【作者】 欧璇；

【导师】 伍欣星；

【作者基本信息】 武汉大学 ， 病原生物学， 2005， 硕士

【摘要】 宫颈癌是危害广大妇女健康的主要恶性肿瘤之一,占癌症造成妇女死亡的第二位。中国在世界上宫颈癌发病率国家中居第二位,其中湖北、陕西和江西三省为中国宫颈癌的高发区。 过去研究认为高危型人乳头瘤病毒（human papillomavirus, HPV）如HPV16、18等型的感染是宫颈癌的主要诱发因素,在90%以上的原发性宫颈癌组织中均发现存在HPV的感染。但有研究表明HPV并不能单独致癌,在体外实验中HPV需协同某些细胞癌基因才能产生转化作用,细胞内的遗传性改变可能是宫颈癌的更主要病因。因此,寻找并确定细胞内宫颈癌相关基因成为宫颈癌研究中的重点。基于此,我科室研究人员通过细胞原癌基因、抑癌基因分类表达谱芯片,分析了原发性宫颈癌细胞中癌相关基因的情况,筛选出存在差异表达的原癌和抑癌基因共11条,其中人类与鼠同源乳腺转化基因（Homo sapiens homolog of mouse MAT-1 oncogene,HMAT1）表达明显上调,极有可能与肿瘤的发生有关,而且目前尚未有其功能的相关报道,因此有必要对其功能进行研究,以确定其在肿瘤发生中的作用。 首先采用生物信息学的方法对HMAT1基因在核酸及氨基酸水平上进行了分析,并对其结构和功能做了初步预测,发现了一条与HMAT1基因同源性较高的基因:Homo sapiens PEA15 protein（PEA15）gene,它与HMAT1基因的序列相似性为99%,HMAT1基因可能与该基因有相似的功能。HMAT1基因编码的75个氨基酸中亮氨酸（10.66%）、赖氨酸（10.66%）、丝氨酸（9.33%）、精氨酸（9.33%）含量较高,对其二级结构分析发现HMAT1基因编码的蛋白质螺旋主要集中在该蛋白的中间区域;而折叠主要集中在该蛋白的两端。在其10-15aa处存在一个跨膜区与4-20aa处疏水区域相符合。利用SignalP V3.0软件对其进行信号肽序列分析,发现在20-21aa处存在一个裂解位点。利用ExPASy Proteomics Server中的Motifscan分析发现在33-35aa,45-47aa,和62-64aa处存在着三个可能的蛋白激酶C磷酸化位点,在22-25aa和62-65aa处,存在两个典型的[ST]-X_（2）-[DE]结构,即酪氨酸蛋白激酶Ⅱ磷酸化位点,该位点可能与HMAT1基因编码蛋白质的功能活化有关。通过生物信息学方法,对HMAT1基因及其编码的蛋白功能的预测为实验室进一步研究其功能奠定了基础。更多还原

【Abstract】 Cervical carcinoma is one of the commonest malignant tumors that have great influence on women’s health, and the incidence and the rate of death caused by cervical carcinoma in developing countries are very high. In China, Hubei, Jiangxi, and Xingjiang. Provinces are areas that have high-incidence of cervical carcinoma,It was previously believed that the infection of the high-risk type human papillomavirus (HPV) such as HPV16 or HPV18 is that the major cause of cervical carcinoma, because this kind of infection could be detected in more than 90% of patients with this cancer. But current research indicates that HPV alone cannot cause the carcinoma, and the experiments carried out in vitro shown that some cellular oncogenes are also needed to tranform the cells in addition to HPV infection. Based on these results, our group analyzed the cervical carcinoma specimen by using the oncogene and suppressor gene cDNA microarrays. Through these experiments, we find that the Homo sapiens homolog of mouse MAT-1 gene (HMAT1) is a candidate tumor oncogene in cervical cancer.In this study, we firstly performed some bioinformatic analysis on the HMAT1 gene to predict its characteristics. Blasting the gene sequence of HMAT1 against Genbank showed that the Homo sapiens PEA15 protein (PEA15) gene has a high identity (99%) with HMAT1. HMAT1 codes for a polypeptide with 75 amino acid residues. Amino acid composition analysis indicated that Lys(10.66%), Leu(10.66%), Arg(9.33%) and Ser(9.33%) have the highest percentage. Secondary structure analysis predicted that the a-helix are almost concentrated in the C-terminal, while the 3 -sheet are almost located in the N-terminal. The prediction of hydrophobicity region in 4-20aa was consistant with the predicted transmembrane region locating in 10-15aa. Signal peptide analysis by using SignalP Version 3 showed that there were potential cleavage sites in 21-22aa. It was predicted that 33-35aa, 45-47aa and 62-64aa were potential protein kinase C phosphorylation sites, and 22-25aa and 62-65aa were potential Casein kinase II phosphorylation sites. It is estimated that these sites may be involved in the regulation of HMAT1 protein.Secondly, in order to study the function of HMATl gene by using RNAi technology, we established a platform for siRNA screen via the vector-based method. Taking the advantage of EGFP gene, we constructed a fusion expression vector for the expression of HMATl and then designed three different siRNA vectors targeting different regions of HMATl. These two kinds of vectors were co-transfected into B16F0 cells. Both of the intensity of EGFP and the RT-PCR were used to evaluate the RNAi’s efficiency of the silencing HMATl. The results revealed that: for the first siRNA, when co-tranfecting cells by using the expression vector and siRNA vector in a ratio of 1:3 or 1:4, the intensity of EGFP was obviously weaker than the control, and the density of the cell were on the same level. For the second and third siRNAs, compared with the controls, there were no significant differences of the intensity of EGFP when they were transfected in serial concentrations. The RT-PCR results indicated that the amount of HMATl mRNAs in the cells tranfected with the first specific siRNA was significant lower (P<0.05) than the controls. For the second and third siRNAs, the amount of the mRNAs did not have significant difference with the controls.This study demonstrated that the siRNA could effectively silence the expression of HMATl. Because the siRNAs used in this study was not chemically synthesized, they had little toxic effects on the cells, which provided an effective tool for the further study of the function of the HMATl gene in cervical cancer. In conclusion, this study successfully established the siRNA expression vectors that target different regions of the HMATl gene, and the EGFP expression system that provided us a convenient and fast platform for the screen of the siRNAs. We also identified a siRNA which can effectively silence the expression of HMATl by using this system.更多还原