【作者】 刘娟；

【导师】 伍欣星；

【作者基本信息】 武汉大学 ， 病原生物学， 2004， 硕士

【摘要】 宫颈癌是发展中国家最常见的妇科恶性肿瘤之一，其发病率和死亡率均很高，已成为威胁女性身体健康的主要杀手。此病的发病率有明显的地理差异，在我国存在宫颈癌的高发区——主要集中在湖北省，江西省及新疆等地区。 过去常认为高危型人乳头瘤病毒（human papillomavirus，HPV）如HPV16，18等型的感染是导致宫颈癌的主要原因之一，但近年来众多的研究表明，细胞内遗传因素的改变也起着重要的作用。因此，确定宫颈癌相关基因，从分子水平上了解细胞癌变机制，为宫颈癌的临床诊断、预防、易感性预测及治疗提供分子标记和靶基因具有十分重要的意义。基于此目的，我科室研究人员采用细胞原癌、抑癌基因分类芯片对原发性宫颈癌标本进行了分析，确定了与宫颈癌相关的11条候选原癌或抑癌基因（其中包括在宫颈癌组织中表达降低的7条基因和表达增高的4条基因），并选取了在宫颈癌组织中表达降低最明显、GenBank ID为NM₀06698的人膀胱癌相关蛋白（Homo sapiens bladder cancer associated protein，BLCAP）基因，进行进一步研究，包括：转染HeLa细胞后的细胞形态观察、HeLa细胞生长曲线的绘制以及集落形成实验等。为明确BLCAP基因与宫颈癌的相关性及其在宫颈癌发病中所起的作用，本研究首先采用生物信息学的方法对其在核酸及氨基酸水平上进行了分析，并对其功能做了初步预测，发现了几条与BLCAP基因同源性较高的基因：Homo sapiens mRNA（DKFZp564M053），BC-10基因等，它们与BLCAP基因的序列相似性分别为：99％、87％，而且不同种属间BLCAP基因较为保守。BLCAP基因编码的87个氨基酸中亮氨酸（19.5％）、脯氨酸（9.19％）、丝氨酸（8.04％）、半胱氨酸（8.04％）含量较高，对其二级结构的分析发现BLCAP基因编码的蛋白质N端以螺旋结构为主，C端以折叠结构为主，亲水性较高，而中间区域则以无规则卷曲为主，疏水性较高。在其45-65aa处存在一个跨膜区，初步预测其为一个Ⅰ型跨膜蛋白。利用SignalP V2.0软件对其进行信号肽序列分析，发现在63-64aa处可能存在一个裂解位点，利用Netpho软件对其进行磷酸化位点分析发现在68位的Tyr，73位和78位的Ser存在着3个可能的磷酸化位点，在78-81aa处发现一个典型的[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BLCAP基因编码蛋白质的功能活化有关。通过生物信息学方法，对BLCAP基因及其编码蛋白功能的预测为实验室进一步研究其功能奠定了基础。 第二，为了证实BLCAP基因为一条宫颈癌相关抑癌基因，我们根据判定抑癌基因的三个标准，即:（l）该基因在恶性肿瘤的相应正常组织中必须正常表达;（2）在恶性肿瘤中该基因出现功能失活或结构改变或表达缺陷;（3）将该基因的野生型导人该基因异常的肿瘤细胞内，可部分或全部改变其恶性表型;进行了相关实验研究，包括:检测了BLCAP基因在人正常细胞和不同肿瘤细胞中表达水平的差异、BLCAP基因裸鼠体内抑瘤实验及DNA Ladder实验，发现:BLCAP基因在人不同肿瘤细胞中表达水平较之在人正常细胞中的表达水平低;同等量HeLa细胞注射裸鼠皮下后，稳定转染了BLCAP基因的HeLa细胞较之未转染BLCAP基因的空HeLa细胞及转染了空载体的HeLa细胞出瘤时间晚，最终成瘤体积小，且肿瘤病理切片结果显示，稳定转染了BLC妙基因的HeLa细胞所成肿瘤组织仅在中央部分出现小的坏死灶，未转染BLCAP基因的空HeLa细胞及转染了空载体的HeLa细胞所成肿瘤组织周边已出现坏死;在DNA Ladder实验中，稳定转染BLCAP基因的HeLa细胞DNA中出现大小约200bp及其倍体的DNA梯带，而转染空载体pLXsN的HeLa细胞及正常培养的HeLa细胞中则未出现，说明HeLa细胞在转染BLCAP基因后出现了凋亡。基于以上实验结果，并结合我科室已有的研究结果，我们认为BLCAP基因为一条宫颈癌相关抑癌基因。 第三，在证实BLCAP基因为一条宫颈癌相关抑癌基因基础上，我们进一步研究了其在宫颈癌发生过程中失活的机制，即:检测了BLCAP基因调控区的单核昔酸多态性（SNP）位点及其不同基因型与宫颈癌的相关性。我们采用病例一对照研究方法及动态等位基因杂交（D AsH）技术，对30例原发性宫颈癌患者和60例正常人BLCAP基因调控区的2个单核昔酸多态性（SNP）位点进行了检测，结果显示:位于BLCAP基因下游调控区的SNP rs3795147位点存在AA、AC和CC三种基因型，且与宫颈癌发病存在显著相关性（P<0.05）。由于SNP rs3795147处在BLC妙基因下游调控区域，其多态类型可能在某种程度上影响BLCAP基因的表达调控，从而进一步支持了:BLCAP基因与宫颈癌的发生、发展可能存在密切关系。 综上所述，本次实验结果表明:BLCAP基因为一条宫颈癌相关抑癌基因，且该基因下游调控区SNP rs3795147位点的不同基因型，与宫颈癌发病存在显著相关性。更多还原

【Abstract】 Cervical carcinoma is one of the most common malignancy among women in developing country. The incidence and the rate of death of cervical carcinoma are very high. There are some high-incidence areas of cervical carcinoma in china, such as Hubei, Jiangxi, and Xinjiang Province. The infection with high-risk type human papillomavirus such as HPV16 or HPV18 was ever regarded as the major etiological factor for cervical carcinoma, but the alter of genetic factors is also important during the occurrence of cervical carcinoma. Therefore, some researchers of our lab analysed the cervical carcinoma specimen with the oncogene and anti-oncogene cDNA microarray. And after doing some experiments, theseresearchers discovered a candidate tumor suppressor gene for cervical carcinoma--Homosapiens bladder cancer associated protein (BLCAP) gene.Since the character and function of BLCAP gene is unknown, we firstly analyzed BLCAP gene through bioinformatic means. The results showed that there were several genes that were highly resembled with BLCAP gene. The comparability between the sequences of BLCAP gene and Homo sapiens mRNA (DKFZp564M053) or BC10 was 99% and 87% respectively. The protein encoded by BLCAP were composite of Leu(19.5%), pro(9.19%), ser(8.04%), cys(8.04%) and other amino acids. The N-terminal of the protein which was encoded by BLCAP gene was an alpha helix. At the C-terminal, it was beta sheet and in the middle, it was coil. The two terminal regions were more hydrophobile than the middle region. Between 45-65aa, there was a transmembrane region. Therefore, we forecasted that BLCAP gene was a member of transmembrane protein I. By analyzing the signal peptide of BLCAP gene with the program of SignalP (V2.0), we found a cleavage site in 63-64aa. By using the program of Netpho, we predicted that there might be three phospholate sites: 68aa, 73aa and 78aa. In 78-8laa, we found a typical [ST]-X(2)-[DE] structure-the phospholate site of Tyrosine protein kinase, Which might be related to its function. Bioinformatic studies of BLCAP gene provided foundation for the functionalresearch of BLCAP gene in laboratory.Secondly, in order to prove that BLCAP gene was a tumor suppressor gene associated with cervical carcinoma, we examined the expressional level of BLACP gene mRNA in human normal cells and different rumor cells, investigated the tumorigenicity in nude mice of HeLa stably-transfected with BLCAP gene, and also took the DNA ladder experiment. The results showed: (a) the expressional level of BLACP gene mRNA in tumor cells was lower than that in human normal cells, (b) BLCAP gene showed suppressor ability in HeLa tumorigenicity. (c) apoptosis could be investigated in HeLa after it was stably-transfected with BLCAP gene. Basing on the data above, we can think that the abnormal expression of BLACP gene was related to malignant proliferation and anchorage independent growth of human cervical carcinoma cells.Finally, we studied two single nucleotide polymorphisms (SNPs) within BLCAP gene in this experiment, in order to make out the function of BLCAP gene in the occurrence of human cervical carcinoma. We analyzed 30 unrelated cervical carcinoma cases and 60 control subjects, which matched to the cervical carcinoma cases in age and residence, by means of dynamic allele specific hybridization (DASH). Significant results were obtained from one SNP site (rs3795147), which was located at 711bp down-stream of BLCAP gene. SNP rs3795147 had three kinds of genotype: AA, AC and CC, and results of x2test showed that the frequencies of different genotypes resulted in cervical carcinoma cases and control subjects were significantly different (P (0.05 ) . The result had proved again that BLCAP gene may play a certain role in the development of cervical carcinoma, for the SNP site rs3795147 may influence the expression of BLCAP gene.In conclusion, BLCAP gene was a tumor suppressor gene associated with cervical carcinoma, and the different genotypes of SNP rs3795147 had a close relationship with the occurrence of cervical carcino更多还原