染色体12q14.2区域重测序筛选卵巢癌易感性SNP位点及其分子机制研究
发布时间：2018-07-20 21:00
【摘要】：目的卵巢癌是一种常见妇科肿瘤,由于缺乏早期相关明显症状而常被喻为“沉默杀手”。其是一种复杂的多基因疾病,遗传因素发挥着重要作用。为了发现中国人特有的遗传易感信息,课题组前期开展的中国汉族女性上皮源性卵巢癌GWAS研究发现了12q14.2区的rs11175194位点与国人卵巢癌发病风险相关,其不同基因型与基因SRGAP1表达相关,基因SRGAP1高表达组卵巢癌患者预后差,提示此高连锁不平衡易感区域重要,可能成为卵巢癌高危筛查、诊断和治疗靶点。本研究拟对该区域进行重测序,对该区域进行精细作图,筛选病例对照有差异的SNP位点,分析SNP基因型与环境因素的交互作用,预测该区域差异SNP卵巢癌易感性位点的功能,进行功能探索,同时结合美国TCGA数据进行验证,为阐明卵巢癌的病因和发生发展机制,筛选高危易感人群,开展早期诊断及预后预测提供新依据,为提高卵巢癌早诊率降低死亡率提供参考。方法基于Hapmap里中国及日本人群的数据,利用Haploiew软件,对中国汉族人群卵巢癌GWAS研究发现的易感性位点rs11175194进行连锁不平衡分析,得到该位点所在的连锁不平衡区域。采用高通量二代测序方法,对192例正常对照人群中该区域进行重测序,分析中国汉族女性该区域中已知和未知变异位点,剔除MAF0.05的位点,筛选与rs11175194高度连锁的位点进行病例对照研究,找出病例对照中SNP频率有差异的位点,利用生物信息学方法进行功能预测。分析有功能SNP位点不同基因型与其所在基因表达量之间的关系,同时利用TCGA公共数据库中卵巢癌患者基因表达数据信息以及相应体外细胞实验进行验证。结果分析得到中国汉人卵巢癌GWAS研究发现的易感性SNP位点rs11175194所在的高度连锁不平衡区域(linkage disequilibrium block,LD block)64215629-64268005之间全长52kb。经过对正常对照人群该区域重测序,筛选与rs11175194高度连锁并且病例对照频率差异有意义的SNP位点rs11175195和rs67799338。这两个位点均位于SRGAP1基因的第一号内含子区域,生物信息学方法预测结果显示,SNP位点rs67799338所在序列存在人源性转录因子Ref1、AP1、SP1结合位点。SNP位点rs67799338不同基因型与其所在基因SRGAP1的mRNA表达相关,C基因型的表达量高于T基因型。TCGA公共数据库卵巢癌患者基因表达数据分析结果显示,转录因子Ref1、AP1、SP1的表达量与基因SRGAP1的表达量呈正相关,其中转录因子SP1的表达量与基因SRGAP1表达量相关性最强,r=0.607,p0.001。CHIP实验结果显示,转录因子SP1可以与rs67799338所在区域结合。过表达SRGAP1可以促进SKOV3细胞进入S期。过表达SRGAP1可以增加总Rho含量,SRGAP1高表达的卵巢癌患者预后差。结论位于SRGAP1基因第一号内含子上卵巢癌易感性SNP位点rs11175195、rs67799338,其不同基因型均影响所在基因SRGAP1的表达量。以上两个SNP位点为C基因型时其所在基因SRGAP1的mRNA表达量高于T基因型。SNP位点rs67799338所在区域具有增强子作用,并与转录因子SP1结合。SRGAP1高表达的卵巢癌患者生存时间短,预后差。SRGAP1有可能成为卵巢癌预后预测的新靶标。
[Abstract]:Objective ovarian cancer is a common gynecologic tumor, which is often referred to as "silent killer" due to the lack of early related obvious symptoms. It is a complex polygenic disease, and the genetic factor plays an important role. In order to find the unique genetic susceptibility information of Chinese people, the Chinese Han women's epithelial ovarian cancer GWA has been developed in the earlier period. The S study found that the rs11175194 loci in the 12q14.2 region are associated with the risk of ovarian cancer in the country. The different genotypes are associated with the gene expression of SRGAP1, and the prognosis of the ovarian cancer patients with the high expression of SRGAP1 is poor, suggesting that the high linkage unbalance susceptible region is important and may be a high risk screening, diagnosis and therapeutic target for ovarian cancer. This study is to be used in this study. The region was re sequenced, the region was carefully plotted, the SNP loci with different case control were screened, the interaction between the SNP genotype and the environmental factors was analyzed, the function of the SNP ovarian cancer susceptibility loci was predicted, and the functional exploration was carried out. At the same time, the TCGA data of the United States were used to verify the etiology and occurrence of ovarian cancer. It provides a new basis for early diagnosis and prognosis prediction of high-risk and susceptible population, and provides a reference for improving the early diagnosis rate of ovarian cancer. Methods based on the data of Chinese and Japanese people in Hapmap, the Haploiew software is used to chain the susceptible locus rs11175194 found in the GWAS study of Chinese Han population. The linkage disequilibrium region of the loci was obtained by unbalanced analysis. A high throughput two generation sequencing method was used to re sequence the region in 192 normal controls. The known and unknown mutation sites in the Chinese Han women were analyzed, the MAF0.05 loci were eliminated and the highly linked rs11175194 loci were screened for case control research. To find out the loci with different frequencies of SNP in the case control and use the Bioinformatics Method to predict the function, the relationship between the different genotypes of the functional SNP loci and the expression of their genes is analyzed, and the data of the gene expression data of the ovarian cancer patients in the TCGA public database and the corresponding in vitro cell experiment are verified. The results were obtained from the high linkage disequilibrium region (linkage disequilibrium block, LD block) 64215629-64268005 of the susceptibility SNP locus (linkage disequilibrium block, LD block) 64215629-64268005, which was found in the Chinese Han Chinese ovarian cancer study (linkage disequilibrium block, LD block). The total length of 52kb. was re sequenced in the normal control population, screening with rs11175194 highly linked and case control frequency difference. The two loci of SNP loci rs11175195 and rs67799338. are located in the first intron of the SRGAP1 gene. The results of bioinformatics methods show that the sequence of SNP loci rs67799338 exists the human source transcription factor Ref1, AP1, SP1 binding site.SNP locus rs67799338 different genotypes and their genes SRGAP1. The expression of A was related, the expression of C genotype was higher than that of the T genotype.TCGA public database of ovarian cancer patients. The expression of Ref1, AP1, SP1 was positively correlated with the expression of gene SRGAP1, and the expression of the transcription factor SP1 was strongest with the expression of gene SRGAP1, r=0.607, p0.001.CHIP experiment The results showed that the transcription factor SP1 could be combined with the region of rs67799338. Overexpression of SRGAP1 could promote the entry of SKOV3 cells into the S phase. The overexpression of SRGAP1 could increase the total Rho content, and the prognosis of ovarian cancer patients with high expression of SRGAP1 was poor. Conclusion the SNP locus of the susceptibility to ovarian cancer in the first intron No. 1 of the SRGAP1 gene was rs11175195, rs67799338, it was not. The same genotype affects the expression of the gene SRGAP1. When the two SNP loci are C genotype, the mRNA expression of the gene SRGAP1 is higher than that of the T genotype.SNP locus rs67799338, and the survival time of the ovarian cancer patients with the high expression of the transcription factor SP1 binding.SRGAP1 is short, and the poor prognosis is possible. It is a new target for prognosis prediction of ovarian cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
本文编号：2134761
[Abstract]:Objective ovarian cancer is a common gynecologic tumor, which is often referred to as "silent killer" due to the lack of early related obvious symptoms. It is a complex polygenic disease, and the genetic factor plays an important role. In order to find the unique genetic susceptibility information of Chinese people, the Chinese Han women's epithelial ovarian cancer GWA has been developed in the earlier period. The S study found that the rs11175194 loci in the 12q14.2 region are associated with the risk of ovarian cancer in the country. The different genotypes are associated with the gene expression of SRGAP1, and the prognosis of the ovarian cancer patients with the high expression of SRGAP1 is poor, suggesting that the high linkage unbalance susceptible region is important and may be a high risk screening, diagnosis and therapeutic target for ovarian cancer. This study is to be used in this study. The region was re sequenced, the region was carefully plotted, the SNP loci with different case control were screened, the interaction between the SNP genotype and the environmental factors was analyzed, the function of the SNP ovarian cancer susceptibility loci was predicted, and the functional exploration was carried out. At the same time, the TCGA data of the United States were used to verify the etiology and occurrence of ovarian cancer. It provides a new basis for early diagnosis and prognosis prediction of high-risk and susceptible population, and provides a reference for improving the early diagnosis rate of ovarian cancer. Methods based on the data of Chinese and Japanese people in Hapmap, the Haploiew software is used to chain the susceptible locus rs11175194 found in the GWAS study of Chinese Han population. The linkage disequilibrium region of the loci was obtained by unbalanced analysis. A high throughput two generation sequencing method was used to re sequence the region in 192 normal controls. The known and unknown mutation sites in the Chinese Han women were analyzed, the MAF0.05 loci were eliminated and the highly linked rs11175194 loci were screened for case control research. To find out the loci with different frequencies of SNP in the case control and use the Bioinformatics Method to predict the function, the relationship between the different genotypes of the functional SNP loci and the expression of their genes is analyzed, and the data of the gene expression data of the ovarian cancer patients in the TCGA public database and the corresponding in vitro cell experiment are verified. The results were obtained from the high linkage disequilibrium region (linkage disequilibrium block, LD block) 64215629-64268005 of the susceptibility SNP locus (linkage disequilibrium block, LD block) 64215629-64268005, which was found in the Chinese Han Chinese ovarian cancer study (linkage disequilibrium block, LD block). The total length of 52kb. was re sequenced in the normal control population, screening with rs11175194 highly linked and case control frequency difference. The two loci of SNP loci rs11175195 and rs67799338. are located in the first intron of the SRGAP1 gene. The results of bioinformatics methods show that the sequence of SNP loci rs67799338 exists the human source transcription factor Ref1, AP1, SP1 binding site.SNP locus rs67799338 different genotypes and their genes SRGAP1. The expression of A was related, the expression of C genotype was higher than that of the T genotype.TCGA public database of ovarian cancer patients. The expression of Ref1, AP1, SP1 was positively correlated with the expression of gene SRGAP1, and the expression of the transcription factor SP1 was strongest with the expression of gene SRGAP1, r=0.607, p0.001.CHIP experiment The results showed that the transcription factor SP1 could be combined with the region of rs67799338. Overexpression of SRGAP1 could promote the entry of SKOV3 cells into the S phase. The overexpression of SRGAP1 could increase the total Rho content, and the prognosis of ovarian cancer patients with high expression of SRGAP1 was poor. Conclusion the SNP locus of the susceptibility to ovarian cancer in the first intron No. 1 of the SRGAP1 gene was rs11175195, rs67799338, it was not. The same genotype affects the expression of the gene SRGAP1. When the two SNP loci are C genotype, the mRNA expression of the gene SRGAP1 is higher than that of the T genotype.SNP locus rs67799338, and the survival time of the ovarian cancer patients with the high expression of the transcription factor SP1 binding.SRGAP1 is short, and the poor prognosis is possible. It is a new target for prognosis prediction of ovarian cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
【参考文献】
相关期刊论文 前1条
1 郭俊;李小燕;蔡伦;王绿娅;杜杰;;医学研究生"高通量测序技术"应用能力的培养[J];现代生物医学进展;2016年31期
，本文编号：2134761
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