rs4969170GG基因型抑制SOCS3基因转录活性促进肝癌发生发展的功能机制研究

Our previous studies demonstrated that SOCS3 rs4969170GG genotype was closely related to the occurrence of HCC, the degree of malignancy and the prognosis of HCC. rs4969170GG genotype could reduce the expression of SOCS3 by transcriptional repression of SOCS. However, the molecular mechanism of the occurrence and development of HCC is still unclear. In this study, we obtained rs4969170A/G different alleles of HCC cells using CRISPR-Cas technology. We will reveal the biological function of rs4969170GG in HCC, by comparing the proliferation, apoptosis, cell cycle, clone formation, migration and in vivo tumorigenicity of the different rs4969170A/G alleles HCC cells. We will study the ability of the tumor formation using the rs4969170GG knock-in mice. We will reveal the mechanism of rs4969170GG in regulating the transcriptional activity of the promoter of SOCS3 using luciferase reporter system, Gel shift Assay and Chromatin Immunoprecipitation Assay. Furthermore, we will detect the expression levels of SOCS3 in hepatocellular carcinoma tissue microarray to analyse their relevance and verify the regulation mechanism of SOCS3 on HCC. We will also reveal the association between the related cell signal pathway and SOCS3 rs4969170A/G SNP using our abundant clinical patient resource. This study will reveal the mechanisms of SOCS3 rs4969170GG genotype in promoting the occurrence of HCC and will be helpful to assess the feasibility of SOCS3 as a potential therapeutic target for HCC therapy.

本课题组前期研究证实rs4969170A/G SNP的rs4969170GG基因型下调SOCS3的表达，并与肝癌的发生发展、临床病理特征及预后密切相关，但其分子机制尚不清楚。本课题拟利用CRISPR-Cas 技术获得rs4969170A/G不同基因型肝癌细胞，比较其增殖、调亡、细胞周期、克隆形成、迁移、侵袭能力及体内成瘤的差异，明确rs4969170GG促进肝癌发生发展的生物学功能；构建rs4969170GG基因型的knock-in小鼠研究SOCS3 rs4969170GG基因型的成瘤能力。利用双荧光报告系统等技术探讨rs4969170GG对SOCS3基因的转录调控机制；利用动物及HCC样本研究rs4969170GG引起HCC有关信号通路的变化。通过本项目阐明rs4969170GG促进肝癌发生发展的分子机制，为针对SOCS3基因 rs4969170A/G多态性的肝癌个性化治疗提供理论基础。

作为全球第五大常见肿瘤之一，原发性肝细胞癌（hepatocellular carcinoma，HCC）占我国恶性肿瘤死亡率的第二位。HCC 早期诊断难、恶性程度高、复发转移率高，大部分患者确诊时已是中晚期，至今没有令人满意的治疗方法。本课题组发现，与健康人群相比，HCC 患者中SOCS3 rs4969170GG 基因型发生率更高，而且在HCC 患者中，SOCS3 rs4969170GG 基因型患者，肿瘤的恶性程度更高、预后更差。SOCS3 rs4969170GG 基因型引起肝癌的机制未见报道。本项目先利用临床标本通过western-blot以及Real-time PCR等技术手段明确了rs4969170A/G SNP的rs4969170GG基因型通过抑制SOCS3基因的表达促进肝癌的发生发展的。进一步体外增殖实验证实含有rs4969170GG基因型的SMCC-7721细胞比其他两种基因型的增殖明显加快，且不易发生凋亡，克隆形成率更高，细胞迁移能力更强，小鼠成瘤能力显著增强。分子机制方面，通过双荧光素酶报告系统明确rs4969170GG通过抑制SOCS3基因的转录减少SOCS3基因的表达；rs4969170A/G SNP 位于SOCS3 基因的启动子区域，应用启动子与转录因子结合位点数据库和预测工具以及生物信息学分析表明，SOCS3 基因的rs4969170A/G SNP 可能影响Sp1，Runx，Sox-9，AP1 等转录因子的作用；进一步通过gel-shift技术证实rs4969170A/G SNP可能直接影响Sp1，Runx，Sox-9这三个转录因子与SOCS3上游启动子区的结合。因为SOCS3是一个著名的免疫负调控者，通过检测相关肝癌的肿瘤样本表明rs4969170GG肝癌体内的炎症信号被激活。上述结果表明rs4969170GG通过抑制SOCSS3基因的表达使SOCS3 免疫负调控作用减弱，加剧了肿瘤细胞的恶性行为，最终导致HCC 发生。已发表相关SCI文章2篇。培养硕士研究生2名。