锌指蛋白307在肝细胞癌发生发展中的初步功能研究

发布时间：2018-05-13 03:23

本文选题：ZNF307 + 肝细胞癌　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景及目的原发性肝细胞癌(hepatocellular carcinoma,HCC;简称肝癌)是全球发病率最高的几大恶性肿瘤之一,其死亡率位居全球恶性肿瘤死亡的第三位。我国是肝癌最高发的国家,据统计,2012年全世界约有782,500例新发肝癌病例和745,500例肝癌死亡病例,中国占了新发病例和死亡总数的50%。近年来,虽然肝癌的治疗手段不断更新(包括手术、肝移植术、放射治疗、介入治疗、射频消融治疗、靶向治疗等),但是肝癌的总体疗效并无明显提高,复发和转移仍是影响肝癌预后的主要因素。因此,阐明肝癌侵袭转移的机制,寻找有意义的肝癌侵袭转移分子标志物,发现新的防治靶点,是当代肝癌研究的重要内容。锌指蛋白家族蛋白是真核细胞中最常见的转录因子家族之一,人类基因组有超过三千种的锌指蛋白。锌指蛋白功能广泛,包括DNA识别,RNA组装,转录激活和凋亡调控等。由于其功能的多样性,一些锌指蛋白被发现在肿瘤的发生发展中起到抑癌或者促癌的作用。锌指蛋白307(Zinc finger protein 307,ZNF307),又名是 ZKSCAN4(zinc finger with KRAB and SCAN domains 4),ZSCAN36andZNF427,是一个锌指蛋白基因,最初是在人类胚胎心脏文库中通过PCR扩增获得。ZNF307 cDNA分布于6号染色体上NT_007592基因组序列中,包含一个1638bp的开放阅读框,编码546个氨基酸组成的蛋白。ZNF307作为锌指蛋白转录因子中的重要成员之一,可能与细胞的增殖、调亡、肿瘤的发生发展等密切相关。JingLi等发现,在细胞HEK-293中ZNF307通过上调 MDM2(p53-binding protein MDM2)和 EP300(E1 A binding protein p300)从而抑制p53和p21的转录。然而关于ZNF307在体内是否具有转录抑制的功能,在肝癌的发生发展中又起到什么作用,目前尚未有研究报道。因此,本课题的研究目的:探讨ZNF307在肝癌的发生发展中所扮演的角色,为肝癌的诊断、治疗提供新的理论依据。研究方法1、荧光定量PCR检测肝癌细胞株和组织标本中ZNF307的表达在正常人肝细胞株L02和肝癌细胞株MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042 中提取细胞 mRNA,荧光定量 PCR检测ZNF307的表达水平。提取33例肝癌及配对癌旁组织标本的mRNA,荧光定量PCR检测组织标本中ZNF307的表达水平。2、Western blot检测肝癌细胞株和组织中ZNF307的表达在正常人肝细胞株L02和肝癌细胞株MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042 中提取细胞总蛋白,Western blot 检测上述8株细胞中ZNF307的表达水平。Western blot检测4对肝癌及癌旁组织标本中ZNF307的表达水平。3、构建ZNF307敲低细胞株并验证转染效率利用购买自上海吉玛公司的含有ZNF307基因干扰片段的shRNA,采用稳定转染法转染相对高表达ZNF307的肝癌细胞,用含空载体的病毒做对照。荧光定量PCR和Western blot检测ZNF307干扰效率。4、构建ZNF307过表达细胞株并验证转染效率利用购买自上海吉玛生物公司的含有ZNF307基因片段的Lentivirus病毒载体转染相对低表达ZNF307基因的肝癌细胞,用含空载体的病毒做对照。荧光定量PCR和Western blot检测ZNF307过表达效率。5、ZNF307基因沉默对肝癌细胞生物学特性的影响利用平板克隆、CCK-8、划痕实验、体外侵袭实验、流式凋亡实验检测ZNF307基因沉默后对肝癌细胞体外增殖、迁移、侵袭、凋亡的影响。6、ZNF307基因过表达对肝癌细胞生物学特性的影响利用平板克隆、CCK-8、划痕实验、体外侵袭实验、流式凋亡实验检测ZNF307基因过表达后对肝癌细胞体外增殖、迁移、侵袭、凋亡的影响。7、体内实验检测ZNF307基因沉默和过表达对肝癌细胞增殖能力的影响应用上述构建的稳定干扰肝癌细胞株MHCC97L和稳定过表达肝癌细胞株Be17402,与对照组细胞分别在裸鼠左右腋下行皮下注射,每5天对裸鼠皮下瘤进行测量及数据分析,体内实验揭示ZNF307基因沉默和过表达对肝癌细胞增殖能力的影响。8、Western blot检测ZNF307基因沉默和过表达的肝癌细胞的凋亡相关蛋白表达的变化Western blot检测ZNF307基因稳定沉默和过表达的肝癌细胞的凋亡相关蛋白(Caspase3、BAX和BCL-2)的表达情况,探讨ZNF307基因影响肝癌细胞凋亡的可能机制。研究结果1、ZNF307在肝癌细胞株中mRNA及蛋白水平表达下调采用荧光定量 PCR 法检测 MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042共7株人肝癌细胞和1株人正常肝细胞L02 ZNF307基因的表达情况。结果显示ZNF307在正常肝癌细胞株L02中表达明显高于 MHCC97L,HCCLM3,Huh7,Be17402 细胞(由高到低)(P0.05,P0.05,P0.05,P0.05),与 QGY7701,QGY7703 和 Be17404 细胞无明显差异,提示ZNF307基因在肝癌细胞中普遍低表达。用Western blot 检测 MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042共7株人肝癌细胞和1株人正常肝细胞L02 ZNF307蛋白的内源性表达,结果显示ZNF307蛋白在正常肝癌细胞株L02中表达明显高于MHCC97L,HCCLM3,Huh7,Be17402 细胞(由高到低),与 QGY7701,QGY7703和Be17404细胞无明显差异,提示ZNF307蛋白在肝癌细胞中普遍低表达。Western blot检测ZNF307蛋白表达水平与荧光定量PCR检测的RNA表达水平趋势基本一致。2、ZNF307在肝癌组织中mRNA及蛋白水平表达下调利用荧光定量PCR检测33对肝细胞癌组织及癌旁正常肝组织ZNF307的mRNA水平表达。结果显示ZNF307在肝癌组织中表达低于癌旁正常肝组织(Z=-4.404,P0.001),ZNF307在肝细胞癌组织转录水平下调。利用Western blot检测4对肝细胞癌组织及癌旁正常肝组织ZNF307的蛋白水平表达。结果显示ZNF307在肝癌组织中的表达明显低于癌旁正常肝组织,ZNF307在肝细胞癌组织蛋白翻译水平下调。3、ZNF307基因沉默对肝癌细胞生物学特性的影响(1)构建ZNF307敲低细胞株并验证转染效率:应用含有ZNF307干扰片段的shRNA稳定转染外源性表达ZNF307较高的肝癌细胞MHCC97L和QGY7701。荧光定量PCR和Western blot分别证实干扰组较对照组ZNF307表达降低,有显著差异(MHCC97L:t=69.640,P0.001;QGY7701:t=43.725,P0.001)。(2)ZNF307干扰后促进肝癌细胞株体外增殖能力:CCK-8实验、平板克隆实验证明,与对照组相比,干扰ZNF307细胞体外增殖能力(MHCC97L:F=62.066,P0.001;QGY7701:F=10.525,P0.001)和克隆能力增强(MHCC97L:t=7.252,P0.05;QGY7701:t=10.713,P0.001)。(3)ZNF307干扰后促进肝癌细胞株体外侵袭能力:Transwell小室检测细胞侵袭能力,实验证明,与对照组相比,干扰ZNF307细胞体外侵袭能力增强(MHCC97L:t=13.976,P0.001;QGY7701:t=25.652,P0.001)。(4)ZNF307干扰后促进肝癌细胞株体外迁移能力:划痕实验检测细胞迁移能力,实验证明,与对照组相比,干扰ZNF307细胞体外迁移能力增强(MHCC97L:t=22.979,P0.001;QGY7701:t=19.489,P0.001)。(5)ZNF307干扰后抑制肝癌细胞株凋亡:流式细胞仪检测细胞凋亡率,实验证明,与对照组相比,干扰ZNF307细胞凋亡率明显降低(MHCC97L:t=8.414,P0.05;QGY7701:t=47.369,P0.001),提示 ZNF307 沉默抑制肝癌细胞凋亡。4、ZNF307基因过表达对肝癌细胞生物学特性的影响(1)构建ZNF307过表达细胞株并验证转染效率:应用含有ZNF307-cDNA的慢病毒稳定转染外源性表达ZNF307较低的肝癌细胞Be17402和HCCLM3。荧光定量PCR和Western blot分别证实过表达组较对照组ZNF307表达升高,有显著差异(Be17402:t=105.364,P0.001;HCCLM3:t=126.165,P0.001)。(2)ZNF307过表达后抑制肝癌细胞株体外增殖能力:CCK-8实验、平板克隆实验证明,与对照组相比,过表达ZNF307细胞体外增殖能力(Be17402:F=27.136,P0.001;HCCLM3:F=4.541,P0.05)和克隆能力降低(Be17402:t=16.670,P0.001;HCCLM3:t=10.169,P0.05)。(3)ZNF307过表达后抑制肝癌细胞株体外侵袭能力:Transwell小室检测细胞侵袭能力,实验证明,与对照组相比,过表达ZNF307细胞体外侵袭能力降低(Be17402:t=8.358,P0.05;HCCLM3:t=24.607,P0.001)。(4)ZNF307过表达后抑制肝癌细胞株体外迁移能力:划痕实验检测细胞迁移能力,实验证明,与对照组相比,过表达ZNF307细胞体外迁移能力降低(Be17402:t=19.176,P0.001;HCCLM3:t=6.031,P0.05)。(5)ZNF307过表达后促进肝癌细胞株凋亡:流式细胞仪检测细胞凋亡率,实验证明,与对照组相比,过表达ZNF307细胞凋亡率明显升高(Be17402:t=7.790,P0.05;HCCLM3:t=17.451,P0.001),提示ZNF307 过表达促进肝癌细胞凋亡。5、ZNF307基因沉默和过表达对肝癌细胞体内增殖的影响裸鼠皮下成瘤实验表明,与对照组相比,ZNF307干扰后皮下成瘤的增殖能力增强(F=31.378,P0.05)。相反,ZNF307过表达后皮下成瘤的增殖能力降低(F=33.629,P0.05)。6、ZNF307基因沉默和过表达对肝癌细胞的凋亡相关蛋白表达的影响Western blot 检测 MHCC97L干扰 ZNF307 和 Be17402 过表达 ZNF307 后凋亡相关蛋白(Caspase3、BAX和BCL-2)的表达情况,发现沉默ZNF307后Caspase3和BAX表达降低,BCL-2表达升高。相反,过表达ZNF307后Caspase3和BAX表达升高,BCL-2表达降低,提示ZNF307基因可能通过调控凋亡通路相关蛋白从而促进肝癌细胞凋亡。结论1、ZNF307基因/蛋白在人肝癌细胞/组织中的表达明显降低。2、ZNF307基因抑制肝癌细胞体内外增殖、侵袭、迁移及促进肝癌细胞凋亡。3、ZNF307基因可能通过调控凋亡通路相关蛋白促进肝癌细胞凋亡。
[Abstract]:Background and objective primary hepatocellular carcinoma (hepatocellular carcinoma, HCC, for short) is one of the most malignant tumors in the world with the highest mortality rate in the world. China is the third most fatal tumor in the world. China is the country with the highest incidence of liver cancer. According to statistics, there are about 782500 cases of new liver cancer and 745500 cases in the world in 2012. In recent years, the number of new cases of liver cancer and the total number of deaths in China have taken up 50%. in recent years. Although the treatment of liver cancer is constantly updated (including surgery, liver transplantation, radiotherapy, interventional therapy, radiofrequency ablation, targeting therapy, etc.), the overall curative effect of liver cancer is not obviously improved, and the recurrence and metastasis are still the main factors affecting the prognosis of liver cancer. Therefore, to elucidate the mechanism of invasion and metastasis of liver cancer, to find a meaningful marker for invasion and metastasis of liver cancer, and to find a new target for prevention and treatment, it is an important content of the study of liver cancer in the present time. The family of zinc finger protein family proteins is one of the most common transcription factor families in eukaryotic cells. There are more than three thousand kinds of zinc finger proteins in human genomes. A wide range of white functions, including DNA recognition, RNA assembly, transcription activation and apoptosis regulation. Due to its functional diversity, some zinc finger proteins are found to inhibit cancer or promote cancer in the occurrence and development of tumors. Zinc finger protein 307 (Zinc finger protein 307, ZNF307), also known as ZKSCAN4 (zinc finger with KRAB and 4), zinc CAN36andZNF427, a zinc finger protein gene, was initially amplified by PCR amplification in the human embryonic heart library by.ZNF307 cDNA distributed in the NT_007592 genome sequence on chromosome 6, containing an open reading frame of 1638bp and encoding a protein.ZNF307 composed of 546 amino acids as an important member of the zinc finger protein transcription factor. First, it may be associated with.JingLi, such as cell proliferation, apoptosis, and tumor development, and so on. In cell HEK-293, ZNF307 inhibits transcription and transcription by up regulation of MDM2 (p53-binding protein MDM2) and EP300 (E1 A binding protein). The purpose of this study is to explore the role of ZNF307 in the development and development of liver cancer, and to provide a new theoretical basis for the diagnosis and treatment of liver cancer. Method 1, the expression of ZNF307 in liver cancer cell lines and tissue specimens by fluorescence quantitative PCR is normal. Human hepatocyte strain L02 and hepatoma cell line MHCC97L, HCCLM3, Huh7, QGY7701, QGY7703, Be17402, and Be174042 were extracted from cell mRNA, and the expression level of ZNF307 was detected by fluorescence quantitative PCR. The expression level of 33 cases of liver cancer and paracancerous tissue specimens was extracted. The expression of ZNF307 in normal human liver cell line L02 and liver cancer cell line MHCC97L, HCCLM3, Huh7, QGY7701, QGY7703, Be17402, Be174042 were extracted from normal human liver cell lines, and Western blot detected the expression level of ZNF307 in the above 8 cells. Knock down the low cell line and verify the transfection efficiency using shRNA, which was purchased from the ZNF307 gene interference fragment of the Jima company in Shanghai, transfected the hepatocellular carcinoma cells with high expression of ZNF307 by stable transfection, and used the virus containing the empty carrier as the control. The fluorescence quantitative PCR and Western blot were used to detect the ZNF307 interference efficiency.4, and the ZNF307 overexpressed cell line was constructed. Transfection efficiency was verified by transfection of liver cancer cells with relatively low expression ZNF307 gene from the Lentivirus virus vector containing ZNF307 gene fragment containing ZNF307 gene from Jima biological Corporation, the virus containing the empty carrier was used as the control. The fluorescence quantitative PCR and Western blot were used to detect the ZNF307 overexpression efficiency.5, and the ZNF307 gene silencing was specific to the hepatoma cell biology. The effect of ZNF307 gene silencing on the proliferation, migration, invasion and apoptosis of hepatoma cells, the effects of.6 on the proliferation, migration, invasion and apoptosis of hepatoma cells, the effect of ZNF307 gene overexpression on the biological characteristics of hepatoma cells,.6, CCK-8, scratch test, invasion test in vitro, flow formula. The effect of ZNF307 gene overexpression on the proliferation, migration, invasion and apoptosis of hepatoma cells in vitro, the effect of.7 on the proliferation, invasion and apoptosis of hepatoma cells was detected. In vivo, the effects of ZNF307 gene silence and overexpression on the proliferation of hepatoma cells were detected by using the above constructed stable interfering hepatocellular carcinoma cell line MHCC97L and the stable overexpressed HCC cell line Be17402, and the control group was fine with the control group. The cells were injected subcutaneously under the armpit of nude mice, and the subcutaneous tumor of nude mice was measured and analyzed every 5 days. In vivo experiments revealed the effect of ZNF307 gene silence and overexpression on the proliferation of hepatoma cells.8. Western blot detected the changes of Western blot in the expression of apoptosis related protein of ZNF307 gene silencing and overexpressed HCC cells. To detect the expression of apoptosis related proteins (Caspase3, BAX and BCL-2) of ZNF307 gene stable and overexpressed, to explore the possible mechanism of ZNF307 gene to affect the apoptosis of hepatoma cells. Results 1, ZNF307 was used to detect MHCC97L, HCCLM3, Huh7 by the fluorescence quantitative PCR method in the expression of mRNA and protein in the liver cancer cell lines. The expression of L02 ZNF307 gene in 7 human hepatoma cells and 1 normal hepatocytes of QGY7701, QGY7703, Be17402 and Be174042. The results showed that the expression of ZNF307 in normal liver cancer cell line L02 was obviously higher than MHCC97L, HCCLM3, Huh7, Be17402 cells (from high to low). The expression of ZNF307 gene was generally low in the hepatoma cells. The endogenous expression of MHCC97L, HCCLM3, Huh7, QGY7701, QGY7703, Be17402, Be174042, 7 human hepatoma cells and 1 normal hepatocytes was detected by Western blot. The results showed that the expression of the protein was significantly higher than that of normal hepatoma cell lines. LM3, Huh7, Be17402 cells (from high to low), not significantly different from QGY7701, QGY7703 and Be17404 cells, suggesting that ZNF307 protein is generally low expression of.Western blot detection ZNF307 protein expression level and RNA expression level is basically consistent with fluorescence quantitative PCR detection. The expression of mRNA in 33 hepatocellular carcinoma tissues and normal liver tissue ZNF307 was detected by fluorescence quantitative PCR. The results showed that the expression of ZNF307 was lower than normal liver tissue (Z=-4.404, P0.001) in the hepatocellular carcinoma tissue (Z=-4.404, P0.001), and the transcription level of ZNF307 in the hepatocellular carcinoma tissue was down regulated. The use of Western blot detected 4 for hepatocellular carcinoma tissue and normal para cancer. The expression of ZNF307 protein in liver tissue. The results showed that the expression of ZNF307 in liver cancer tissue was significantly lower than that of normal liver tissue, ZNF307 was down regulated by.3, and the effect of ZNF307 gene silencing on the biological characteristics of hepatoma cells (1) construction of ZNF307 knockout low cell lines and validation of transfection efficiency: the application containing ZNF307 ShRNA stable transfection with exogenous expression of exogenous ZNF307, MHCC97L and QGY7701. fluorescence quantitative PCR and Western blot respectively confirmed that the ZNF307 expression in the interference group was lower than that of the control group (MHCC97L:t=69.640, P0.001; QGY7701:t=43.725, P0.001). (2) the proliferation of hepatocellular carcinoma cell lines was promoted after interference. K-8 experiments, plate cloning experiments showed that compared with the control group, interference in vitro proliferation of ZNF307 cells (MHCC97L:F=62.066, P0.001; QGY7701:F=10.525, P0.001) and the enhancement of cloning ability (MHCC97L:t=7.252, P0.05; QGY7701:t=10.713, P0.001). (3) ZNF307 interference promotes the invasiveness of hepatocellular carcinoma cells in vitro: cell invasion by Transwell cells Ability, the experiment proved that the invasion ability of ZNF307 cells in vitro was enhanced (MHCC97L:t=13.976, P0.001; QGY7701:t=25.652, P0.001). (4) ZNF307 interference promoted the ability of cell migration in vitro after ZNF307 interference: the scratch test was used to detect cell migration ability, and it was proved that the migration ability of ZNF307 cells in vitro was increased compared with the control group. Strong (MHCC97L:t=22.979, P0.001; QGY7701:t=19.489, P0.001). (5) ZNF307 interference inhibited the apoptosis of liver cancer cell line: flow cytometry was used to detect the apoptosis rate. The experiment proved that the apoptosis rate of ZNF307 cells decreased significantly (MHCC97L:t=8.414, P0.05; QGY7701:t=47.369, P0.001) compared with the control group (MHCC97L:t=8.414, P0.05; QGY7701:t=47.369, P0.001), suggesting that ZNF307 silence inhibited the apoptosis of hepatoma cells. The effect of.4, ZNF307 gene overexpression on the biological characteristics of hepatoma cells (1) construction of ZNF307 overexpressed cell lines and validation of transfection efficiency: the use of lentivirus stable transfection with ZNF307-cDNA, Be17402 and HCCLM3. fluorescent quantitative PCR and Western blot, respectively, confirmed that the overexpressed group was compared with the control group. There were significant differences (Be17402:t=105.364, P0.001; HCCLM3:t=126.165, P0.001). (2) ZNF307 overexpression inhibited the proliferation of hepatoma cell lines in vitro: CCK-8 experiment, and flat cloning experiments showed that the proliferation energy of ZNF307 cells in vitro (Be17402:F=27.136, P0.001; HCCLM3:F=4.541, P0.05) and the decrease of cloning ability were compared with the control group. (Be17402:t=16.670, P0.001; HCCLM3:t=10.169, P0.05). (3) ZNF307 overexpression inhibits the invasiveness of liver cancer cells in vitro: Transwell chamber to detect cell invasiveness. Experiments show that the invasion ability of overexpressed ZNF307 cells in vitro decreased (Be17402:t=8.358, P0.05; HCCLM3:t=24.607, P0.001) compared with the control group. (4) inhibition of ZNF307 after expression. In vitro migration ability of liver cancer cell line: scratch test to detect cell migration ability. Experiments showed that the ability of overexpressed ZNF307 cells in vitro migration decreased (Be17402:t=19.176, P0.001; HCCLM3:t=6.031, P0.05). (5) ZNF307 overexpression promoted the apoptosis of liver cancer cell line: flow cytometry was used to detect the apoptosis rate of cells. The experiment proved that the cell apoptosis rate was detected by flow cytometry Compared with the control group, the apoptosis rate of overexpressed ZNF307 cells was significantly increased (Be17402:t=7.790, P0.05, HCCLM3:t=17.451, P0.001), suggesting that ZNF307 overexpression promoted the apoptosis of hepatoma cells.5. The effect of ZNF307 gene silencing and overexpression on the proliferation of hepatoma cells in nude mice showed that the subcutaneous formation of ZNF307 interference was compared with the control group. The proliferation ability of the tumor was enhanced (F=31.378, P0.05). On the contrary, the proliferation ability of subcutaneous tumor after ZNF307 overexpression was reduced (F=33.629, P0.05).6. The effect of ZNF307 gene silence and overexpression on the expression of apoptosis related proteins in liver cancer cells Western blot detection MHCC97L interference ZNF307 and Be17402 overexpressed apoptosis related proteins The expression of BAX and BCL-2 showed that the expression of Caspase3 and BAX decreased and the expression of BCL-2 increased. On the contrary, the expression of Caspase3 and BAX increased after overexpression of ZNF307, and the expression of BCL-2 decreased, suggesting that ZNF307 gene might promote apoptosis of liver cancer cells by regulating apoptosis pathway related proteins. Conclusion 1, ZNF307 gene / protein is fine in human liver cancer. The expression in cell / tissue is obviously reduced by.2. ZNF307 gene inhibits the proliferation, invasion, migration and apoptosis of hepatoma cells in vivo and in vitro, and promotes the apoptosis of hepatoma cells. The ZNF307 gene may promote the apoptosis of hepatoma cells by regulating the apoptosis pathway related proteins.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

【参考文献】