胰腺癌易感基因LINC00673的发现及其功能研究

发布时间：2018-04-21 20:12

  本文选题：胰腺癌 + 遗传易感性　； 参考：《北京协和医学院》2015年博士论文

【摘要】：全基因组关联研究(GWAS)作为一种高通量的研究方法,近年来已帮助生物医学研究者们发现了一些与胰腺癌易感性相关的基因及遗传位点,然而影响胰腺癌发生、发展的遗传易感因素仍未被完全阐明。本研究利用中国人群胰腺癌全基因关联研究数据进行进一步挖掘,鉴定新的与胰腺癌易感性相关的基因及单核苷酸多态(SNP),并运用生物化学、分子生物学、细胞学等手段,对与胰腺癌易感性相关的基因及其SNP进行生物学功能研究,以阐明其生物学作用机制。关联研究采用全基因组筛查和验证两阶段设计。探讨胰腺癌遗传易感因素采用病例-对照研究策略。全基因组筛查阶段包含981个病例和1,991个对照,验证阶段包含2,869个病例和3,207个对照。胰腺癌全基因组筛查阶段共发现78个P10-5的SNP,本研究筛选出10-6P10-5的46个SNP进行独立验证(P10-6的32个SNP的验证结果已经发表)。我们发现位于17q24.3的rs11655237与胰腺癌发病风险显著相关(P=3.95×10-11),与rs11655237G等位基因相比较,rs11655237A等位基因显著增加了胰腺癌的发病风险(OR=1.26,95% CI=1.15-1.38)。基因定位分析发现,该位点位于LINC00673(基因间长链非编码RNA)基因的第4个外显子区。生物学功能研究表明,rs11655237A等位基因向G等位基因的转变,干扰了miRNA-1231与LINC00673的结合,进而导致LINC00673表达升高。运用LINC00673过表达和敲降的研究策略,以及RNA-Pulldown、RNA免疫共沉淀等实验方法,我们的研究结果证实,LINC00673能够与蛋白酪氨酸磷酸酶SHP2结合,并促进其通过泛素化途径降解,从而,一方面抑制具有促癌活性的SRC/ERK信号通路的活化,另外一方面激活STAT1依赖性的抗肿瘤应答反应信号通路。表型研究结果显示,在LINC00673高表达的胰腺癌细胞系中,其细胞增殖、克隆形成以及裸鼠移植瘤生长受到明显的抑制；而在LINC00673低表达的胰腺癌细胞系中,这些恶性表型则得到了显著的增强。上述结果表明,LINC00673作为一种抑癌基因在胰腺癌的发生、发展中发挥重要的调控作用,而位于其外显子区的遗传变异rs11655237则可能通过某些精细而复杂调节机制来影响其表达,进而修饰个体对胰腺癌的遗传易感性。我们的研究结果对于阐明胰腺癌病因、鉴定高危个体、进行早期诊断和早期干预具有重要理论意义和潜在的应用价值。
[Abstract]:Genome-wide association study (GWAS), as a high-throughput method, has helped biomedical researchers in recent years to identify some genes and genetic loci associated with the susceptibility to pancreatic cancer, while affecting the occurrence of pancreatic cancer. The genetic susceptibility to development has not yet been fully elucidated. In this study, we further excavated the data of the whole gene association of pancreatic cancer in Chinese population, identified new genes and single nucleotide polymorphisms (SNPs) related to the susceptibility of pancreatic cancer, and used biochemistry, molecular biology, cytology and other methods. The biological function of genes and SNP associated with pancreatic cancer susceptibility were studied in order to elucidate its biological mechanism. The whole genome screening and validation two-stage design was used in the association study. To investigate the genetic predisposing factors of pancreatic cancer, a case-control study was used. The whole genome screening phase consisted of 981 cases and 1991 controls, while the validation phase consisted of 2 869 cases and 320 7 controls. A total of 78 SNPs of P10-5 were found in the whole genome screening stage of pancreatic cancer. In this study, 46 SNP of 10-6P10-5 were screened for independent validation of 32 SNP of P10-6. We found that the rs11655237 located in 17q24.3 was significantly associated with the risk of pancreatic cancer. Compared with the rs11655237G allele, the rs1165237A allele significantly increased the risk of pancreatic cancer by 1.2695% CI 1.15-1.38. The locus was located in the fourth exon of LinC00673 (intergenic long chain noncoding RNAs) gene. The study of biological function showed that the transformation of rs11655237A allele to G allele interfered with the binding of miRNA-1231 to LINC00673 and led to the increase of LINC00673 expression. Using the strategies of overexpression and knockdown of LINC00673 and RNA-Pulldown RNA immunoprecipitation, our results show that LinC00673 can bind to protein tyrosine phosphatase SHP2 and promote its degradation through ubiquitin pathway. On the one hand, the activation of SRC/ERK signaling pathway with carcinogenic activity was inhibited; on the other hand, the STAT1-dependent anti-tumor response signaling pathway was activated. Phenotypic studies showed that the proliferation, clone formation and growth of xenografts in nude mice were significantly inhibited in pancreatic cancer cell lines with high expression of LINC00673, but in pancreatic cancer cell lines with low expression of LINC00673. These malignant phenotypes were significantly enhanced. These results suggest that LinC00673 plays an important regulatory role in the development of pancreatic cancer as a tumor suppressor gene, while the genetic variation rs11655237 located in its exon may affect its expression through some fine and complex regulatory mechanisms. And then modify the individual genetic susceptibility to pancreatic cancer. Our results have important theoretical significance and potential application value in elucidating the etiology of pancreatic cancer, identifying high-risk individuals, early diagnosis and early intervention.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.9
，

本文编号：1783931