【作者】 李波；

【导师】 杨家印；

【作者基本信息】 四川大学 ， 外科学（肝脏）， 2022， 博士

【摘要】 目的:肝细胞肝癌(HCC)切除术后复发率高,尤其是术后2年内早期复发率占比70%,严重影响患者预后。丝裂原活化蛋白激酶(MAPK)信号通路是参与肝癌增殖、转移等生物学行为调控的主要通路之一。双特异性磷酸酶家族(DUSPs)是MAPK信号通路关键的负调控分子。目前未见DUSPs在HCC早期复发中的研究报道。本研究对DUSPs在HCC早期复发中的作用及其机制进行探索,以便为肝癌早期复发防治策略的制定提供帮助。材料和方法:首先,在临床样本方面,本研究收集我院HCC复发患者原发癌及其非癌肝组织等新鲜冰冻样本57对。选取HCC早期复发与晚期复发患者原发癌组织各3例,提取总核糖核酸(RNA),予以信使RNA(mRNA)转录组测序分析,富集DUSPs差异表达谱。在我中心57对样本中,利用实时荧光定量聚合酶链反应(q RT-PCR)技术和免疫印迹(WB)技术对差异表达基因在mRNA表达及蛋白水平的验证。同时,结合临床病理资料进行单、多因素生存分析及spearman相关关系分析。进一步,选用Hep G2、HCCLM3、Huh7等肝癌细胞构建慢病毒稳转细胞株。同时,利用裸鼠肝癌皮下瘤模型、肝内转移模型、尾静脉肺转移模型以及肝癌复发模型进行肝癌体内生长、转移及复发等功能验证。最后,利用免疫共沉淀(Co-IP)、WB及免疫荧光(IF)共定位技术进行机制研究。结果:1、由HCC早期复发与晚期复发患者原发癌组织mRNA转录组测序数据,富集到差异表达基因:双特异性磷酸酶9(DUSP9)。经临床大样本验证,发现DUSP9在HCC原发癌组织中高表达;另外,其表达水平越高,患者早期复发风险越高,预后越差。同时,DUSP9与AFP具有较强的正相关关系。2、通过细胞蛋白核质分离技术、WB技术,发现DUSP9在细胞浆与细胞核均有分布。另外,通过构建DUSP9敲低、过表达稳转肝癌细胞株,在体外细胞功能实验中,发现DUSP9促进HCC细胞增殖、迁移与侵袭;在体内动物实验中证明了DUSP9促进肝癌的生长、转移和复发。3、通过对MAPK经典信号通路的验证,发现DUSP9下调p38 MAPK磷酸化水平。进一步,对p38 MAPK信号通路经典调控分子进行验证,发现DUSP9上调c AMP反应元件结合蛋白(CREB)磷酸化水平。另外,结合DUSP9特殊的激酶互作结构域(KIM)以及STRING蛋白互作在线数据库预测提示,利用Co-IP技术及IF共定位技术初步证明了DUSP9与p38可能存在结合互作。结论:本研究通过HCC早期复发与晚期复发患者原发癌组织转录组测序数据,分析得到mRNA差异表达谱。经临床大样本、体内外功能实验多维度验证了DUSP9在HCC中预测患者早期复发以及促进肝癌细胞增殖、迁移、侵袭等方面的价值及作用。另外,在一定程度上对DUSP9促进肝癌复发的分子机制进行了探索。得出如下结论:1、DUSP9在HCC肝癌中高表达,其表达水平越高,患者早期复发风险越高,是预测HCC早期复发的重要指标。2、DUSP9促进肝癌细胞增殖、迁移及侵袭等生物学行为,进而促进肝癌的生长、转移和早期复发。3、DUSP9可能通过CREB MAPK信号轴调控肝癌细胞的增殖、转移和复发等恶性生物学行为。更多还原

【Abstract】 Objective:The recurrence rate of hepatocellular carcinoma(HCC)after resection is high,especially the early recurrence rate within 2 years after operation accounts for 70%,which seriously affects the prognosis of patients.Mitogen activated protein kinase(MAPK)signaling pathway is one of the most important pathways involved in the regulation of biological behavior such as proliferation and metastasis of liver cancer.Dual specificity phosphatases(DUSPs)are key negative regulators of MAPK signaling pathway.At present,there are no reports on the relationship between DUSPs and early recurrence of liver cancer.To help formulate strategies of prevention and treatment for the early recurrence of HCC,this study explored the role and mechanism of DUSPs in the early recurrence of HCC.Materials and Methods:Firstly,in terms of clinical samples,this study collected 57 pairs of fresh frozen samples of primary cancer and non cancerous liver tissue from patients with recurrent HCC in our hospital.Three primary cancer tissues of patients with early recurrence and late recurrence of HCC were selected.Total RNA was extracted and analyzed by messenger RNA(mRNA)transcriptome sequencing to enrich the differential expression profile of DUSPs.In 57 pairs of samples in our center,the mRNA expression and protein level of differentially expressed genes were verified by quantitive reverse transcription polymerase chain reaction(q RT-PCR)and Western blotting(WB).At the same time,combined with clinicopathological data,univariate and multivariate survival analysis and spearman correlation analysis were carried out.Further,Hep G2,HCCLM3 and Huh7 were selected to construct lentivirus stably transfected cells,and cell proliferation,migration and invasion were verified in vitro.In addition,the subcutaneous tumor model,intrahepatic metastasis model,caudal vein lung metastasis model and liver cancer recurrence model of nude mice were used to verify the function of liver cancer growth,metastasis and recurrence in vivo.Finally,the regulatory mechanism was studied by Co-immunoprecipitation(Co-IP),WB and immunofluorescence(IF)co-localization techniques.Results:1.We sequenced the mRNA transcriptome of primary cancer tissues from patients with early and late recurrence of HCC to enrich the differentially expressed gene of DUSPs family-dual specificity phosphatase 9(DUSP9).Furthermore,in 57 pairs of HCC clinical samples,the high expression of DUSP9 in HCC primary cancer tissues was verified from the mRNA expression and protein level.The higher expression level,the higher risk of early recurrence and the worse prognosis.In addition,DUSP9 has a strong positive correlation with alpha fetoprotein.2.We found that the protein of DUSP9 was distributed in both cytoplasm and nucleus.In addition,we found that knockdown and overexpression of DUSP9 could promote the proliferation and migration of HCC cells in vitro.Furthermore,the animal experiments have proved that DUSP9 could promote the growth,metastasis and recurrence of liver cancer in vivo.3.By verifying the classical signaling pathways of MAPK,we found that DUSP9 down-regulated the phosphorylation level of p38 MAPK.Further,the classical regulatory molecules of p38 MAPK signaling pathway were verified,and it was found that DUSP9 up-regulated the phosphorylation level of c AMP response element binding protein(CREB).In addition,combined with the kinase interacting motif(KIM)of DUSP9 and prediction of string protein-protein interaction online database,we preliminarily proved the possible binding interaction between DUSP9 and p38 by using Co-IP and IF co-localization techniques.Conclusion:In this study,mRNA differential expression profiles were obtained by analyzing the transcriptome sequencing data of primary cancer tissues in patients with early recurrence and late recurrence of HCC.The value and role of DUSP9 in predicting early recurrence and promoting the proliferation,migration and invasion of liver cancer cells in HCC were verified by large clinical samples and multi-dimensional functional experiments in vivo and in vitro.In addition,the molecular mechanism of DUSP9 promoting the recurrence of liver cancer was explored to a certain extent.The following conclusions are drawn:1.DUSP9 is highly expressed in HCC primary cancer.The higher expression of DUSP9,the higher risk of early recurrence.It is an important parameter to predict the early recurrence of HCC.2.DUSP9 promotes the proliferation,migration and invasion of liver cancer cells,and then promotes the growth,metastasis and early recurrence of liver cancer.3.DUSP9 may regulate the proliferation,metastasis and recurrence of hepatoma cells through CREB MAPK signal axis.更多还原