【作者】 宋伟；

【导师】 樊红；

【作者基本信息】 东南大学 ， 生物学， 2019， 博士

【摘要】 肝细胞癌(hepatocellular carcinoma,HCC)是一种具有高复发率和高死亡率的恶性肿瘤疾病。乙型肝炎病毒(Hepatitis B virus,HBV)编码的X蛋白(HBx)以多种方式,包括基因畸变和失调、表观遗传学修饰改变及非编码RNA(non-coding RNA,nc RNA)调控等在HCC细胞增殖和转移等过程中发挥重要生物学作用。长链非编码RNA(long non-coding RNA,lncRNA)是一类长度超过200 bp不编码蛋白质的RNA分子,通过挥信号、诱饵、指导或支架的作用方式参与肿瘤的多种生物学过程。HCC中lncRNA的研究结果提示lncRNA可影响HCC的增殖、转移、凋亡等多种功能,HBx亦可通过调控相关分子表达,特别是表观遗传调控方式参与肝癌的发生发展。进一步探究HBx诱导lncRNA及其在HCC恶性进展中的作用机制,将有助于肝癌的有效诊断和靶向治疗策略的制定。本研究应用lncRNA芯片和m RNA芯片分析HBx影响HCC细胞差异表达的lncRNA表达谱和m RNA表达谱;lncRNA TRERNA1因其在过表达HBx细胞中差异表达显著而作为进一步研究的候选分子;通过HBx质粒转染以及RNA干扰(RNAi)方法检测HBx对lncRNA TRERNA1的表达水平的关系;实时定量PCR(Quantitative Real-time PCR,q-PCR)方法检测TRERNA1与HBx在HCC病例组织和HCC细胞中表达水平相关性;细胞划痕实验,Transwell迁移和Matrigel侵袭实验检测TRERNA1对HCC细胞迁移和侵袭能力的影响;裸鼠尾静脉注射建立肿瘤转移模型,以苏木精-伊红染色(hematoxylin-eosin staining,H&E)染色方法检测TRERNA1在体内转移结节的形成;Q-PCR、Western blot分别检测TRERNA1对可能靶基因CDH1 m RNA和蛋白表达水平的影响;RNA免疫共沉淀(RNA Immunoprecipitation,RIP)、免疫共沉淀(Immunoprecipitation,IP)实验检测TRERNA1与EHMT2、SNAI1三者之间的相互结合。染色质免疫共沉淀(Chromatin Immunoprecipitation,Ch IP)方法分析EHMT2与CDH1启动子区的结合;Q-PCR法检测CDH1在HCC病例中的表达水平及其与TRERNA1转录水平之间的关系;RNA原位杂交方法评估TRERNA1在HCC病例中的表达情况及其对HCC预后不良的关系;通过GO和KEGG Pathway分析TRERNA1可能参与的生物学功能和信号通路;CCK-8增殖实验、平板克隆形成、流式细胞术以及裸鼠皮下成瘤实验检测TRERNA1对HCC细胞增殖能力的影响;Western blot用于检测细胞周期相关蛋白的表达;CCK-8法用于检测TRERNA1对索拉菲尼治疗HCC敏感性的影响,同时Western blot检测TRERNA1对Raf-MEK-ERK磷酸化通路的影响。通过以上研究获得如下研究结果:1 HBx上调了lncRNA TRERNA1的表达,且TRERNA1表达水平与HBx表达存在剂量依赖关系;2 HCC组织中的研究发现TRERNA1的表达水平与HCC转移和分期密切相关,且与HCC患者预后不良有关;3体内外实验研究结果表明,表达上调的TRERNA1不仅促进HCC细胞的迁移和侵袭能力,TRERNA1高表达促进了HCC细胞的增殖和裸鼠移植瘤的生长;4高表达的TRERNA1降低了索拉非尼对HCC细胞的敏感性,与HCC预后不良有关;5结合GO和KEGG Pathway分析过表达TRERNA1表达诱导的差异基因表达谱,发现TRERNA1参与调控的基因主要集中在调控细胞增殖、细胞周期等相关功能;6 TRERNA1以分子支架功能,通过招募EHMT2/SNAI1复合体使CDH1启动子区发生H3K9二甲基化从而抑制CDH1表达,同时TRERNA1发挥增强子样功能激活其邻近基因SNAI1的表达,促进HCC细胞的侵袭转移能力;7 TRERNA1抑制p21蛋白的表达,上调细胞周期调控分子Cyclin D1、CDK2和CDK4的表达,促进HCC细胞G1/S期的转换,增强HCC细胞的增殖能力;8 TRERNA1通过激活磷酸化Raf-MEK-ERK信号通路降低了HCC靶向药物索拉菲尼对HCC治疗的敏感性。综上所述,我们通过研究首次揭示,受HBx诱导的lncRNA TRERNA1通过招募EHMT2/SNAI1复合体抑制了CDH1的表达从而促进HCC的转移。TRERNA1通过激活磷酸化Raf-MEK-ERK通路,降低了索拉菲尼对HCC细胞的敏感性。深入研究lncRNA TRERNA1作用将有助于揭示促进HCC进展的分子机制,并有利于评估HCC靶向药物治疗疗效,将为HCC患者的诊断和治疗提供新的研究策略。更多还原

【Abstract】 Hepatocellular carcinoma(HCC)is a malignant tumor disease with high recurrence rate and high mortality.The X protein(HBx)encoded by HBV(Hepatitis B virus)plays various biological roles in the process of proliferation and metastasis of hepatocarcinoma through genetic aberrations and disorders,epigenetic modification,non-coding RNA(nc RNA)regulation.Long non-coding RNA(lncRNA)defined as being longer than 200 nucleotidesis that does not encode protein.It may participate in the biological processes of various tumors by means of signal,decoy,guide or scaffold.The results of lncRNA in HCC suggest that lncRNA can affect the proliferation,metastasis,apoptosis and other functions of HCC.HBx can also participate in the development of liver cancer by regulating the expression of related molecules,especially epigenetic regulation.Further exploration of lncRNAs induced by HBx and its mechanism of action in the malignant progression of tumors will contribute to the effective diagnosis and targeted therapeutic strategies in HCC.The differentially expressed lncRNAs and m RNAs induced by HBx in HCC cells were screened using Human Lnc RNA Array and Human m RNA Array analysis.TRERNA1 was considered as a candidate because of its relatively high expression level after transfection with HBx using Lnc RNA Array.The regulation of HBx to lncRNA TRERNA1 was detected by HBx plasmid transfection and RNAi.Quantitative Real-time PCR(q-PCR)assay showed a negative correlation between TRERNA1 and HBx in liver cancer tissues and cells.The effect of TRERNA1 on cell migration and invasion was analyzed by wound healing assay,transwell migration assay and matrigel invasion assay.Metastatic model of tail vein injection and H&E staining in nude mice were used to detect the effect of TRERNA1 on metastasis in vivo.The potential regulatory mechanisms of TRERNA1 to its target gene CDH1 were explored by q PCR and WB assays.RIP and IP assay were employed to detect the physical association among TRERNA1,EHMT2 and SNAI1.The binding of EHMT2 to CDH1 promoter region was analyzed by Ch IP assay.Q-PCR was used to detect the expression of CDH1 in clinical cases and evaluate the relationship between CDH1 and TRERNA1.RNA in situ hybridization was used to assess the prognosis of TRERNA1 in HCC cases.The biological functions and signaling pathways that TRERNA1 may participate in were analyzed by GO and KEGG Pathway.CCK-8 assay,plate colony formation,flow cytometry and xenograft mice tumorigenicity assay were used to assess the proliferation of HCC cells.Cell cycle associated proteins were detected by western blot.CCK-8 assay was performed to determine the impact of TRERNA1 expression on the sensitivity of HCC cells to sorafenib.At the same time,western blot was used to detect the effect of TRERNA1 on Raf-MEK-ERK phosphorylation pathway.Through the present study,the results showed as following:1)LncRNA TRERNA1 was up-regulated by HBx,and there was a dose-dependent manner between TRERNA1 expression and HBx expression.2)The expression of TRERNA1 was not only positively correlated with tumor metastasis and staging grade,but also with poor prognosis in HCC cases.3)In vitro and in vivo,increased TRERNA1 expression promoted cell migration and invasion.TRERNA1 also promoted the proliferation of hepatoma cells and the tumorigenicity of xenografts in nude mice.4)High expression of TRERNA1 reduced the sensitivity of sorafenib to HCC cells,it was associated with poor prognosis of HCC.5)Combined with GO and KEGG Pathway analysis,the differential genes regulated by TRERNA1 were involved in cell proliferation and cell cycle.6)As a scaffold,TRERNA1 repressed CDH1 expression via recruiting EHMT2/SNAI1 complex to di-methylate H3K9 of CDH1 promoter region.In the meantime,TRERNA1 regulated its neighboring gene SNAI1 as an enhancer-like lncRNA to promote HCC migration and invasion.7)TRERNA1 inhibited the activity of p21 protein and enhanced the expression of cell cycle regulatory molecules Cyclin D1,CDK2 and CDK4,which accelerated cell cycle progression of G1/S phase and promoted the proliferation of HCC.8)TRERNA1 reduced the sensitivity of sorafenib to HCC cells by activating the phosphorylated Raf-MEK-ERK signaling pathway.Taken together,we have revealed for the first time that lncRNA TRERNA1 induced by HBx inhibits the expression of CDH1 by recruiting the EHMT2/SNAI1 complex to promote HCC metastasis.TRERNA1 reduces the sensitivity of sorafenib to HCC cells by activating the phosphorylated Raf-MEK-ERK signaling pathway.The study of lncRNA TRERNA1 will help reveal HCC progression and evaluate the efficacy of HCC-targeted drug therapy,which will provide new research strategies for the diagnosis and treatment of HCC patients.更多还原