【作者】 张雯；

【导师】 赵春华；

【作者基本信息】 上海大学 ， 细胞生物学， 2024， 硕士

【摘要】 肝细胞癌(Hepatocellular carcinoma,HCC)是一种具有多种遗传变异的异质性疾病,包括缺失、插入、重复和拷贝数变异(Copy number variation,CNV)。大量研究表明,从最常见的CNV基因座转录而来的长链非编码RNA(Long non-coding RNA,Lnc RNA)已被证明对肿瘤的恶性进展起关键作用,Lnc RNA的差异表达与肿瘤的发生发展密切相关。本课题旨在寻找位于拷贝数扩增区域内且在肝癌组织中显著高表达的Lnc RNA,通过分析TCGA数据库里50对肝癌及配对癌旁组织的RNA测序数据以及58对肝癌和配对癌旁组织的SNP6.0芯片数据(拷贝数分析),我们发现定位于1号染色体长臂的21区1带的Lnc RNA LINC02802,发生显著的拷贝数扩增,且在肝癌组织中的表达水平显著上调。进一步我们对LINC02802在肝癌生长与转移中的功能、分子机制、表达调控及临床意义作了深入研究。首先,在80对肝癌和配对癌旁组织的基因组DNA样本中,通过q PCR检测LINC02802的拷贝数水平,发现LINC02802在肝癌组织中存在拷贝数的显著扩增。在130对肝癌及相应癌旁组织的c DNA样本中检测LINC02802的表达水平,发现LINC02802的RNA水平在肝癌组织中显著升高。通过分析LINC02802的表达水平与临床病理特征的关联性,发现LINC02802高表达的肝癌患者肿瘤体积较大,且预后较差。其次,通过体内和体外实验对LINC02802的生物学功能进行研究,发现过表达LINC02802可以促进肝癌细胞体外的增殖、侵袭、迁移能力,以及肝癌细胞的裸鼠皮下成瘤能力和裸鼠肝内转移能力。而敲除肝癌细胞中的LINC02802则具有截然相反的效果,这提示LINC02802在肝癌的发生发展过程中起到癌基因的作用。最后,采用RNA pull down和RNA免疫共沉淀实验,鉴定LINC02802的相互作用蛋白,探索其在肝癌中发挥功能的分子机制。结果表明,LINC02802可以直接结合含有IQ基序的GTP酶激活蛋白1(IQ motif-containing GTPase-activating protein 1,IQGAP1)使自身的RNA稳定性增加,从而使HCC细胞中的LINC02802维持在较高水平。高表达的LINC02802可以通过抑制IQGAP1与其E3泛素连接酶TRIM56的结合来抑制IQGAP1蛋白的泛素化降解,使得癌基因IQGAP1的蛋白产物在HCC细胞中积累,最终促进肝癌细胞生长和转移。综上所述,LINC02802可以作为肝癌患者临床诊断和预后的分子标志物。更多还原

【Abstract】 Hepatocellular carcinoma(HCC)is a heterogeneous disease with a variety of genetic variations,including deletion,insertion,duplication,and copy number variation(CNV).A large number of studies have shown that long non-coding RNA(Lnc RNA)transcribed from the most common CNV loci has been proven to play a key role in the malignant progression of tumors,and the differential expression of Lnc RNA is closely related to the occurrence and development of tumors.The purpose of this study was to find Lnc RNAs located in the region of copy number amplification and significantly highly expressed in liver cancer tissues.By analyzing the RNA sequencing data of 50pairs of liver cancer and paired adjacent tissues in the TCGA database and the SNP6.0chip data of 58 pairs of liver cancer and paired adjacent tissues(copy number analysis),we found that the Lnc RNA LINC02802,located in the 21 region 1 band of the long arm of chromosome 1,had significant copy number amplification,and its expression level in liver cancer tissues was significantly upregulated.Firstly,the copy number of LINC02802 was detected by q PCR in 80 pairs of genomic DNA samples of liver cancer and matched adjacent tissues,and it was found that the copy number of LINC02802 was significantly amplified in liver cancer tissues.The expression level of LINC02802 was detected in 130 pairs of c DNA samples of liver cancer and corresponding adjacent tissues,and it was found that the RNA level of LINC02802 was significantly increased in liver cancer tissues.By analyzing the correlation between the expression level of LINC02802 and clinicopathological characteristics,it was found that the tumor volume of liver cancer patients with high expression of LINC02802 was larger and the prognosis was poor.Secondly,the biological function of LINC02802 was studied by vivo and vitro experiments,and it was found that overexpression of LINC02802 can promote the proliferation,invasion and migration ability of liver cancer cells in vitro,as well as the subcutaneous tumorigenic ability of liver cancer cells in nude mice and intrahepatic metastasis ability of liver cancer cells in nude mice.However,the knockout of LINC02802 in liver cancer cells has the opposite effect,which suggests that LINC02802 plays an oncogene role in the occurrence and development of liver cancer.Finally,RNA pull down and RNA immunoprecipitation experiments were used to identify the interaction protein of LINC02802,and to explore the molecular mechanism of its function in liver cancer.The results showed that LINC02802 can directly bind to IQ motif-containing GTPase-activating protein 1(IQGAP1)to increase its RNA stability,so as to maintain a high level of LINC02802 in HCC cells.The overexpression of LINC02802 can inhibit the ubiquitination degradation of IQGAP1protein by inhibiting the binding of IQGAP1 and its E3 ubiquitin ligase TRIM56,which makes the protein products of oncogene IQGAP1 accumulate in HCC cells and ultimately promote the growth and metastasis of liver cancer cells.In summary,LINC02802 can be used as a molecular marker for clinical diagnosis and prognosis of patients with liver cancer.更多还原