利用深度测序技术筛选膀胱癌突变基因及对HECW1基因突变的验证

发布时间：2018-11-23 17:28

【摘要】：目的：利用外显子组深度测序技术发现和筛选膀胱移行细胞癌(Transitional CellCarcinoma Of Bladder, TCCB)中的致病候选基因，并通过Sanger测序技术进行验证，为膀胱癌病因学研究提供一定的理论依据，并探寻理想的肿瘤诊断标志物和有效的治疗靶点。方法：第一部分，取苏州大学附属第一医院2012年4月手术治疗的4例膀胱移行细胞癌患者癌及癌旁组织，其中2例为非肌层浸润性（非浸润组），2例为肌层浸润性（浸润组）。采用SOLID深度基因测序的方法进行全外显子组测序，寻找突变基因，并通过特定的筛选条件进行过滤，选出其中发生错义突变的基因。第二部分，取苏州大学附属第一医院病理科提供的2011年9月至2012年9月膀胱移行细胞癌术后腊片标本84例，其中浸润性54例，非浸润性30例，及2013年3月至5月本院手术治疗的膀胱移行细胞癌患者肿瘤组织15例，其中浸润性7例，非浸润性8例和10例癌旁组织作为对照，将此25例组织制作成蜡块。使用OMEGA试剂盒法提取癌及癌旁组织中基因组DNA，经聚合酶链反应（Polymerase ChainReaction，PCR）扩增后，应用Sanger测序技术验证深度测序中的突变结果。取制作的25例蜡块，采用免疫组化Envision二步法，观察HECW1蛋白在癌组织和癌旁组织中的表达差异。结果：第一部分：深度测序结果数据质量满意，，发现众多突变基因，筛选出其中10个出现错义突变的基因，其中非浸润组4个，为IVL,RGPD5,ZNF79,SRRM5；浸润组3个，为CPNE7,RBMXL3,ACSM2A；两组共有3个，为HECW1,ZNF273,TCHH。第二部分：99例膀胱癌组织中发现6例HECW1基因突变，比例为6.06%，均为点突变，位于第11号外显子，且与深度测序突变位点相同或在附近区域。其中，38例非浸润性膀胱癌组织DNA中，1例同义突变，比例为2.63%；61例浸润性膀胱癌组织DNA中，4例错义突变和1例无义突变（即突变为终止密码子），比例为8.20%，两者突变比例无统计学意义（P0.05），对照组10例未发现突变。免疫组化中，15例肿瘤组织9例阳性，阳性率60.0%，10例对照组1例阳性，阳性率10%，差异有统计学意义（P0.05）。结论：（1）深度测序技术，特别是全外显子组测序，可以作为泌尿系肿瘤病因学研究中行之有效的实验手段。（2）Sanger测序是检验深度测序结果的金标准。（3）HECW1可能是与膀胱癌发生发展有关的候选基因。（4）HECW1蛋白在膀胱癌组织中的表达高于癌旁组织。（5）HECW1基因突变在膀胱癌中的作用机制及是否影响蛋白的表达和功能还有待深入研究。
[Abstract]:Objective: to identify and screen candidate genes for (Transitional CellCarcinoma Of Bladder, TCCB) in bladder transitional cell carcinoma (TCC) by using exon group deep sequencing technique, and to provide a theoretical basis for the etiological study of bladder transitional cell carcinoma (TCC). And to explore the ideal tumor diagnosis markers and effective therapeutic targets. Methods: in the first part, 4 patients with bladder transitional cell carcinoma (TCC) were treated by surgery in the first affiliated Hospital of Suzhou University in April 2012, and 2 of them were non-myometrial infiltrating (non-invasive group). 2 cases were myometrial infiltration (infiltrating group). The SOLID deep gene sequencing method was used to sequence the whole exon group to search for the mutant gene, and the missense mutation gene was selected by specific screening conditions. In the second part, 84 specimens of transitional cell carcinoma of bladder were collected from Department of Pathology of the first affiliated Hospital of Suzhou University from September 2011 to September 2012. Among them, 54 cases were invasive and 30 cases were non-invasive. From March to May 2013, 15 cases of bladder transitional cell carcinoma (TCC) were treated by operation in our hospital, including 7 cases of infiltrating, 8 cases of non-invasive and 10 cases of paracancerous tissue as control. Genomic DNA, extracted from cancer and adjacent tissues was amplified by polymerase chain reaction (Polymerase ChainReaction,PCR) using OMEGA kit method. The mutation results in deep sequencing were verified by Sanger sequencing technique. The expression of HECW1 protein in cancer tissues and paracancerous tissues was observed by immunohistochemical Envision two-step method. Results: the first part: the quality of deep sequencing data was satisfactory, many mutation genes were found, and 10 of them had missense mutation, including 4 non-invasive genes, which were IVL,RGPD5,ZNF79,SRRM5;. Infiltration group 3, CPNE7,RBMXL3,ACSM2A; group 3, HECW1,ZNF273,TCHH. The second part: 6 cases (6.06%) of HECW1 gene mutations were found in 99 cases of bladder cancer, all of them were point mutations, located in exon 11, and were the same as or in the vicinity of deep sequencing mutation. Of 38 cases of non-invasive bladder cancer, 1 case had synonymous mutation (2.63%). In 61 cases of invasive bladder cancer DNA, 4 missense mutations and 1 nonsense mutation (i.e. mutation as termination codon) were found in 4 cases (8.20%). There was no significant difference between the two mutations (P0.05), while no mutation was found in 10 cases in the control group. In immunohistochemical staining, 9 cases were positive in 15 cases of tumor tissue, and 1 case was positive in 10 cases of control group. The difference was statistically significant (P0.05). Conclusion: (1) Deep sequencing, especially total exon sequencing, (2) Sanger sequencing is the gold standard for testing the results of deep sequencing. (3) HECW1 may be a candidate gene related to the occurrence and development of bladder cancer. The expression of HECW1 protein in bladder cancer tissues is higher than that in paracancerous tissues. (5) the mechanism of HECW1 gene mutation in bladder cancer and whether it affects the expression and function of HECW1 protein remains to be further studied.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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