长链非编码RNA—HNF1A-AS1对肝癌的抑制作用及其分子机制

Hepatocyte nuclear factor 1α (HNF1α) is one of the key transcription factors which plays a critical role in hepatocyte differentiation and liver function maintenance. Our previous studies showed that forced expression of HNF1α induced the differentiation of hepatocellular carcinoma (HCC) cells into hepatocytes and significantly inhibited the growth of the orthotopic HCC nodules in the liver. HNF1α antisense RNA 1 (HNF1A-AS1) is a long noncoding RNA located on the 6.5kb upstream of HNF1α gene, which function remains largely enigmatic. Our previous microarray analysis indicated that HNF1α induced the expression of HNF1A-AS1 in HCC cells. Overexpression of HNF1A-AS1 suppressed the proliferation, migration and colony formation of HCC cells in accompanied with the up-regulation of HNF1α. These data suggest a bidirectional regulation between HNF1A-AS and HNF1α which may affect HCC progression. Based on our previous findings, the present project aims to analyze the relationship between HNF1A-AS1 levels and the clinical malignant phenotypes as well as the prognosis of human HCC patients. In addition, we will evaluate the impact of HNF1A-AS1 on the malignant behaviors of HCC cells both in vitro and in vivo as well as its therapeutic efficacy on experimental HCC in mice. Moreover, the molecular mechanism of bidirectional regulation between HNF1A-AS1 and HNF1α will be clarified. Finally, the binding protein of HNF1A-AS1 and the signal pathway affected by HNF1A-AS1 will be identified to further elucidate the molecular mechanism by which HNF1A-AS1 inhibits the development and progression of HCC. This study will shed new light on the molecular mechanism of hepatocarcinogenesis and provide novel targets for the diagnosis and treatment of HCC.

肝细胞核因子1α(HNF1α)系维持肝细胞分化和功能的重要转录因子。我们研究发现HNF1α可诱导肝癌细胞向肝细胞分化，显著抑制肝脏种植瘤生长。HNF1A-AS1基因为一功能尚不明确的长链非编码RNA，位于HNF1α基因上游6.5kb。我们前期发现HNF1α可上调肝癌细胞HNF1A-AS1的表达，过表达HNF1A-AS1可抑制肝癌细胞增殖、迁移和克隆形成，并促进HNF1α的表达，提示HNF1A-AS1与HNF1α二者之间存在相互调控并影响肝癌发生发展。本课题拟分析HNF1A-AS1表达与肝癌患者临床恶性表型和预后的关系，体内外实验观察HNF1A-AS1对肝癌细胞恶性生物学行为的影响及实验性肝癌的治疗效果，阐明HNF1A-AS1与HNF1α的相互调控机制，确定HNF1A-AS1的相互作用蛋白，明确HNF1A-AS1抑制肝癌的分子机制。研究成果将进一步揭示肝癌发生发展机制，为肝癌诊治提供新靶点。

肝细胞核因子1α(HNF1α)系维持肝细胞分化和功能的重要转录因子。我们既往研究发现HNF1α可诱导肝癌细胞向肝细胞分化，显著抑制肝脏种植瘤生长。本课题利用基因芯片技术发现长链非编码RNA HNF1A-AS1是HNF1α的下游基因，进一步克隆了HNF1A-AS1的全长RNA并明确了HNF1α对HNF1A-AS1的转录调控作用；功能研究发现HNF1A-AS1可抑制肝癌细胞的增殖、迁移和侵袭以及成瘤性，同时动物实验表明尾静脉注射过表达HNF1A-AS1的慢病毒可抑制小鼠肝癌原位种植瘤的生长及转移。进一步研究发现HNF1A-AS1可与非离子性酪氨酸磷酸酶SHP-1结合，二者的结合常数为59.57±14.29 nM，相关的酶活实验表明HNF1A-AS1与SHP-1结合可促进SHP-1酪氨酸磷酸酶活性，而HNF1α可通过调控HNF1A-AS1影响SHP-1活性，抑制SHP-1酶活可阻断HNF1α和HNF1A-AS1对肝癌细胞恶性表型的抑制作用。本课题同时明确了SHP-1对肝癌的发生发展影响。发现SHP-1在肝癌组织中较癌旁表达明显下调，肝癌组织中SHP-1的表达与肝癌病人的生存期呈显著负相关；在肝癌细胞中过表达SHP-1显著抑制细胞的增殖、迁移和侵袭以及肝癌细胞的成瘤能力，利用小干扰RNA抑制SHP-1表达，促进肝癌细胞的增殖和转移能力；尾静脉注射过表达SHP-1的腺病毒AdSOX9对肝癌细胞小鼠原位种植瘤具有治疗作用。肝脏特异性敲除SHP-1基因显著增加二乙基亚硝胺（DEN）诱导的小鼠原发性肝癌的发生及肺转移，尾静脉注射AdSOX9对小鼠原发性肝癌具有明显的治疗作用。这些研究表明SHP-1对肝癌的发生发展具有抑制作用，进一步的机制研究发现SHP-1可能通过抑制STAT3、NFκB及AKT等肿瘤相关信号通路在肝癌细胞中的活化抑制肝癌的发生发展。我们的研究进一步明确了HNF1α抑制肝癌的分子机制，阐明了HNF1α/HNF1A-AS1/SHP-1信号通路对肝癌发生发展影响，为肝癌治疗提供了新靶点。