【作者】 赵志兰；

【导师】 程牛亮；

【作者基本信息】 山西医科大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 研究背景:大肠癌(Colorectal cancer)是严重威胁人类生存的消化系统恶性肿瘤之一,在西方国家恶性肿瘤的发病率中居第2位,在我国居常见恶性肿瘤第4位。大肠癌总的5年存活率较低,因此早期发现、早期诊断与治疗大肠癌是提高生存率的关键。传统的治疗方法对肿瘤特异性和敏感性差,副作用大,效果欠佳,近年来,由于分子生物学技术的发展,大肠癌发生发展的分子机制研究取得了突破性进展,因此探寻大肠癌的基因治疗已成为目前研究的热点。转录因子特化蛋白1(Sp1)是一种核转录因子,其在肿瘤组织和细胞中的异常表达和活化,可通过促进肿瘤相关生长因子和血管生成基因表达等机制,营造适宜肿瘤生长的局部微环境,正向调节肿瘤增殖、转移潜能和血管生成。目前研究发现转录因子Sp1的异常表达与肝癌、乳腺癌、胰腺癌、胃癌等肿瘤的发生、发展、及其预后密切相关。但Sp1基因在大肠癌中转录调控作用及其分子机制等研究工作国内尚无开展。因此探讨大肠癌中Sp1基因的表达及其对大肠癌细胞生物学行为的影响,将有助于进一步明确Sp1基因在大肠癌发生、发展中的作用及其可能机制,为大肠癌的预防和治疗提供切入点,在大肠癌的基因治疗方面有较大的应用前景。目的:1.构建针对Sp1基因的RNA干扰真核表达载体,观察其对大肠癌SW620细胞Sp1基因表达及活性的抑制作用。2.探讨Sp1基因沉默后对大肠癌SW620细胞增殖转移的影响及相关基因的表达改变情况,初步分析其发挥作用的可能机制。方法:1.设计合成针对Sp1基因的有短发夹结构的DNA序列,退火后克隆到RNA干扰表达载体pGenesil-1质粒上,构建pGenesil-1-Sp1(+)和pGenesil-1-Sp1(-)重组质粒,并进行酶切鉴定和DNA测序分析;脂质体介导转染SW620细胞后,提取总RNA及蛋白质,应用实时荧光定量PCR、Western blot检测其对Sp1 mRNA及蛋白质表达水平的影响,凝胶迁移率变动分析(EMSA)检测Sp1活性的变化。2.采用细胞计数法绘制生长曲线、MTT法、流式细胞术、细胞划痕实验分别观察Sp1下调后对SW620细胞增殖、细胞周期及凋亡和细胞迁移能力等生物学行为的影响。3.脂质体介导pGenesil-1-Sp1(+)及pGenesil-1-Sp1(-)质粒分别转染SW620细胞,实时荧光定量PCR、Western blot、免疫荧光细胞化学技术检测Sp1沉默对VEGF mRNA和蛋白表达水平的影响,EMSA检测Sp1与VEGF启动子结合活性的变化;结果:1.成功构建针对Sp1基因的RNA干扰真核表达载体,转染SW620细胞后可以抑制Sp1mRNA及蛋白质的表达。pGenesil-1-Sp1(+)-SW620组Sp1 mRNA的相对表达量在24h、48h、72h分别为0.549±0.034、0.311±0.075、0.406±0.041,抑制率分别约为42.44%、68.47%、57.70%;Sp1蛋白抑制率在48h约为56.82%;EMSA结果显示Sp1的活性明显下降,统计分析表明,组间差异有统计学意义(P<0.05)。提示转染Sp1干扰质粒可以有效地抑制SW620细胞中Sp1的表达及活性。2.生长曲线、MTT法、流式细胞术、细胞划痕实验结果表明转染Sp1干扰质粒后SW620细胞增殖迁移能力受到明显抑制,细胞周期表现为干扰实验组G0/G1期细胞比例为(67.47±2.51)%,而SW620空白组和阴性对照组G0/G1期细胞比例分别为(42.19±2.13)%、(48.52±1.86)%;实验组细胞凋亡率约为22.54%。而阴性对照组细胞凋亡率约为4.31%,两组有显著差异(P<0.05)。3.实时荧光定量PCR、Western blot和免疫荧光结果表明转染pGenesil-1-Sp1(+)质粒后,SW620细胞中VEGF mRNA和蛋白表达水平显著下调,EMSA结果显示Sp1基因沉默可降低其与VEGF的结合活性。结论:1.成功构建针对Sp1基因的RNA干扰质粒,并证实其在大肠癌SW620细胞中可下调Sp1基因的表达及活性,为利用RNA干扰技术探索Sp1基因在大肠癌中的作用奠定了基础。2.转录因子Sp1基因沉默可以有效抑制人大肠癌SW620细胞体外恶性增殖及迁移能力,促进凋亡。其作用机制与Sp1基因沉默降低其转录活性进而下调VEGF基因的表达有关,为大肠癌基因治疗提供新的思路和方法。更多还原

【Abstract】 Backgroud:Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies that seriously threaten the human’s health. In western countries, the morbidity of colorectal cancer ranks the second among the cancer-related death, which is next to lung cancer. In China, it ranks from the 4th to 6th among all the cancer-related death. Because of the 5 years survival rates of CRC was lower, it is important to improve CRC patient survival through early detection and early therapy. It is bad in tumor specificity and sensitivity with traditional ways, and it has serious side-effect and bad effect. In recent years, a breakthrough has been made on the molecular mechanisms of colorectal cancer with the development of molecular biology technology. Therefore, the gene therapy for colorectal cancer has been a research hotspot.Transcription factor special protein1 (Sp1) is a nuclear transcription factor. Its abnormal expression and activation in the tumor tissues and cells. Sp1 can improve the mechanisms of tumor-associated growth factor gene expression and angiogenesis which can create the suitable micro-environment for the tumor growth and regulate the proliferation, metastasis potential and angiogenesis of tumor. The current study showed that transcription factor Sp1 was abnormal expression in liver cancer, breast cancer, pancreatic cancer, gastric cancer and other tumor. It is closely related to their occurrence, development, and prognosis of tumor. Howerve, in our country, there is no report on Sp1 expression and its function in CRC. Thus, the research of Sp1 gene expression and biological function in CRC could contribute to clarify the role of Sp1 in colorectal carcinogenesis,which maybe beneficial to the diagnosis and treatment of CRC. It has great application prospects in the Cancer gene therapy.Objective:1. To construct RNA inference eukaryotic expression vectors targeting to transcription factor special protein 1(Sp1) gene and investigate its inhibitory effects on Sp1 expression and its activity in SW620 cell line.2. To study the effects of down-regulation of Sp1 on biological behaviors of colorectal cancer SW620 cells and detect the states of the related proliferation genes after transfected recombinant plasmid.Methods:1. Sense and antisense oligonucleotides targeting to transcription factor special protein 1(Sp1) gene were designed and synthesized. After annealing, it was inserted into pGenesil-1 vector, the recombined plasmid was identified by enzyme digestion and DNA sequence analysis. The mRNA and protein levels of Sp1 was detected by real time PCR and Western blot respectively after recombinants transfected into SW620 cell.The transcriptional activity of Sp1 was analyzed by EMSA after 48h transfection.2. The effects of down-regulation Sp1 on cell proliferation, cell cycle and apoptosis, migration were assayed by the cell growth curve, MTT, flow cytometry (FCM) and scratch method respectively.3. The recombinant plasmid of Sp1 RNAi was transfected into SW620 cells by Lipofectamin, The mRNA and protein expression levels of VEGF were measured by real time PCR、Western blotting and Immunofluoresence staining respectively. Nuclear factors binding to the VEGF promoter were analyzed by electrophoretic mobility shift assay (EMSA).Results:1. The expression plasmid against Sp1 was successfully constructed. Recombinant vectors could reduce the expressions of Sp1 mRNA and protein in SW620 cell, the relative expression of Sp1 mRNA were 0.549±0.034、0.311±0.075、0.406±0.041 in 24h, 48h, and 72h. the ratio of inhibition were 42.44%、68.47%、57.70% respectively, the ratio of inhibition against the expression of Sp1 protein was 56.82%in 48th hour. EMSA in our study showed that the transcriptional activity of Sp1 was decreased after 48h transfection. Compared with control group, the difference was significant (P<0.05). RNAi expression vectors can effectively inhibit the expression and activity of Sp1 in SW620 cell line.2. The proliferation and migration ability of transfected cells was inhibited which was detected by the cell growth curve, MTT analysis, flow cytometry (FCM) and scratch method respectively. Cell proportion of G0/G1 phase were (67.47±2.51)%、(42.19±2.13)% and (48.52±1.86)% in experimental group, negative controlled group and blank cell group, The apoptosis rates of SW620 cells was 22.54% and 4.31% in experimental group and controlled group respectively, the difference was significant (P<0.05).3. The expression of VEGF in the transfected cells was significantly down-regulated at both mRNA and protein levels(P<0.05). The capacity of nuclear factors Sp1 binding to the VEGF promoter was decreased.Conclusion:1. RNA interference expression vector of targeting to Sp1is successfully constructed and it can inhibite the expression and activity of Sp1, it prepared for exploring the function of Sp1 gene in CRC with RNA interference(RNAi) technique. 2. Silencing the Sp1 expression by RNAi could inhibit the proliferation and promote apoptosis of SW620 cells, which was probably related with Down-regulating the expression of VEGF due to the decrease of its transcriptional activity. It may provide a novel therapeutic approach for color cancer therapy.更多还原