002mbe.2 in the HCC cell lines will be used to study it effects on gene expression and to assess its effect on biological behavior both in vitro and in vivo. The results generated from the proposed study lays a novel foundation for the role of uc002mbe.2 as a regulator of apoptosis and offer new avenues for therapeutic intervention against HCC.

长链非编码RNA（long non-coding RNA，lncRNA）表达水平改变与肝癌发生发展密切相关，但其异常表达的机制尚不清楚。我们前期研究发现lncRNA-uc002mbe.2在肝癌组织和多株肝癌细胞系中显著下降，但组蛋白去乙酰化酶抑制剂TSA能诱导uc002mbe.2表达，敲除uc002mbe.2能显著降低TSA的凋亡效应，并且能促进肝癌细胞增殖，未见其相关报道。由此我们推测：uc002mbe.2是一个新的、有抑制肝癌作用的lncRNA，其在肝癌中表达下降与组蛋白乙酰化调控紊乱有关。本课题将研究组蛋白乙酰化修饰调控肝癌细胞uc002mbe.2表达机制；并通过干预表达，在细胞和裸鼠模型中探索uc002mbe.2对肝癌细胞生物学行为的调控功能；寻找其相关的靶基因，探索其调控机制。研究有助于阐明uc002mbe.2在肝癌中发挥功能的分子机制，并为肝癌治疗提供新的分子靶点。

研究表明长链非编码RNA参与调控肝癌的发生进展。我们前期以组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）诱导肝癌细胞凋亡为模型，发现lncRNA-uc002mbe.2表达水平与TSA诱导的凋亡水平正相关，但TSA诱导uc002mbe.2表达上调的机制尚未阐明。因此，本研究旨在探讨uc002mbe.2在TSA诱导肝癌细胞凋亡中的作用及其机制。研究证实TSA明显增加肝癌细胞胞浆中uc002mbe.2的表达，uc002mbe.2表达沉默抑制TSA诱导的细胞周期G2/M期阻滞、p21表达及细胞凋亡的发生，并且能够逆转TSA介导的p-AKT表达水平减少。RNA pull-down和RIP结果显示TSA促进Huh7细胞uc002mbe.2与核糖核蛋白A2B1(hnRNPA2B1)相互作用。uc002mbe.2与hnRNPA2B1相互作用导致AKT失活并诱导p21表达。在裸鼠实验中，uc002mbe.2表达沉默明显抑制TSA减少肿瘤大小及肿瘤重量的作用。且TSA减少裸鼠体内肿瘤的hnRNPA2B1表达、p-AKT水平及诱导p21表达的作用能够被uc002mbe.2表达沉默阻止。上述结果提示肝癌细胞中TSA对肝癌细胞的杀伤作用与uc002mbe.2和hnRNPA2B1的相互作用密切相关。在相关拓展领域中，本项目还发现：（1）SUZ12在肝癌中低表达，其表达水平与肝癌患者的不良预后负相关。SUZ12通过影响肝癌细胞的迁移及侵袭发挥其生物学功能，并且ERK1/2信号通路参与调控SUZ12对肝癌细胞的侵袭迁移作用。（2）黄连素联合索拉菲尼治疗能够增加肝癌细胞对索拉菲尼的敏感性，联合用药组肝癌细胞的增殖活性、克隆形成能力明显受抑制，联合治疗诱导肝癌细胞发生凋亡，并且具有潜在抑制血管生成的作用。（3）通过芯片技术和生物信息学分析筛选参与调控小鼠非酒精性脂肪肝（NAFLD）的lncRNA-AK012226，结果显示NAFLD肝组织中AK012226表达上调，干扰AK012226表达明显减少NAFLD细胞模型中脂肪蓄积。我们的研究通过探讨uc002mbe.2在肝癌中的生物学功能及作用机制，明确SUZ12在肝癌中的的功能及分子机制及黄连素联合索拉菲尼在肝癌治疗中的作用，寻找参与NAFLD发生进展的lncRNA-AK012226，为寻找肝癌及NAFLD分子标志物和靶向治疗提供新的思路及可靠的理论依据。