UHRF1在肝细胞癌中的表达及其功能的初步研究

发布时间：2018-05-09 00:03

本文选题：肝细胞癌 + UHRF1　；参考：《南方医科大学》2016年硕士论文

【摘要】：背景与目的原发性肝癌(Primary carcinoma of the liver)是目前世界上最常见的恶性肿瘤之一,最近一份调查报告表明：我国原发性肝癌年发病率为26.39/10万,占恶性肿瘤发病率第4位；死亡率为23.93/10万,占恶性肿瘤死亡率第2位。原发性肝癌按组织学分型可分为肝细胞型、胆管细胞型和混合型,其中以肝细胞型最为多见,约占原发性肝癌的90%,又称肝细胞癌(Hepatocellular carcinoma, HCC)。在我国,肝细胞癌发生的主要病因为乙型肝炎病毒感染,我们前期的研究显示,85%以上的肝细胞癌患者同时合并有乙型肝炎病毒感染。近年来,由于各种肿瘤标志物的临床应用和影像学技术的进步,特别是甲胎蛋白(alpha fetoprotein, AFP)和超声显像用于肝癌高危人群的监测,使肝癌能够在无明显症状或体征的亚临床期被诊断,加上外科手术技术的成熟,以及各种局部治疗等非手术治疗方法的发展,肝癌患者的总体预后较过去有了明显改善,但仍不尽如人意。肝癌具有高复发和高转移的生物学特征,有资料显示肝癌患者在接受根治性切除手术后五年内的复发率高达50-70%。如此高的术后复发率可能是由于患者手术前己合并存在目前医疗手段尚不能检测到的微转移灶。有研究发现,近一半接受肝癌切除术的患者在术前已合并有肝内微血管侵犯,进一步分析提示肝癌术前合并微血管侵犯是影响术后患者无瘤生存时间的独立危险因素。肝癌在早期缺乏典型症状,而患者一旦出现明显临床表现时,肝癌往往已经进展为晚期。因此,早发现、早诊断、早治疗是肝癌防治的关键。目前,研究者一致认为肿瘤是多因素、多步骤、多基因、多突变的结果,肝癌亦不例外。随着分子生物学等技术的进步,分子治疗也应运而生；近年来,科学家们发现了许多与肝癌患者预后相关的分子标志物,这些研究成果让我们对肝癌的发病机制及其治疗有了新的认识,为肝癌患者的预后评估及管理提供了新的思路和方法。然而,目前我们仍未能找到一种能够有效监测肝癌微转移灶的指标或系统。为此,我们有必要继续努力去发现与肝癌转移及复发相关的生物标志物。含植物同源结构和环指域泛素样蛋白1(ubiquitin-like with PHD and ring finger domains 1, UHRF1),又称为Np95或ICBP90,是新近发现的一种与肿瘤发生发展有关的核蛋白基因,定位于染色体19p13.3。研究表明,UHRF1通过与DNA甲基转移酶(DNA methyltransferase 1, DNMT1)以及组蛋白去乙酰化酶(histone deacetylase 1, HDAC1)等相互作用,参与基因表达调控及染色质修饰(如调节DNA甲基化,抑制抑癌基因的表达等)。同时,研究者还发现UHRF1在多种肿瘤组织中均存在过表达现象,且与肿瘤患者的预后呈负相关。此外,UHRF1的表达被敲低后,肿瘤细胞的增殖及侵袭能力等明显下降。这提示UHRF1有可能成为一个全新的阻止肿瘤细胞迁移和入侵的药物靶点。然而截至目前,UHRF1在肝细胞癌中尚未有一个较全面且深入的研究。本课题通过荧光定量PCR技术和免疫组织化学染色了解UHRF1在肝细胞癌组织中的表达情况及其与肝细胞癌生物学特征的关系,并探讨其在肝细胞癌发生、发展中可能的作用。此外,为进一步研究UHRF1在肝细胞癌中的作用,我们采用RNA干扰技术特异性敲低肝癌细胞株中UHRF1的表达,并通过体内和体外两个水平研究UHRF1在肝细胞癌发生、发展中的作用,为将来肝细胞癌患者的预后评估及个体化治疗等提供新的理论依据。第一章UHRF1 mRNA在肝细胞癌中的表达目的探索UHRF1 mRNA在肝细胞癌组织中的表达情况及其与肝细胞癌生物学特征和肝细胞癌患者预后的关系。方法1.实验组织标本的获取：组织标本来自于2010年11月至2014年11月期间在南方医科大学南方医院肝胆外科接受根治性手术治疗的80例经病理证实的肝细胞癌患者,所有患者术前血清学检查均提示有乙型肝炎病毒感染；2.实时荧光定量PCR检测UHRF1 mRNA在肝细胞癌组织及对应非癌组织中的表达情况；3.采用统计学方法分析UHRF1 mRNA在肝细胞癌患者癌组织中的表达水平及其与临床病理参数和预后的关系。结果1.在80例肝细胞癌患者中,54例患者癌组织中UHRF1 mRNA的表达水平高于非癌组织,高表达率约为67.5%(P0.05)；2.经统计分析,肝细胞癌组织中UHRF1 mRNA的表达水平与肝细胞癌患者肿瘤的大小、TNM分期、病理分化程度、微癌栓、转移、术后复发及术后无瘤生存时间等病理参数相关(P0.05)；3. Kaplan-Meier生存分析表明：高表达UHRF1 mRNA的肝细胞癌患者术后无瘤生存时间显著缩短(P=0.002)。结论1.UHRF1 mRNA在肝细胞癌组织中的表达显著高于非癌组织；2. UHRF1 mRNA在肝细胞癌组织中的表达水平与肝细胞癌患者肿瘤的大小、TNM分期、微癌栓、术前转移、术后复发及术后无瘤生存时间等病理参数相关；3. UHRF1 mRNA高表达的肝细胞癌患者术后无瘤生存时间较低表达的患者显著缩短；4.肝细胞癌患者癌组织中UHRF1 mRNA高表达提示预后不良。第二章UHRF1蛋白在肝细胞癌中的表达目的探索UHRF1蛋白在肝细胞癌组织中的表达水平及其与肝细胞癌生物学特征和肝细胞癌患者预后的关系。方法1.实验组织标本的获取：组织标本来自于2010年11月至2014年11月期间在南方医科大学南方医院肝胆外科接受根治性手术治疗的102例经病理证实的肝细胞癌患者,所有患者术前血清学检查均提示有乙型肝炎病毒感染；2.免疫组织化学染色检测’UHRF1蛋白在肝细胞癌组织及非癌组织中的表达情况;3.采用统计学方法分析肝细胞癌患者癌组织中UHRF1蛋白阳性表达与临床病理参数及预后的关系。结果1.在102例肝细胞癌患者中,癌组织中UHRF1蛋白阳性表达率为57.8%；而在非癌组织中为32.7%,进一步分析表明,肝细胞癌组织中UHRF1蛋白的阳性表达率显著高于非癌组织(P0.05)。2.经统计分析,肝细胞癌组织中UHRF1蛋白阳性表达与肝细胞癌患者的肿瘤大小、病理分化程度、微癌栓、TNM分期、术前转移、术后复发及术后无瘤生存时间等病理参数相关(P0.05)；3. Kaplan-Meier生存分析表明：UHRF1蛋白阳性表达组肝细胞癌患者的术后无瘤生存时间显著缩短(P0.05)结论1. UHRF1蛋白在肝细胞癌组织中的阳性表达率显著高于非癌组织；2. UHRF1蛋白阳性表达与肝细胞癌患者肿瘤的大小、病理分化程度、微癌栓、TNM分期、术前转移、术后复发及术后无瘤生存时间等病理参数相关；3. UHRF1蛋白阳性表达的肝细胞癌患者术后无瘤生存时间较阴性表达的患者显著缩短；4.肝细胞癌患者癌组织UHRF1蛋白阳性表达可能提示预后不良。第三章UHRF1在肝细胞癌中作用的体外研究目的研究UHRF1表达敲低后肝癌细胞的增殖、迁移及侵袭能力以及细胞周期和凋亡等的变化：同时探讨mTOR抑制剂AZD2014对肝癌细胞中UHRF1表达的影响。方法1.实时荧光定量PCR检测UHRF1 mRNA在不同肝癌细胞株及L02中的表达情况；2. siRNA特异性敲低肝癌细胞HepG2及HCCLM3中UHRF1的表达；3.CCK-8法检测UHRF1表达敲低后肝癌细胞增殖能力的变化；4.流式细胞术和Western Blot检钡UHRF1表达敲低后肝癌细胞中细胞周期及细胞周期相关蛋白表达的变化；5.流式细胞术检测UHRF1表达敲低后肝癌细胞中细胞凋亡的变化;6. Transwell法检测UHRF1表达敲低后肝癌细胞迁移及侵袭能力的变化,Western Blot检测肝癌细胞中EMT相关蛋白表达的变化；7.实时荧光定量PCR及Western Blot检测]mTOR抑制剂AZD2014对肝癌细胞HCCLM3中UHRF1表达的影响。结果1. UHRF1 mRNA在肝癌细胞株中的表达水平较L02高,且UHRF1 mRNA在HepG2、HCCLM3及Hep3B中的表达显著高于L02(P0.05)；2.实时荧光定量PCR及Western Blot结果显示我们选取的的两条siRNA可显著敲低HepG2和HCCLM3细胞中UHRF1的表达；3. UHRF1表达敲低后可以显著抑制HepG2和HCCLM3细胞的增殖能力(P0.05);4.UHRF1表达敲低后,HepG2和HCCLM3细胞中的G2/M期细胞比例显著升高(P0.05),G1期细胞比例明显降低(P0.05),S期细胞比例无明显变化；Western Blot检测结果显示：UHRF1表达敲低后,cyclinB1表达上调,cyclinD1表达下调；5. Annexin V-FITC/PI流式检测显示,UHRF1表达敲低后,HepG2和HCCLM3细胞中各组间出现凋亡的细胞数无明显区别；6. Transwell迁移及侵袭实验发现,UHRF1表达敲低后,HepG2和HCCLM3细胞穿过Transwell小室的细胞数较对照组均显著减少(P0.05)；Western Blot结果显示：UHRF1表达敲低后,HepG2和HCCLM3细胞中上皮表型标志物E-cadherin表达上调,而间质表型标志物N-cadherin、β-cateni、Vimenti、Slug和Snail表达下调；7.实时荧光定量PCR及Western Blot结果显示,mTOR抑制剂AZD2014可以降低HCCLM3细胞中UHRF1的表达水平。结论1.敲低肝癌细胞HepG2和HCCLM3中UHRF1的表达可显著抑制细胞的增殖、迁移和侵袭能力,诱导细胞出现G2/M期阻滞,抑制EMT进程；2. UHRF1可能是mTOR抑制剂AZD2014发挥抗肿瘤作用的药物靶点。第四章UHRF1在肝细胞癌中作用的体内研究目的研究UHRF1表达敲低后对肝癌细胞在体内的生物学功能的影响,并进一步探讨UHRF1在肝细胞癌发生、发展中的作用。方法1.采用腺病毒介导的shRNA稳定敲低肝癌细胞HCCLM3中UHRF1的表达；2.将稳定转染sh-NC和sh-UHRF1的HCCLM3细胞接种至裸鼠皮下,建立荷瘤模型；3.采用苏木素-伊红及免疫组织化学染色对裸鼠皮下肿瘤进行进一步检测。结果1.荧光倒置显微镜下观察及流式细胞术检测均提示腺病毒介导的sh-NC和sh-UHRF1转染细胞后具有理想的转染效率,荧光定量PCR提示：腺病毒介导的sh-UHRF1可显著敲低HCCLM3中UHRF1的表达；2.裸鼠成瘤试验结果显示：裸鼠皮下均可见肿瘤形成,但sh-UHRF1组裸鼠皮下形成的肿瘤体积明显小于sh-NC组,皮下形成的肿瘤重量也显著小于sh-NC组(P0.05)；肿瘤生长曲线提示sh-UHRF1组裸鼠的肿瘤生长显著受到抑制(P0.05)；荧光定量PCR结果显示sh-UHRF1组瘤块中UHRF1 mRNA的表达量也显著低于sh-NC组(P0.05);3.苏木素-伊红染色显示：sh-NC组和sh-UHRF1组裸鼠皮下形成的肿瘤均可见明显的异型细胞；免疫组织化学染色结果提示：sh-UHRF1组裸鼠皮下肿瘤中UHRF1、Ki-67、N-cadherin和Vimentin的表达较sh-NC组降低,E-cadherin的表达升高。结论UHRF1表达敲低后可显著抑制肿瘤的生长和EMT进程。
[Abstract]:Background and objective primary liver cancer (Primary carcinoma of the liver) is one of the most common malignant tumors in the world. The latest report shows that the annual incidence of primary liver cancer in China is 26.39/10 million, accounting for fourth of the malignant tumor incidence, the mortality rate is 23.93/10 million, and the mortality rate of malignant tumor is second. The histological type can be divided into hepatocyte type, bile duct cell type and mixed type, among which hepatocyte type is the most common, about 90% of primary liver cancer, also known as Hepatocellular carcinoma (HCC). In China, the main disease of hepatocellular carcinoma is hepatitis B virus infection. Our previous study showed that more than 85% of liver cells were thin. In recent years, the clinical application of various tumor markers and advances in imaging techniques, especially the alpha fetoprotein (AFP) and ultrasound imaging, have been used to monitor the high risk population of liver cancer in recent years, so that the liver cancer can be diagnosed in subclinical stage without obvious symptoms or signs, plus The development of surgical techniques, as well as the development of non-surgical methods, such as local treatment, has improved the overall prognosis of the patients with liver cancer than in the past, but it is still unsatisfactory. The biological characteristics of high recurrence and high metastasis of the liver cancer show that the recurrence rate of the liver cancer patients within five years after the radical resection is high. The recurrence rate of such a high 50-70%. may be due to the presence of the micrometastases that are not yet detected by the medical treatment before the operation. The independent risk factors for the free survival time of the patients. There is a lack of typical symptoms in the early stage of the liver cancer, and the liver cancer is often advanced in the case of obvious clinical manifestation. Therefore, early detection, early diagnosis and early treatment are the key to the prevention and treatment of liver cancer. As a result, liver cancer is no exception. Molecular therapy has also emerged as molecular biology advances. In recent years, scientists have discovered a number of molecular markers related to the prognosis of patients with liver cancer. These results have given us a new understanding of the pathogenesis and treatment of liver cancer, and for the evaluation of the prognosis of the liver cancer patients and Management provides new ideas and methods. However, we are still unable to find an indicator or system that can effectively monitor liver cancer micrometastases. To this end, it is necessary to continue our efforts to identify biomarkers associated with metastasis and recurrence of liver cancer. Containing plant homologous structure and ubiquitin-like with PHD a Nd ring finger domains 1, UHRF1), also known as Np95 or ICBP90, is a newly discovered nuclear protein gene associated with the development of tumor, and is located in chromosome 19p13.3. studies indicating that UHRF1 passes through the DNA methyltransferase (DNA methyltransferase 1) and histone deacetylase (1). It is involved in the regulation of gene expression and chromatin modification (such as regulating DNA methylation and inhibiting the expression of tumor suppressor genes, etc.). At the same time, the researchers also found that UHRF1 has overexpression in various tumor tissues and has a negative correlation with the prognosis of tumor patients. In addition, the proliferation and invasion ability of the tumor cells after the knockout of UHRF1 This suggests that UHRF1 may be a new drug target to prevent tumor cell migration and invasion. However, up to now, UHRF1 has not been in a more comprehensive and in-depth study in hepatocellular carcinoma. This topic is to understand the expression of UHRF1 in hepatocellular carcinoma by fluorescence quantitative PCR and immunohistochemical staining. In addition, in order to further study the role of UHRF1 in hepatocellular carcinoma, we used RNA interference to specifically knock down the expression of UHRF1 in the hepatocellular carcinoma cell lines, and study the UHRF1 in the liver by two levels in vivo and in vitro. The role of cell carcinogenesis and development to provide a new theoretical basis for the prognosis assessment and individualized treatment of patients with hepatocellular carcinoma in the future. Chapter 1 UHRF1 mRNA expression in hepatocellular carcinoma to explore the expression of UHRF1 mRNA in hepatocellular carcinoma and its relationship with the biological characteristics of hepatocellular carcinoma and the prognosis of hepatocellular carcinoma patients Methods 1. experimental tissue specimens were obtained from 80 cases of hepatocellular carcinoma confirmed by pathology at the Department of hepatobiliary surgery of the Southern Hospital of Southern Medical University from November 2010 to November 2014. All the patients were diagnosed with hepatitis B virus infection before the preoperative serological examination; 2. real time fluorescence determination. The expression of UHRF1 mRNA in the tissues of hepatocellular carcinoma and corresponding non cancer tissues was measured by PCR. 3. the expression of UHRF1 mRNA in the cancer tissues of patients with hepatocellular carcinoma and its relationship with the clinicopathological parameters and prognosis were analyzed by statistical method. Results 1. the expression of UHRF1 mRNA in 80 cases of hepatocellular carcinoma and 54 cases of cancer tissues were expressed. The high expression rate was about 67.5% (P0.05). 2. by statistical analysis, the expression level of UHRF1 mRNA in HCC tissues was related to the tumor size, TNM staging, pathological differentiation, microcarcinoma thrombus, metastasis, postoperative recurrence and postoperative tumor free survival (P0.05), and 3. Kaplan-Meier survival points. The analysis showed that the tumor free survival time of HCC patients with high expression of UHRF1 mRNA was significantly shorter (P=0.002). Conclusion the expression of 1.UHRF1 mRNA in HCC tissues was significantly higher than that of non cancer tissues; the expression level of 2. UHRF1 mRNA in HCC tissues was associated with the size of tumor, TNM staging, microcarcinoma thrombus, and preoperative metastasis in hepatocellular carcinoma. The postoperative recurrence and postoperative tumor free survival time were related to pathological parameters. 3. UHRF1 mRNA high expression of hepatocellular carcinoma patients were significantly shorter than those with low expression of tumor free time. 4. UHRF1 mRNA expression in the carcinoma tissue of patients with hepatocellular carcinoma showed poor prognosis. The expression of second chapter UHRF1 egg white in hepatocellular carcinoma was UHRF The expression level of 1 protein in hepatocellular carcinoma and its relationship with the biological characteristics of hepatocellular carcinoma and the prognosis of hepatocellular carcinoma. Method 1. experimental tissue specimens: tissue specimens from 102 cases of radical surgery in Department of hepatobiliary surgery, Southern Hospital of Southern Medical University from November 2010 to November 2014. In all patients with hepatocellular carcinoma, all patients showed hepatitis B virus infection before operation; 2. immunohistochemical staining was used to detect the expression of UHRF1 protein in the tissues of hepatocellular carcinoma and non cancer tissues; 3. the positive expression of UHRF1 protein in the cancer tissues of patients with hepatocellular carcinoma and clinical disease were analyzed by statistical method. Results 1. of the 102 patients with hepatocellular carcinoma, the positive expression rate of UHRF1 protein was 57.8% and 32.7% in non cancer tissue. The further analysis showed that the positive rate of UHRF1 protein in the hepatocellular carcinoma tissue was significantly higher than that of non cancer tissue (P0.05).2., and the UHRF1 egg in the hepatocellular carcinoma tissue was UHRF1 The white positive expression was related to the tumor size, the degree of pathological differentiation, the microcarcinoma thrombus, TNM staging, preoperative metastasis, postoperative recurrence and postoperative tumor free survival time (P0.05). 3. Kaplan-Meier survival analysis showed that the tumor free survival time of the patients with UHRF1 protein positive expression of HCC was significantly shortened (P0.05 Conclusion the positive expression rate of 1. UHRF1 protein in hepatocellular carcinoma was significantly higher than that of non cancer tissue, and the positive expression of 2. UHRF1 protein was related to the size of tumor, the degree of pathological differentiation, the microcarcinoma thrombus, TNM staging, preoperative metastasis, postoperative recurrence and postoperative tumor free survival. The positive expression of 3. UHRF1 protein was positive. The tumor free survival time of the patients with hepatocellular carcinoma was significantly shorter than those with negative expression; 4. the positive expression of UHRF1 protein in the carcinoma tissue of the patients with hepatocellular carcinoma may indicate poor prognosis. The purpose of the third chapter in the study of hepatocellular carcinoma in vitro is to study the proliferation, migration and invasion ability of hepatoma cells and the cells after UHRF1 knockout. Changes in cycle and apoptosis, and the effect of mTOR inhibitor AZD2014 on the expression of UHRF1 in hepatoma cells. Method 1. real-time quantitative PCR PCR was used to detect the expression of UHRF1 mRNA in different hepatocellular carcinoma cell lines and L02; the expression of UHRF1 in the HepG2 and HCCLM3 in the 2. siRNA specific knockout of hepatoma cells; The changes in the proliferation ability of HCC cells, the changes in cell cycle and cell cycle related protein expression in hepatoma cells after 4. flow cytometry and Western Blot examination of barium UHRF1, and 5. flow cytometry to detect the changes of apoptosis in the hepatoma cells after the UHRF1 expression was knocked low; and the 6. Transwell method was used to detect the liver cancer after the UHRF1 knockout. Changes in cell migration and invasion ability, Western Blot detection of EMT related protein expression in hepatoma cells; 7. real time fluorescence quantitative PCR and Western Blot to detect the effect of]mTOR inhibitor AZD2014 on the expression of UHRF1 in liver cancer cell HCCLM3. Results the expression level of 1. UHRF1 mRNA in liver cancer cell lines is higher. The expression in CLM3 and Hep3B was significantly higher than that of L02 (P0.05); 2. real-time fluorescent quantitative PCR and Western Blot results showed that our selected two siRNA could significantly knock down the UHRF1 expression in the HepG2 and HCCLM3 cells, and the 3. UHRF1 decreased after knockout. The proportion of G2/M cells in CCLM3 cells increased significantly (P0.05), the proportion of G1 cells decreased significantly (P0.05), and there was no significant change in the proportion of cells in the S phase. The Western Blot detection results showed that the cyclinB1 expression was up and the cyclinD1 expression was down after the low expression of UHRF1 expression. There was no significant difference in the number of apoptotic cells in the cells. 6. Transwell migration and invasion experiments found that after UHRF1 knockout, the number of HepG2 and HCCLM3 cells passing through the Transwell cells decreased significantly (P0.05). The Western Blot results showed that the epithelial phenotype in HepG2 and HCCLM3 cells after the UHRF1 table was low. The expression of E-cadherin was up and the expression of interstitial phenotypes N-cadherin, beta -cateni, Vimenti, Slug and Snail were down regulated. 7. real time fluorescent quantitative PCR and Western Blot results showed that mTOR inhibitor AZD2014 could reduce the expression level in HCCLM3 cells. Conclusion 1. knockout low liver cancer cells and the expression of Western are significantly inhibited. The proliferation, migration and invasion of cells, inducing G2/M phase block and inhibiting EMT process; 2. UHRF1 may be a drug target for anti-tumor effect of mTOR inhibitor AZD2014. In vivo study on the role of UHRF1 in hepatocellular carcinoma in vivo And further explore the role of UHRF1 in the development of hepatocellular carcinoma (HCC). Method 1. the expression of UHRF1 in HCCLM3 cells was stabilized by adenovirus mediated shRNA; 2. the HCCLM3 cells transfected with sh-NC and sh-UHRF1 were inoculated subcutaneously into nude mice, and the tumor bearing model was established; 3. using hematoxylin eosin and immunohistochemical staining. Further detection of subcutaneous tumor in nude mice. Results 1. fluorescence inverted microscope observation and flow cytometry showed that adenovirus mediated sh-NC and sh-UHRF1 transfected cells had ideal transfection efficiency. Fluorescence quantitative PCR suggested that adenovirus mediated sh-UHRF1 could significantly reduce the expression of UHRF1 in HCCLM3, and 2. nude mice were tumorigenic test. The results showed that tumor formation was observed in the subcutaneous tissue of nude mice, but the volume of tumor subcutaneously formed in nude mice in group sh-UHRF1 was significantly smaller than that in group sh-NC.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

【相似文献】