本文選題：肝細(xì)胞癌 + HRC��； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：目的：肝癌發(fā)病率在所有惡性腫瘤中排名第五,但肝癌致死率卻在腫瘤致死率中排名第二;肝癌惡性程度高、易早期轉(zhuǎn)移是導(dǎo)致其致死率高的主要原因。目前,基于肝癌分子機(jī)制研發(fā)的抗癌藥物索拉菲尼已成功運(yùn)用于臨床,但經(jīng)索拉非尼治療的肝癌患者的中位生存期并沒有顯著提高；因此,進(jìn)一步探究肝癌發(fā)生發(fā)展的分子機(jī)制或可發(fā)現(xiàn)更多潛在的干預(yù)靶點(diǎn)。研究表明,鈣信號(hào)參與腫瘤發(fā)生、發(fā)展的各個(gè)環(huán)節(jié)。HRC (histidine-rich calcium binding protein)是一種新近發(fā)現(xiàn)的鈣離子結(jié)合蛋白,在維持細(xì)胞內(nèi)鈣平衡及鈣信號(hào)傳導(dǎo)過程中發(fā)揮重要的調(diào)控作用。本研究擬探討HRC在肝癌侵襲轉(zhuǎn)移中的作用及機(jī)制。方法：通過實(shí)時(shí)熒光定量PCR (RT-qPCR)、免疫印跡(western blot)與免疫組織化學(xué)(IHC)方法檢測(cè)83對(duì)肝癌及相應(yīng)癌旁組織中HRC的定位及表達(dá)情況,并比較不同轉(zhuǎn)移潛能肝癌細(xì)胞株與正常肝細(xì)胞中HRC的表達(dá)差異。采用HRC真核表達(dá)載體(pcDNA3.1-Flag-HRC)轉(zhuǎn)染構(gòu)建過表達(dá)HRC的SMMC-7721細(xì)胞株,而采用siRNA干擾技術(shù)(siHRC)沉默Sk-hep-1細(xì)胞中HRC的表達(dá);通過體外Transwell小室侵襲遷移與Wound healing實(shí)驗(yàn)探索過表達(dá)/沉默HRC后肝癌細(xì)胞侵襲、遷移能力的變化；通過裸鼠人肝癌轉(zhuǎn)移模型體內(nèi)驗(yàn)證HRC對(duì)肝癌侵襲轉(zhuǎn)移的影響。流式細(xì)胞術(shù)、免疫熒光與免疫共沉淀檢測(cè)過表達(dá)／沉默HRC對(duì)鈣信號(hào)、粘著斑更新的調(diào)控。最后,采用RT-qPCR、western blot、熒光素酶報(bào)告基因、凝膠遷移實(shí)驗(yàn)(EMSA)與染色質(zhì)免疫沉淀(CHIP)等方法闡明引起肝癌細(xì)胞中HRC表達(dá)上調(diào)的相關(guān)機(jī)制。結(jié)果：HRC主要定位于細(xì)胞質(zhì),在83對(duì)肝癌組織樣本中均可檢測(cè)到其表達(dá),肝癌組織中HRC的表達(dá)水平明顯高于相應(yīng)的癌旁組織(56/83,67.47%),且臨床病理特征分析顯示,HRC的表達(dá)水平與肝癌的大小(p=0.026)、轉(zhuǎn)移(p=0.004)密切相關(guān)；HRC在高轉(zhuǎn)移潛能的肝癌細(xì)胞Sk-hep-1、HCC-LM3及MHCC-97H中的表達(dá)明顯高于轉(zhuǎn)移潛能相對(duì)較低的肝癌細(xì)胞MHCC-97L、Huh-7及SMMC-7721,而在正常肝細(xì)胞Chang Liver中表達(dá)最低。增強(qiáng)肝癌細(xì)胞SMMC-7721中HRC表達(dá)后,細(xì)胞侵襲、遷移能力增強(qiáng),裸鼠肝內(nèi)、肺轉(zhuǎn)移灶增加：而干擾肝癌細(xì)胞Sk-hep-1中HRC表達(dá)后,細(xì)胞侵襲、遷移能力明顯減弱。過表達(dá)／沉默HRC表達(dá)后,鈣離子濃度、鈣調(diào)蛋白(CaM)表達(dá)及粘著斑激酶(FAK)的磷酸化水平隨之升高／降低;采用鈣離子螯合劑毒胡蘿卜素thapsigargin (TG)、CaM拮抗劑TFP預(yù)處理后,HRC促FAK活化、粘著斑更新、細(xì)胞遷移的作用明顯減弱。沉默肝癌細(xì)胞Sk-hep-1中HRC表達(dá)后,鈣泵SERCA2表達(dá)水平顯著升高,而鈣通道RyR與NCX無明顯改變；且在6種不同轉(zhuǎn)移潛能的肝癌細(xì)胞中,SERCA2與HRC的表達(dá)水平呈負(fù)相關(guān)(相關(guān)系數(shù)R=-0.74)。在肝癌組織樣本中,SATB1與HRC的表達(dá)水平呈正相關(guān)(相關(guān)系數(shù)R=0.494)；在肝癌細(xì)胞中,核基質(zhì)結(jié)合蛋白SATB1可誘導(dǎo)HRC表達(dá)上調(diào);過表達(dá)SATB1后,肝癌細(xì)胞侵襲、遷移能力增強(qiáng),但在SMMC-7721-SATB1細(xì)胞中干擾HRC表達(dá)后,SATB1促肝癌細(xì)胞侵襲、遷移作用顯著減弱；過表達(dá)/沉默SATB1后,MEK/ERK與JNK/c-Jun信號(hào)通路關(guān)鍵蛋白的表達(dá)、轉(zhuǎn)錄因子AP-1及HRC啟動(dòng)子的活性隨之發(fā)生相應(yīng)變化；SP600125(JNK抑制劑)而非U0126(MEK抑制劑)預(yù)處理可減弱SATB1對(duì)HRC的上調(diào)作用：過表達(dá)/沉默c-Jun后,HRC啟動(dòng)子活性隨之增強(qiáng)/減弱,且轉(zhuǎn)錄因子AP-1可直接與HRC啟動(dòng)子結(jié)合；在SMMC-7721-SATB1細(xì)胞中干擾c-Jun表達(dá)后,SATB1激活HRC啟動(dòng)子并誘導(dǎo)其表達(dá)的作用明顯減弱。結(jié)論：HRC通過激活Ca2+/CaM信號(hào)誘導(dǎo)FAK活化,調(diào)控粘著斑更新,進(jìn)而促進(jìn)肝癌侵襲轉(zhuǎn)移：而HRC促肝癌侵襲轉(zhuǎn)移這一作用又受到SATB1調(diào)控,其機(jī)制可能是與激活JNK/c-Jun信號(hào)通路、上調(diào)轉(zhuǎn)錄因子AP-1表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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