本文選題：ZNF307 + 肝細(xì)胞癌　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景及目的原發(fā)性肝細(xì)胞癌(hepatocellular carcinoma,HCC;簡(jiǎn)稱肝癌)是全球發(fā)病率最高的幾大惡性腫瘤之一,其死亡率位居全球惡性腫瘤死亡的第三位。我國是肝癌最高發(fā)的國家,據(jù)統(tǒng)計(jì),2012年全世界約有782,500例新發(fā)肝癌病例和745,500例肝癌死亡病例,中國占了新發(fā)病例和死亡總數(shù)的50%。近年來,雖然肝癌的治療手段不斷更新(包括手術(shù)、肝移植術(shù)、放射治療、介入治療、射頻消融治療、靶向治療等),但是肝癌的總體療效并無明顯提高,復(fù)發(fā)和轉(zhuǎn)移仍是影響肝癌預(yù)后的主要因素。因此,闡明肝癌侵襲轉(zhuǎn)移的機(jī)制,尋找有意義的肝癌侵襲轉(zhuǎn)移分子標(biāo)志物,發(fā)現(xiàn)新的防治靶點(diǎn),是當(dāng)代肝癌研究的重要內(nèi)容。鋅指蛋白家族蛋白是真核細(xì)胞中最常見的轉(zhuǎn)錄因子家族之一,人類基因組有超過三千種的鋅指蛋白。鋅指蛋白功能廣泛,包括DNA識(shí)別,RNA組裝,轉(zhuǎn)錄激活和凋亡調(diào)控等。由于其功能的多樣性,一些鋅指蛋白被發(fā)現(xiàn)在腫瘤的發(fā)生發(fā)展中起到抑癌或者促癌的作用。鋅指蛋白307(Zinc finger protein 307,ZNF307),又名是 ZKSCAN4(zinc finger with KRAB and SCAN domains 4),ZSCAN36andZNF427,是一個(gè)鋅指蛋白基因,最初是在人類胚胎心臟文庫中通過PCR擴(kuò)增獲得。ZNF307 cDNA分布于6號(hào)染色體上NT_007592基因組序列中,包含一個(gè)1638bp的開放閱讀框,編碼546個(gè)氨基酸組成的蛋白。ZNF307作為鋅指蛋白轉(zhuǎn)錄因子中的重要成員之一,可能與細(xì)胞的增殖、調(diào)亡、腫瘤的發(fā)生發(fā)展等密切相關(guān)。JingLi等發(fā)現(xiàn),在細(xì)胞HEK-293中ZNF307通過上調(diào) MDM2(p53-binding protein MDM2)和 EP300(E1 A binding protein p300)從而抑制p53和p21的轉(zhuǎn)錄。然而關(guān)于ZNF307在體內(nèi)是否具有轉(zhuǎn)錄抑制的功能,在肝癌的發(fā)生發(fā)展中又起到什么作用,目前尚未有研究報(bào)道。因此,本課題的研究目的:探討ZNF307在肝癌的發(fā)生發(fā)展中所扮演的角色,為肝癌的診斷、治療提供新的理論依據(jù)。研究方法1、熒光定量PCR檢測(cè)肝癌細(xì)胞株和組織標(biāo)本中ZNF307的表達(dá)在正常人肝細(xì)胞株L02和肝癌細(xì)胞株MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042 中提取細(xì)胞 mRNA,熒光定量 PCR檢測(cè)ZNF307的表達(dá)水平。提取33例肝癌及配對(duì)癌旁組織標(biāo)本的mRNA,熒光定量PCR檢測(cè)組織標(biāo)本中ZNF307的表達(dá)水平。2、Western blot檢測(cè)肝癌細(xì)胞株和組織中ZNF307的表達(dá)在正常人肝細(xì)胞株L02和肝癌細(xì)胞株MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042 中提取細(xì)胞總蛋白,Western blot 檢測(cè)上述8株細(xì)胞中ZNF307的表達(dá)水平。Western blot檢測(cè)4對(duì)肝癌及癌旁組織標(biāo)本中ZNF307的表達(dá)水平。3、構(gòu)建ZNF307敲低細(xì)胞株并驗(yàn)證轉(zhuǎn)染效率利用購買自上海吉瑪公司的含有ZNF307基因干擾片段的shRNA,采用穩(wěn)定轉(zhuǎn)染法轉(zhuǎn)染相對(duì)高表達(dá)ZNF307的肝癌細(xì)胞,用含空載體的病毒做對(duì)照。熒光定量PCR和Western blot檢測(cè)ZNF307干擾效率。4、構(gòu)建ZNF307過表達(dá)細(xì)胞株并驗(yàn)證轉(zhuǎn)染效率利用購買自上海吉瑪生物公司的含有ZNF307基因片段的Lentivirus病毒載體轉(zhuǎn)染相對(duì)低表達(dá)ZNF307基因的肝癌細(xì)胞,用含空載體的病毒做對(duì)照。熒光定量PCR和Western blot檢測(cè)ZNF307過表達(dá)效率。5、ZNF307基因沉默對(duì)肝癌細(xì)胞生物學(xué)特性的影響利用平板克隆、CCK-8、劃痕實(shí)驗(yàn)、體外侵襲實(shí)驗(yàn)、流式凋亡實(shí)驗(yàn)檢測(cè)ZNF307基因沉默后對(duì)肝癌細(xì)胞體外增殖、遷移、侵襲、凋亡的影響。6、ZNF307基因過表達(dá)對(duì)肝癌細(xì)胞生物學(xué)特性的影響利用平板克隆、CCK-8、劃痕實(shí)驗(yàn)、體外侵襲實(shí)驗(yàn)、流式凋亡實(shí)驗(yàn)檢測(cè)ZNF307基因過表達(dá)后對(duì)肝癌細(xì)胞體外增殖、遷移、侵襲、凋亡的影響。7、體內(nèi)實(shí)驗(yàn)檢測(cè)ZNF307基因沉默和過表達(dá)對(duì)肝癌細(xì)胞增殖能力的影響應(yīng)用上述構(gòu)建的穩(wěn)定干擾肝癌細(xì)胞株MHCC97L和穩(wěn)定過表達(dá)肝癌細(xì)胞株Be17402,與對(duì)照組細(xì)胞分別在裸鼠左右腋下行皮下注射,每5天對(duì)裸鼠皮下瘤進(jìn)行測(cè)量及數(shù)據(jù)分析,體內(nèi)實(shí)驗(yàn)揭示ZNF307基因沉默和過表達(dá)對(duì)肝癌細(xì)胞增殖能力的影響。8、Western blot檢測(cè)ZNF307基因沉默和過表達(dá)的肝癌細(xì)胞的凋亡相關(guān)蛋白表達(dá)的變化Western blot檢測(cè)ZNF307基因穩(wěn)定沉默和過表達(dá)的肝癌細(xì)胞的凋亡相關(guān)蛋白(Caspase3、BAX和BCL-2)的表達(dá)情況,探討ZNF307基因影響肝癌細(xì)胞凋亡的可能機(jī)制。研究結(jié)果1、ZNF307在肝癌細(xì)胞株中mRNA及蛋白水平表達(dá)下調(diào)采用熒光定量 PCR 法檢測(cè) MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042共7株人肝癌細(xì)胞和1株人正常肝細(xì)胞L02 ZNF307基因的表達(dá)情況。結(jié)果顯示ZNF307在正常肝癌細(xì)胞株L02中表達(dá)明顯高于 MHCC97L,HCCLM3,Huh7,Be17402 細(xì)胞(由高到低)(P0.05,P0.05,P0.05,P0.05),與 QGY7701,QGY7703 和 Be17404 細(xì)胞無明顯差異,提示ZNF307基因在肝癌細(xì)胞中普遍低表達(dá)。用Western blot 檢測(cè) MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be174042共7株人肝癌細(xì)胞和1株人正常肝細(xì)胞L02 ZNF307蛋白的內(nèi)源性表達(dá),結(jié)果顯示ZNF307蛋白在正常肝癌細(xì)胞株L02中表達(dá)明顯高于MHCC97L,HCCLM3,Huh7,Be17402 細(xì)胞(由高到低),與 QGY7701,QGY7703和Be17404細(xì)胞無明顯差異,提示ZNF307蛋白在肝癌細(xì)胞中普遍低表達(dá)。Western blot檢測(cè)ZNF307蛋白表達(dá)水平與熒光定量PCR檢測(cè)的RNA表達(dá)水平趨勢(shì)基本一致。2、ZNF307在肝癌組織中mRNA及蛋白水平表達(dá)下調(diào)利用熒光定量PCR檢測(cè)33對(duì)肝細(xì)胞癌組織及癌旁正常肝組織ZNF307的mRNA水平表達(dá)。結(jié)果顯示ZNF307在肝癌組織中表達(dá)低于癌旁正常肝組織(Z=-4.404,P0.001),ZNF307在肝細(xì)胞癌組織轉(zhuǎn)錄水平下調(diào)。利用Western blot檢測(cè)4對(duì)肝細(xì)胞癌組織及癌旁正常肝組織ZNF307的蛋白水平表達(dá)。結(jié)果顯示ZNF307在肝癌組織中的表達(dá)明顯低于癌旁正常肝組織,ZNF307在肝細(xì)胞癌組織蛋白翻譯水平下調(diào)。3、ZNF307基因沉默對(duì)肝癌細(xì)胞生物學(xué)特性的影響(1)構(gòu)建ZNF307敲低細(xì)胞株并驗(yàn)證轉(zhuǎn)染效率:應(yīng)用含有ZNF307干擾片段的shRNA穩(wěn)定轉(zhuǎn)染外源性表達(dá)ZNF307較高的肝癌細(xì)胞MHCC97L和QGY7701。熒光定量PCR和Western blot分別證實(shí)干擾組較對(duì)照組ZNF307表達(dá)降低,有顯著差異(MHCC97L:t=69.640,P0.001;QGY7701:t=43.725,P0.001)。(2)ZNF307干擾后促進(jìn)肝癌細(xì)胞株體外增殖能力:CCK-8實(shí)驗(yàn)、平板克隆實(shí)驗(yàn)證明,與對(duì)照組相比,干擾ZNF307細(xì)胞體外增殖能力(MHCC97L:F=62.066,P0.001;QGY7701:F=10.525,P0.001)和克隆能力增強(qiáng)(MHCC97L:t=7.252,P0.05;QGY7701:t=10.713,P0.001)。(3)ZNF307干擾后促進(jìn)肝癌細(xì)胞株體外侵襲能力:Transwell小室檢測(cè)細(xì)胞侵襲能力,實(shí)驗(yàn)證明,與對(duì)照組相比,干擾ZNF307細(xì)胞體外侵襲能力增強(qiáng)(MHCC97L:t=13.976,P0.001;QGY7701:t=25.652,P0.001)。(4)ZNF307干擾后促進(jìn)肝癌細(xì)胞株體外遷移能力:劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力,實(shí)驗(yàn)證明,與對(duì)照組相比,干擾ZNF307細(xì)胞體外遷移能力增強(qiáng)(MHCC97L:t=22.979,P0.001;QGY7701:t=19.489,P0.001)。(5)ZNF307干擾后抑制肝癌細(xì)胞株凋亡:流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率,實(shí)驗(yàn)證明,與對(duì)照組相比,干擾ZNF307細(xì)胞凋亡率明顯降低(MHCC97L:t=8.414,P0.05;QGY7701:t=47.369,P0.001),提示 ZNF307 沉默抑制肝癌細(xì)胞凋亡。4、ZNF307基因過表達(dá)對(duì)肝癌細(xì)胞生物學(xué)特性的影響(1)構(gòu)建ZNF307過表達(dá)細(xì)胞株并驗(yàn)證轉(zhuǎn)染效率:應(yīng)用含有ZNF307-cDNA的慢病毒穩(wěn)定轉(zhuǎn)染外源性表達(dá)ZNF307較低的肝癌細(xì)胞Be17402和HCCLM3。熒光定量PCR和Western blot分別證實(shí)過表達(dá)組較對(duì)照組ZNF307表達(dá)升高,有顯著差異(Be17402:t=105.364,P0.001;HCCLM3:t=126.165,P0.001)。(2)ZNF307過表達(dá)后抑制肝癌細(xì)胞株體外增殖能力:CCK-8實(shí)驗(yàn)、平板克隆實(shí)驗(yàn)證明,與對(duì)照組相比,過表達(dá)ZNF307細(xì)胞體外增殖能力(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過表達(dá)后抑制肝癌細(xì)胞株體外侵襲能力:Transwell小室檢測(cè)細(xì)胞侵襲能力,實(shí)驗(yàn)證明,與對(duì)照組相比,過表達(dá)ZNF307細(xì)胞體外侵襲能力降低(Be17402:t=8.358,P0.05;HCCLM3:t=24.607,P0.001)。(4)ZNF307過表達(dá)后抑制肝癌細(xì)胞株體外遷移能力:劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力,實(shí)驗(yàn)證明,與對(duì)照組相比,過表達(dá)ZNF307細(xì)胞體外遷移能力降低(Be17402:t=19.176,P0.001;HCCLM3:t=6.031,P0.05)。(5)ZNF307過表達(dá)后促進(jìn)肝癌細(xì)胞株凋亡:流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率,實(shí)驗(yàn)證明,與對(duì)照組相比,過表達(dá)ZNF307細(xì)胞凋亡率明顯升高(Be17402:t=7.790,P0.05;HCCLM3:t=17.451,P0.001),提示ZNF307 過表達(dá)促進(jìn)肝癌細(xì)胞凋亡。5、ZNF307基因沉默和過表達(dá)對(duì)肝癌細(xì)胞體內(nèi)增殖的影響裸鼠皮下成瘤實(shí)驗(yàn)表明,與對(duì)照組相比,ZNF307干擾后皮下成瘤的增殖能力增強(qiáng)(F=31.378,P0.05)。相反,ZNF307過表達(dá)后皮下成瘤的增殖能力降低(F=33.629,P0.05)。6、ZNF307基因沉默和過表達(dá)對(duì)肝癌細(xì)胞的凋亡相關(guān)蛋白表達(dá)的影響Western blot 檢測(cè) MHCC97L干擾 ZNF307 和 Be17402 過表達(dá) ZNF307 后凋亡相關(guān)蛋白(Caspase3、BAX和BCL-2)的表達(dá)情況,發(fā)現(xiàn)沉默ZNF307后Caspase3和BAX表達(dá)降低,BCL-2表達(dá)升高。相反,過表達(dá)ZNF307后Caspase3和BAX表達(dá)升高,BCL-2表達(dá)降低,提示ZNF307基因可能通過調(diào)控凋亡通路相關(guān)蛋白從而促進(jìn)肝癌細(xì)胞凋亡。結(jié)論1、ZNF307基因/蛋白在人肝癌細(xì)胞/組織中的表達(dá)明顯降低。2、ZNF307基因抑制肝癌細(xì)胞體內(nèi)外增殖、侵襲、遷移及促進(jìn)肝癌細(xì)胞凋亡。3、ZNF307基因可能通過調(diào)控凋亡通路相關(guān)蛋白促進(jìn)肝癌細(xì)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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相關(guān)期刊論文 前2條

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2 ;Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells[J];Acta Pharmacologica Sinica;2006年02期

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本文編號(hào)：1881440