lncRNA-2012介导hnRNPAB在肝癌侵袭转移中的作用及其分子机制研究

Long chain non encoding RNA (lncRNA),a class of non encoding RNA with transcription length of more than 200 nucleotides, plays an important role in tumor progression. In our previous study, we found that over-expression of heterogeneous nuclear ribonucleoprotein AB (HNRNP AB) in hepatocellular carcinoma (HCC) cells promoted cell invasion in vitro and lung metastasis in vivo; this effect may be associated with its interaction with Kap1 and its transcriptional regulation of lncRNA2012 expression. Further study shows that over-expression of lncRNA-2012 can inhibit the migration and invasion of HCC, suggesting that lncRNA-2012 can promote HCC metastasis, which might be regulated by HNRNP AB. In this study, through short hairpin RNA (shRNA) interference or up-regulation of hnRNP AB and lncRNA2012 expression in HCC cell lines with various metastatic potentials, we will assess their roles in tumor invasion and in vivo metastasis and determine the role of lncRNA2012 in HNRNP AB-enhanced HCC invasion and metastasis. Furthermore, chromatin immunoprecipitation (ChIP) and luciferase assays will be used to analyze whether hnRNP AB is involved in the transcriptional regulation of lncRNA2012 expression in HCC cells. Subsequently, western blotting and co-immunoprecipitation will be used to determine the interaction between hnRNP AB and Kap1. Through shRNA-mediated down-regulation of Kap1 in HCC cells, we will determine the changes on cellular location of hnRNP AB and its transcriptional activity. We will also assess its roles on regulation of lncRNA2012 expression induced by hnRNP AB and determine its roles on tumor invasion and in vivo metastasis. The relationship between the expression of these genes and the invasive phenotype of HCC cells will be studied. We will determine whether these genes could be important independent factors in determining clinical outcomes of HCC. This study will provide a biological foundation for the discovery of new potential therapeutic targets for HCC.

长链非编码RNA（lncRNA）在肿瘤恶化中发挥重要作用。前期工作中，我们发现上调hnRNPAB能促进肝癌侵袭转移，且与Kap1蛋白互作和lncRNA2012表达调控密切相关；进一步发现过表达lncRNA2012可抑制肝癌细胞运动和侵袭。本课题通过共转染hnRNPAB和lncRNA2012过表达质粒或shRNA，进行体内外功能分析，明确lncRNA2012介导的hnRNPAB在肝癌侵袭转移中的作用；应用染色质免疫沉淀、荧光素报告基因分析hnRNPAB表达对lncRNA2012转录调控的影响；通过免疫共沉淀和免疫印迹明确hnRNP AB和Kap1的互作，观察shRNA-Kap1干预前后肝癌细胞内hnRNPAB亚细胞定位和转录活性的改变及其对调控lncRNA2012表达和肝癌侵袭转移的影响，同时分析三者在肝癌组织中的表达及与肝癌转移复发间的关系。本研究为探讨肝癌治疗中新的干预靶点提供实验依据。

我们利用lncRNA芯片筛选hnRNPAB调控的LncRNA，发现LncELF2-209（探针名称：pl112012）在肝癌细胞中的表达与hnRNPAB的表达关系最显著。在肝癌细胞中过表达LncELF2-209可导致间质型标志物(N-cadherin,vimentin和snail)的表达下调，同时两个上皮标志物(E-cadherin和ZO-1)表达上调，显著降低肝癌细胞侵袭转移能力，该作用可能与磷酸丙糖异构酶(TPI)的活化相关。进一步发现，hnRNPAB通过负调控lnc-ELF209促进上皮间质转化(EMT)而增加肝癌细胞的侵袭转移能力，Kap1与hnRNPAB之间的互作在其中发挥作用。该成果以通讯作者，发表在Int J Cancer杂志(2020年,IF=5.15)和《中华肝脏病杂志》(2017年)。我们还探讨miRNA在肝癌侵袭转移中的作用，发现miR-561-5p调控靶基因CX3CL1的表达促进肝癌侵袭转移;还发现miR-29a通过拮抗TET-SOCS1-Stat3-MMP9信号通路在肝癌侵袭转移中发挥重要作用，这些研究成果以通讯作者，分别发表在“Theranostics”(2019年,IF=8.06)和“Cell Death Dis”(2017年,IF=5.64)。此外，我们发现m6ARNA调节基因的突变和拷贝数变异与肝癌患者的不良预后密切相关；PDEF下调可促进肝癌细胞体内外生长和转移；我们鉴定出与患者预后相关的含有9个基因标签的诊断模型；建立了由6个基因构建的患者预后预测价值的干性标签，这些诊断模型的建立有助于肝癌患者预后的评估。这些成果以通讯作者发表在“Front Oncol”(2020年,IF=4.85),“Carcinogenesis”(2017年,IF=4.0)，“J Transl Med”(2019年,IF=4.10)和“Expert Rev Gastroenterol Hepatol”(2018年,IF=3.51)。以通讯作者发表与本课题相关的论文15篇(SCI收录9篇)。2019年荣获华夏医学科技奖一等奖和第三十一届上海市优秀发明选拔赛优秀发明金奖。入选科技部“创新人才推进计划重点领域创新团队”核心成员。作为大会秘书长，组织5次大型全国学术会议。在该项目资助下，共培养研究生6人，其中,已毕业博士和硕士研究生分别为2人和4人。

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