【作者】 张静；

【导师】 陈川； 周钢桥；

【作者基本信息】 河北大学 ， 生物学， 2022， 硕士

【摘要】 肝细胞癌(Hepatocellular carcinoma,HCC)是全球最常见的原发性恶性肿瘤之一,起病隐匿且预后差。我国是HCC高发国家,HCC相关死亡率呈逐年上升趋势。尽管目前HCC的防诊治技术已取得了很大进展,但晚期HCC患者的治疗方案仍非常有限。因此,发现新的HCC相关基因并解析其作用机制,对于制定高效的预防和诊疗方案具有重要的意义。长链非编码RNA(Long noncodingRNA,lncRNA)是一类长度大于200个核苷酸的RNA分子,几乎不具备蛋白质编码能力,常以RNA形式直接行使功能。LncRNA可与DNA、RNA和蛋白质分子等发生相互作用,进而在表观遗传、转录、转录后修饰、翻译及翻译后修饰等多个水平行使功能。多项研究已显示,lncRNA与HCC的发生发展密切相关。然而,大部分lncRNA在HCC中的作用及其分子机制并未被详细阐明。前期本课题组鉴定到一个与HCC相关的全新的lncRNA——IGFR-inducing lncRNA(命名为IGFRIL)。IGFRIL全长为3329个核苷酸、蛋白编码能力较低、具有多聚腺苷酸(PolyA)尾。体外和体内初步的功能实验均显示,IGFRIL可显著促进HCC细胞的生长和转移;IGFRIL与多聚嘧啶区结合蛋白1(Polypyrimidine tract-binding protein 1,PTBP1)存在相互作用,可形成RNA-蛋白质复合体机器。此外,IGFRIL在肝癌组织中显著上调表达,且其高表达与患者较短的总体生存期及无病生存期显著相关。本论文拟在前期工作基础上,深入探讨IGFRIL-PTBP1复合体机器在促进HCC进展中的作用和分子机理,为HCC新的临床治疗策略的制定提供理论依据。首先,本论文证实IGFRIL-PTBP1复合体机器可通过激活AKT-mTOR信号通路促进HCC进展。我们通过在HCC细胞系HepG2中敲低IGFRIL或PTBP1的转录组测序(RNA-seq)分析发现,IGFRIL和PTBP1可共同干扰RTK-AKT信号通路。进一步的生化实验证实IGFRIL和PTBP1均可显著激活HCC细胞中的AKT-mTOR信号通路。IGFRIL可显著抑制m TOR介导的HCC细胞的自噬过程,并且,经m TOR激活剂MHY1485及抑制剂雷帕霉素处理HCC细胞后,我们证实IGFRIL发挥促癌功能依赖于mTOR通路。其次,我们发现IGFRIL-PTBP1复合机器可通过减弱IGFBP3 mRNA的稳定性促进IGFR-AKT-mTOR通路活性。我们应用紫外交联免疫沉淀结合高通量测序(Crosslinking-immunprecipitation and high-throughput sequencing,CLIP-seq)技术发现,在HCC细胞中敲低IGFRIL可减弱PTBP1与靶mRNA的结合能力;综合RNA-seq及CLIP-seq数据,我们发现敲低IGFRIL及PTBP1后IGFBP3的mRNA表达显著上调,且敲低IGFRIL可显著减弱PTBP1与IGFBP3 mRNA的结合能力。进一步发现IGFRIL和PTBP1均可显著降低IGFBP3 mRNA的稳定性。此外,体外功能实验证实IGFBP3可通过抑制IGFR-AKT-mTOR通路来抑制HCC细胞的生长、迁移及侵袭能力。IGFRIL发挥促癌功能及对AKT-mTOR通路的激活作用均依赖于IGFBP3及其竞争性结合的IGFR。综上,IGFRIL与PTBP1相互作用形成的复合体通过降低IGFBP3 mRNA的稳定性,下调IGFBP3的表达,进而激活IGFR-AKT-mTOR信号通路并扰乱细胞自噬过程,最终促进HCC的进展。上述结果提示,IGFRIL-PTBP1-IGFBP3轴可能是未来HCC新的潜在的生物标志物和/或治疗靶点。更多还原

【Abstract】 Hepatocellular carcinoma(HCC)is one of the most common cancers worldwide,especially with high incidence in China.Although great progress has been made in the prevention and treatment of HCC,the prognosis of patients is still limited.Thus,it is necessary to identify HCC-related genes and further reveal the underlying molecular mechanism,which will be helpful for developing new prevention and treatment strategies.Long noncodingRNAs(lncRNAs)are a class ofRNAs with length greater than 200nucleotides,which lacks coding potential and functions in the form ofRNAs.LncRNAs are able to interact with DNAs,RNAs,and/or proteins,and exert their functions at multi-layers,including epigenetic,transcriptional,post-transcriptional,translational and post-translational modulation.Accumulating studies have revealed several HCC-assocaied lncRNAs.However,the functions and molecular mechanisms of most lncRNAs in HCC have not been comprehensively dissected.Our team previously identified an HCC-related lncRNA,IGFR-inducing lncRNA(IGFRIL).IGFRIL is a transcript with length of 3,329 nucleotides,poor coding potential and a poly A tail.The in vitro and in vivo functional assays demonstrated that IGFRIL promotes the HCC cells growth and migration.Furthermore,we found that IGFRIL directly interacts with theRNA binding protein polypyrimidine tract-binding protein 1(PTBP1).IGFRIL was also up-regulated in HCC tumor tissues,and its high expression was significantly associated with shorter overall survival and disease-free survival periods.In this study,we aimed to explore the function and mechanism of the IGFRIL-PTBP1 complex in the development of HCC.First,we demonstrated that the IGFRIL-PTBP1 complex promotes HCC progression by activating the AKT-mTOR signaling pathway.By usingRNA-sequencing(RNA-seq)analyses in IGFRIL-or PTBP1-depleted Hep G2 cells,we found that both IGFRIL and PTBP1are correlated with the activation of RTK-AKT signaling.Biochemical assays consistently confirmed that both IGFRIL and PTBP1 significantly activate AKT-mTOR signal pathway.Further,IGFRIL could inhibit the mTOR-mediated autophagy of HCC cells.After treating HCC cells with mTOR activator MHY1485 or inhibitor rapamycin,we demonstrated that IGFRIL’s oncogenic function depends on the mTOR pathway.Secondly,we revealed that the IGFRIL-PTBP1 complex promotes the IGFR-AKT-mTOR signaling by reducing the stability of IGFBP3 mRNA.By using crosslinking-immunprecipitation and high-throughput sequencing(CLIP-seq)analyses,we found that knockdown of IGFRIL attenuates the binding ability of PTBP1 to its target mRNAs in HCC cells.Combing theRNA-seq with CLIP-seq data,we found that the expression of IGFBP3 is up-regulated after knockdown of IGFRIL or PTBP1,and the binding ability of PTBP1 to IGFBP3 mRNA is significantly weakened by IGFRIL depletion.Furthermore,we observed that both IGFRIL and PTBP1 reduce the stability of IGFBP3 mRNA after treating HCC cells with actinomycin D.Additionaly,functional assays showed that IGFBP3 exerts its tumor suppressive role by inhibiting IGFR-AKT-mTOR pathway.The oncogenic function of IGFRIL and its effect on the activation of AKT-mTOR pathway depend on IGFBP3 and the IGFR signaling.In summary,IGFRIL,coupled with its interactor PTBP1,reduces the stability of IGFBP3 mRNA,which in turn promotes the IGFR-AKT-mTOR signaling pathway and HCC progression.These findings indicate that IGFRIL-PTBP1-IGFBP3 axis might be the candidate biomarkers and therapeutic targets for the prognosis and/or treatment of HCC.更多还原