SATB1-HRC：一条促肝癌侵袭转移的新途径

发布时间：2018-06-16 01:09

本文选题：肝细胞癌 + HRC　；参考：《华中科技大学》2016年博士论文

【摘要】：目的：肝癌发病率在所有恶性肿瘤中排名第五,但肝癌致死率却在肿瘤致死率中排名第二;肝癌恶性程度高、易早期转移是导致其致死率高的主要原因。目前,基于肝癌分子机制研发的抗癌药物索拉菲尼已成功运用于临床,但经索拉非尼治疗的肝癌患者的中位生存期并没有显著提高；因此,进一步探究肝癌发生发展的分子机制或可发现更多潜在的干预靶点。研究表明,钙信号参与肿瘤发生、发展的各个环节。HRC (histidine-rich calcium binding protein)是一种新近发现的钙离子结合蛋白,在维持细胞内钙平衡及钙信号传导过程中发挥重要的调控作用。本研究拟探讨HRC在肝癌侵袭转移中的作用及机制。方法：通过实时荧光定量PCR (RT-qPCR)、免疫印迹(western blot)与免疫组织化学(IHC)方法检测83对肝癌及相应癌旁组织中HRC的定位及表达情况,并比较不同转移潜能肝癌细胞株与正常肝细胞中HRC的表达差异。采用HRC真核表达载体(pcDNA3.1-Flag-HRC)转染构建过表达HRC的SMMC-7721细胞株,而采用siRNA干扰技术(siHRC)沉默Sk-hep-1细胞中HRC的表达;通过体外Transwell小室侵袭迁移与Wound healing实验探索过表达/沉默HRC后肝癌细胞侵袭、迁移能力的变化；通过裸鼠人肝癌转移模型体内验证HRC对肝癌侵袭转移的影响。流式细胞术、免疫荧光与免疫共沉淀检测过表达／沉默HRC对钙信号、粘着斑更新的调控。最后,采用RT-qPCR、western blot、荧光素酶报告基因、凝胶迁移实验(EMSA)与染色质免疫沉淀(CHIP)等方法阐明引起肝癌细胞中HRC表达上调的相关机制。结果：HRC主要定位于细胞质,在83对肝癌组织样本中均可检测到其表达,肝癌组织中HRC的表达水平明显高于相应的癌旁组织(56/83,67.47%),且临床病理特征分析显示,HRC的表达水平与肝癌的大小(p=0.026)、转移(p=0.004)密切相关；HRC在高转移潜能的肝癌细胞Sk-hep-1、HCC-LM3及MHCC-97H中的表达明显高于转移潜能相对较低的肝癌细胞MHCC-97L、Huh-7及SMMC-7721,而在正常肝细胞Chang Liver中表达最低。增强肝癌细胞SMMC-7721中HRC表达后,细胞侵袭、迁移能力增强,裸鼠肝内、肺转移灶增加：而干扰肝癌细胞Sk-hep-1中HRC表达后,细胞侵袭、迁移能力明显减弱。过表达／沉默HRC表达后,钙离子浓度、钙调蛋白(CaM)表达及粘着斑激酶(FAK)的磷酸化水平随之升高／降低;采用钙离子螯合剂毒胡萝卜素thapsigargin (TG)、CaM拮抗剂TFP预处理后,HRC促FAK活化、粘着斑更新、细胞迁移的作用明显减弱。沉默肝癌细胞Sk-hep-1中HRC表达后,钙泵SERCA2表达水平显著升高,而钙通道RyR与NCX无明显改变；且在6种不同转移潜能的肝癌细胞中,SERCA2与HRC的表达水平呈负相关(相关系数R=-0.74)。在肝癌组织样本中,SATB1与HRC的表达水平呈正相关(相关系数R=0.494)；在肝癌细胞中,核基质结合蛋白SATB1可诱导HRC表达上调;过表达SATB1后,肝癌细胞侵袭、迁移能力增强,但在SMMC-7721-SATB1细胞中干扰HRC表达后,SATB1促肝癌细胞侵袭、迁移作用显著减弱；过表达/沉默SATB1后,MEK/ERK与JNK/c-Jun信号通路关键蛋白的表达、转录因子AP-1及HRC启动子的活性随之发生相应变化；SP600125(JNK抑制剂)而非U0126(MEK抑制剂)预处理可减弱SATB1对HRC的上调作用：过表达/沉默c-Jun后,HRC启动子活性随之增强/减弱,且转录因子AP-1可直接与HRC启动子结合；在SMMC-7721-SATB1细胞中干扰c-Jun表达后,SATB1激活HRC启动子并诱导其表达的作用明显减弱。结论：HRC通过激活Ca2+/CaM信号诱导FAK活化,调控粘着斑更新,进而促进肝癌侵袭转移：而HRC促肝癌侵袭转移这一作用又受到SATB1调控,其机制可能是与激活JNK/c-Jun信号通路、上调转录因子AP-1表达有关。
[Abstract]:Objective: the incidence of liver cancer ranked fifth in all malignant tumors, but the death rate of liver cancer ranked second in the death rate of cancer; high malignancy of liver cancer and early metastasis are the main causes of high mortality. At present, Sola Feeney, a cancer drug based on the molecular mechanism of liver cancer, has been successfully applied to the clinic, but it has been treated by sorafer. The median survival of the liver cancer patients treated with Nepal did not increase significantly; therefore, further exploring the molecular mechanism of the development of liver cancer or the discovery of more potential intervention targets. Studies have shown that calcium signals are involved in the development of tumor, and the development of.HRC (histidine-rich calcium binding protein) is a newly discovered calcium ionization. Sub binding proteins play an important role in maintaining intracellular calcium balance and calcium signal transduction. The purpose of this study is to explore the role and mechanism of HRC in the invasion and metastasis of liver cancer. Methods: 83 liver cancer and corresponding para cancer were detected by real time fluorescence quantitative PCR (RT-qPCR), Western blot (Western blot) and immunofluorescence histochemistry (IHC). The localization and expression of HRC in the tissue and the difference of the expression of HRC in the hepatocellular carcinoma cell lines with different metastatic potential and normal liver cells were compared. The expression of SMMC-7721 cell lines expressing HRC was constructed by HRC eukaryotic expression vector (pcDNA3.1-Flag-HRC), and the siRNA interference technique (siHRC) was used to silence the expression of HRC in Sk-hep-1 cells; Transwe in vitro Ll cell invasion migration and Wound healing experiment explored the invasion and migration of hepatoma cells after expression / silence HRC; the effect of HRC on the invasion and metastasis of hepatocellular carcinoma in nude mice was verified by human hepatoma metastasis model in nude mice. Flow cytometry, immunofluorescence and immunoprecipitation were used to detect the calcium signal and the alteration of adhesion plaque. Finally, RT-qPCR, Western blot, luciferase reporter gene, gel migration test (EMSA) and chromatin immunoprecipitation (CHIP) were used to elucidate the mechanism of the up regulation of HRC expression in hepatoma cells. Results: HRC is mainly located in the cytoplasm and can be detected in 83 hepatoma tissue samples, and the table of HRC in liver cancer tissue The level of HRC was significantly higher than that of the corresponding para cancerous tissue (56/83,67.47%), and the analysis of clinicopathological features showed that the expression level of HRC was closely related to the size of liver cancer (p=0.026) and metastasis (p=0.004). The expression of HRC in high metastatic potential hepatoma cells, HCC-LM3 and MHCC-97H was significantly higher than that of liver cancer cells with relatively low metastatic potential. The expression of 7L, Huh-7 and SMMC-7721 is the lowest in the normal hepatocytes, Chang Liver. After HRC expression in SMMC-7721, the cell invasion, migration ability is enhanced, and the lung metastasis in nude mice is increased: after the interference of HRC expression in the liver cancer cell Sk-hep-1, the invasion and migration ability of the hepatoma cells is obviously weakened. After overexpression / silencing HRC expression, calcium ionization The level of phosphorylation of calmodulin (CaM) and adhesion kinase (FAK) increased / decreased; HRC promoted FAK activation after TFP pretreatment with calcium ion chelating agent, carotene thapsigargin (TG), CaM antagonist TFP, and the migration of cell migration was significantly weakened. After HRC expression in the Sk-hep-1 cell Sk-hep-1, the calcium pump SERC The expression level of A2 was significantly higher, but the calcium channel RyR and NCX were not significantly changed, and the expression level of SERCA2 and HRC was negatively correlated (correlation coefficient R=-0.74) in 6 different metastatic potential hepatoma cells. In the liver cancer tissue samples, the expression level of SATB1 and HRC was positively correlated (the number of phases R=0.494); in the hepatoma cells, the nuclear matrix combined the egg. White SATB1 can induce up regulation of HRC expression, after overexpression of SATB1, hepatoma cells invade and migrate, but after HRC expression in SMMC-7721-SATB1 cells, SATB1 promotes hepatoma cells to invade and migrate significantly; after overexpression / silence SATB1, the expression of key proteins in MEK/ERK and JNK/c-Jun signal pathway, AP-1 and HRC of transcription factors The activity of SP600125 (JNK inhibitor) rather than U0126 (MEK inhibitor) preconditioning can weaken the up regulation of SATB1 to HRC: after overexpression / silencing of c-Jun, the activity of HRC promoter is enhanced / weakened, and the transcriptional factor AP-1 can be directly associated with HRC promoter, and after interference of c-Jun expression in SMMC-7721-SATB1 cells, B1 activates the HRC promoter and induces its expression. Conclusion: HRC regulates the activation of FAK by activating Ca2+/CaM signal, regulates the regeneration of the adhesion plaque, and then promotes the invasion and metastasis of liver cancer, and the effect of HRC to promote the invasion and metastasis of liver cancer is regulated by SATB1. The mechanism may be the activation of JNK/c-Jun signaling pathway and the up-regulation of the transcription factor AP-. 1 expression is related.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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