【作者】 蒋鹏程；

【导师】 赵浩亮；

【作者基本信息】 山西医科大学 ， 外科学， 2013， 博士

【摘要】 背景与目的：由于与功能基因的紧密相关性以及在基因组进化过程中的重要性,假基因功能已引起了国内外学者的广泛注意。我们前期研究发现Cripto-1基因在许多恶性肿瘤中高表达,且呈现出大肠癌器官特异性肝转移的特性,但是其假基因Cripto-3(Cr-3)在大肠癌中的作用和机制尚不清楚。本研究首先观察并初步了解假基因Cripto-3在人大肠癌发生发展中的作用,并探讨Cripto-3表达对大肠癌细胞生长、克隆形成及侵袭的影响。并进一步从基质金属蛋白酶角度探讨了Cr-3调控癌细胞生物学行为的分子机制。方法：1,收集人大肠癌和正常大肠黏膜组织,采用荧光实时定量PCR方法检测组织中mRNA水平,并分析其表达水平与淋巴结转移、临床分期的相关性。培养大肠癌细胞株,采用荧光实时定量PCR方法检测各细胞株中mRNA不同表达水平,并选择Cripto-3表达最高者SW-620细胞和最低者HCT-116细胞为下一步研究对象。2,构建Cr-3真核表达质粒和Cr-3小干扰RNA(siRNA)。HCT-116细胞分为3组：空白对照组(Con-A)、空载质粒对照组(Con-B)和Cr-3过表达质粒组(Cr-3),其中,Cr-3组以Cr-3真核表达质粒转染处理。分别用MTT法和平板克隆试验检测各组癌细胞生长能力,以Transwell方法检测癌细胞侵袭能力,并作统计学处理。3,构建并用化学方法合成Cr-3siRNA。SW-620细胞分为3组：空白对照组(con-a)、空载质粒对照组con-b和Cr-3siRNA组(siRNA),其中,siRNA组以Cr-3siRNA转染处理。分别用MTT法和平板克隆试验检测各组癌细胞生长能力,以Transwell方法检测癌细胞侵袭能力,并作统计学处理。4,为进一步深入探讨Cr-3在大肠癌发生发展中的作用和分子机制,本课题组进一步采用荧光实时定量PCR方法和蛋白质印迹方法检测2、3各实验组基质金属蛋白酶家族中MMP-2, MMP-7和MMP-9基因mRNA和蛋白水平。结果：1,在政策大肠黏膜组织中,Cr-3mRNA水平极低,在大肠癌组织中,Cr-3mRNA呈高表达,统计学分析,差异十分显著(P<0.01);Cr-3mRNA高表达与癌细胞侵袭和淋巴结转移密切相关,而与组织类型无关；在4株大肠癌细胞株中,Cr-3mRNA均有不同程度的表达,其中HCT-116中Cr-3mRNA最低,SW-620细胞中最高；2,以Cr-3真核表达质粒转染Cr-3低表达者大肠癌HCT-116细胞株,MTT结果显示,72h Con-A、Con-B和Cr-3组相对增值率分别为0.761±0.026、0.764±0.034和0.848±0.008(P<0.05);平板克隆结果显示,Con-A、Con-B和Cr-3组分别为75±6、76±5和119±3(P<0.05)；Transwell结果显示,Con-A、Con-B和Cr-3组穿膜细胞数分别为444±4、45±5和76±6(P<0.05);3,以Cr-3siRNA转染大肠癌SW620细胞株后,MTT结果显示,72h con-a、con-b和siRNA组相对增殖率分别为0.927±0.037、0.922±0.035和0.663±0.018(P<0.05)；平板克隆结果显示,con-a、con-b和siRNA组分别为23±3、24±3和12±3(P<0.05)；Transwell结果显示,con-a、con-b和siRNA组穿膜细胞数分别为38±4、40±3和8±1(P<0.05)；4,荧光实时定量PCR和蛋白质印迹结果显示,与大肠癌HCT-116细胞Con-A组比较,Cr-3过表达组MMP-2、MMP-7和MMP-9mRNA和蛋白水平明显升高；,与大肠癌SW620细胞con-a组比较,siRNA转染组MMP-2、MMP-7和MMP-9mRNA和蛋白水平明显均明显下调。结论：1,假基因Cr-3在大肠癌中过表达,并与癌细胞的侵袭、转移密切相关；2, MMPs家族成员MMP-2、-7和-9在Cr-3调控大肠癌细胞侵袭和转移中发挥着重要作用；3,假基因Cr-3可能是大肠癌基因诊断和治疗中重要靶点之一,深入探讨其作用和分子机制,将有助于提高转移性大肠癌的综合诊疗水平。更多还原

【Abstract】 Background and Objective:Because of its close correlation with functional gene and the importance in process of human genome evolution, pseudogene function has attracted wide attention of scholars both at home and abroad. Our previous study found that Cripto-1(Cr-1) is highly expressed in colorectal cancer and many other malignancies and is closely related to organ-specific liver metastases. We found that the the Cripto-3(Cr-3), a pseudogene of cripto-1, is highly expressed in colorectal cancer cells, but we do not know its carcinogenesis and mechanisms. in the development of colorectal cancer. Firstly, this study observed and studied the effects of Cr-3in the development of colorectal cancer; Secondly, and the effect of Cr-3expression on the growth, colony formation and invasion of colorectal cancer cells were explored; Thirdly, from the matrix metalloproteinase aspects, the molecular mechanism of regulation of cancer biological behavior of Cr-3cells were furtherly determined.Method:1. After human colorectal normal tissues and colorectal tumoral tissues were collected, the mRNA levels of these tissues were determined by real time PCR. Furthermore, the correlations of its expression level to the lymph node metastasis and the clinical stage were analyzed. Colorectal cancer cell lines were cultivated and the different expression levels of mRNA in each cell line were detected by real time PCR,respectively. The SW-620cell line with the highest expression of Cr-3and the HCT-116cells with the lowest expression of Cr-3were selected for further study.2. Cr-3eukaryotic expression plasmid and Cr-3small interfering RNA (siRNA) were built. The HCT-116cells were classified into three groups:blank control group (Con-A), the empty plasmid group (Con-B) and Cr-3over-expression plasmid group (Cr-3). Among them, the Cr-3group was transfected with Cr-3eukaryotic expression plasmid. The cell viability of each group was measured by MTT assay and plate cloning, respectively. The invasion ability was evaluated by Transwell method.All the above were for statistical analysis.3. Cr-3siRNA were built and synthesized by using chemical methods. The SW-620cells were classified into three groups:blank control group (con-a), the empty plasmid group (con-b) and Cr-3siRNA group (siRNA). Among them, the siRNA group was transfected with Cr-3siRNA. The cell viability of each group was measured by MTT assay and plate cloning, respectively. The invasion ability was evaluated by Transwell method.All the above were for statistical analysis.4. To further explore the role and molecular mechanism of Cr-3in the development of colorectal cancer, the mRNA and protein levels of MMP-2,MMP-7,MMP-9in2,3above each experimental group were determined by real time PCR and Western blot, respectively.Result:1. The expression level of Cr-3mRNA was significantly low in normal colon mucosa and adjacent tissues, while was significantly high in colorectal tumoral tissues(P<0.01). The high expression of Cr-3mRNA was closely correlated to the cancer cell invasion and lymph node metastasis, but had nothing to do with the tissue types. In the four colorectal cancer cell lines, Cr-3mRNA was expressed in different degrees, in which the HCT-116cells in the lowest while the SW-620cells in the highest.2. After Cr-3low expression of HCT-116cell line was transfected by Cr-3eukaryotic expression plasmid, the results of the MTT assay showed that the relative proliferation rates of Con-A, Con-B and Cr-3group in72h were0.761±0.026,0.764±0.034and0.848±0.008(OD), respectively (P<0.05); the results of the plate cloning showed that the relative proliferation rates of Con-A,Con-B and Cr-3group in72h were75±6,76±5and119±3, respectively (P<0.05); the results of the Transwell showed that the membrane cell number of Con-A, Con-B and Cr-3group were44±4,45±5and76±6respectively(P<0.05).3. After colorectal cancer SW620cell line was transfected by Cr-3siRNA, the results of the MTT assay showed that the relative proliferation rates of con-a, con-b and Cr-3group in72h were0.927±0.037、0.922±0.035and0.663±0.018(OD) respectively (P<0.05); the results of the plate cloning showed that the relative proliferation rates of con-a, con-b and Cr-3group in72h were23±3、24±3and12±3respectively (P<0.05); the results of the Transwell showed that the membrane cell number of con-a, con-b and Cr-3group were38±4、40±3and8±1respectively (P<0.05).4. The results of the real time PCR and Western blot showed that, the mRNA and protein levels of MMP-2, MMP-7and MMP-9of the Cr-3over-expression group increased significantly when compared with the HCT-116Con-A group. Conversely, the mRNA and protein levels of MMP-2, MMP-7and MMP-9of the SW-620were down-regulated significantly.Conclusion:1. Pseudogene Cr-3was over-expression in colorectal cancer and was closely related to invasion and metastasis of cancer cells.2. MMPs family members:MMP-2,-7and-9might playe an important role in the Cr-3regulation of invasion and metastasis of colorectal cancer cells.3. Pseudogene Cr-3might be one of the important targets in the gene diagnosis and treatment of colorectal cancer. Exploring its role and molecular mechanism further, will help raise the level of comprehensive diagnosis and treatment of metastatic colorectal cancer.更多还原