HIF-1α－UPK1A-AS1－UPK1A正反馈环路对肝癌发生发展的影响及机制

We have demonstrated that long noncoding RNAs (lncRNA) play an important role in initiation and progression of hepatocellular carcinoma (HCC) (Gastroenterology，2015). Recently, our preliminary data showed that lncRNA UPK1A-AS1 and its sense strand UPK1A were regulated by HIF-1α, while HIF-1α was regulated by UPK1A-AS1 and UPK1A. The positive correlation among HIF-1α, UPK1A-AS1, and UPK1A were found in HCC. UPK1A-AS1 was highly expressed in HCC tissues compared with non-tumor tissues and was associated with shorter overall survival of HCC patients. Ectopic expression of UPK1A-AS1 in HCC cells promoted cell proliferation and glycolysis. Thus, based on our data, we proposed that HIF-1α, UPK1A-AS1, and UPK1A may form a positive feedback to promote HCC initiation and progression. To confirm the hypothesis, firstly, the expression and clinical significance of HIF-1α, UPK1A-AS1, and UPK1A will be identified by clinical multicenter HCC sample. Secondly, biological functions of HIF-1α, UPK1A-AS1, and UPK1A will be investigated by in vitro and in vivo tumor assays. Lastly, we will explore regulatory interrelation among HIF-1α, UPK1A-AS1, and UPK1A and provide a framework for understanding the functional properties of these important targets. In conclusion, HIF-1α, UPK1A-AS1, and UPK1A may act as biomarkers for poor prognosis in HCC and confer a malignant phenotype to tumor cells. The positive feedback of HIF-1α-UPK1A-AS1-UPK1A may contribute to a better understanding of liver carcinogenesis and tumor progression.

我们已发现长链非编码RNA（lncRNA）在肝癌发生发展中发挥重要的调控功能(Gastroenterology，2015)。预实验结果表明：lncRNA UPK1A-AS1和其正义链UPK1A均受HIF-1α调控，二者又可以调控HIF-1α表达，并且三者在肝癌中的表达呈正相关关系；UPK1A-AS1促进肝癌细胞增殖和细胞糖酵解代谢，其在肝癌组织中高表达与患者不良预后相关。基于前期研究结果，我们提出HIF-1α、UPK1A-AS1、UPK1A三者形成正反馈环路促进肝癌发生发展的科学假说。为验证假说，本项目拟首先利用临床多中心大样本探讨三者在肝癌中的表达，并探讨它们与肝癌发生发展及预后的关系；其次通过肿瘤体内外实验探讨它们的生物学功能及对肿瘤代谢的影响；最后深入探讨三者之间互为调控的关系。据此，阐明HIF-1α-UPK1A-AS1-UPK1A环路在肝癌中确切的生物学功能及机制。

lncRNA UPK1A-AS1和其正义链UPK1A均受HIF-1α调控，二者又可以调控HIF-1α表达。本课题探讨UPK1A-AS1、UPK1A 及HIF-1α在肝癌中的生物学功能及三者之间的调控关系。我们利用qRT-PCR方法检测了17对肝癌和癌旁组织UPK1A-AS1的表达并用原位杂交的方法对UPK1A-AS1进行细胞定位，分析UPK1A-AS1表达与肝癌患者预后的关系。接着，构建UPK1A-AS1稳定过表达的肝癌细胞株，利用CCK-8、EdU、裸鼠皮下成瘤等实验明确UPK1A-AS1在肝癌中的生物学功能，检测糖酵解相关指标的表达，最后采用双荧光素酶报告实验、泛素化蛋白质免疫共沉淀法探究UPK1A-AS1调控肝癌糖酵解的分子机制。结果表明，UPK1A-AS1在肝癌中高表达，其高表达与肝癌患者不良预后相关。过表达UPK1A-AS1能促进肝癌细胞的增殖和糖酵解，并通过减少HIF-1α的泛素化进而稳定HIF-1α的表达。敲除HIF-1α能逆转过表达UPK1A-AS1对肝癌细胞糖酵解的促进作用。同时，HIF-1α可通过结合到UPK1A-AS1的启动子区的HRE位点促进其转录。上述结果表明，UPK1A-AS1可与HIF-1α形成正反馈环路调节肝癌细胞的糖酵解水平。此外，体外实验表明，UPK1A-AS1可吸附miR-138-5p或结合EZH2蛋白，从而促进肝癌细胞的增殖。. 同时我们发现，UPK1A-AS1的正义链UPK1A，其在肝癌中也高表达且与肝癌患者的不良预后相关。随后我们利用qRT-PCR、EdU、葡萄糖消耗及乳酸生成实验等探讨UPK1A在肝癌中的生物学功能，并用ChIP、western blot等实验阐明UPK1A促进肝癌糖酵解及增殖的分子机制。结果表明UPK1A能通过调控HIF-1α促进肝癌细胞糖酵解及增殖，同时HIF-1α可通过结合UPK1A启动子区的HRE位点调控UPK1A的表达。上述结果表明UPK1A与HIF-1α形成正反馈环路调控肝癌糖酵解及增殖。. 本课题阐明了UPK1A-AS1、UPK1A 及HIF-1α 在肝癌糖酵解及增殖中的调控作用，揭示了UPK1A-AS1-HIF-1α 、UPK1A-HIF-1α两条正反馈环路在调控肝癌进展的重要作用，这为理解肝癌发生发展的机制提供了新观点，有望为肝癌治疗提供新靶点。