PRKAA1基因遗传变异与胃癌的相关性及其功能机制研究

Gastric cancer(GC) is one of the most common malignant tumors. The PRKAA1 protein, the product of PRKAA1 gene (protein kinase, AMP-activated, alpha 1 catalytic subunit), is one of the key subunits of the mammalian 5′-AMP–activated protein kinase (AMPK), which has been reported to be involved in the development of tumors through various signaling ways. Genome-wide association analysis located one of the susceptibility genes of GC in 5p13.1 (PRKAA1 gene region). By bioinformatics analysis,we found that rs461404 and rs10035235，which are both located in the promoter of PRKAA1 gene，lie respectively in combination region of transcription factor NF-κB1and RFX3. Meanwhile，SNP rs13361707 located in the first intron of the PRKAA1 lies just in the combination region of transcription factor GATA1 and PRKAA1. Lacoperator-containing luciferase reporter assay also demonstrated the significant differences of luciferase expression level driven by different sequences containing various alleles. Based on the fore-working, we can pinpoint the function domain of PRKAA1 gene, analyze its functional mechanism, and explore the relationship between the function domain and tumor susceptibility, clinical phenitype, survival time. The study will establish analysis process of functional tumor-associated SNPs and provide a kind of important theory basis for individualized therapy and efficacy prediction.

胃癌是最常见的恶性肿瘤之一。PRKAA1基因编码AMPK激酶的关键催化亚单位，而研究表明AMPK通过多种信号通路参与调控肿瘤的发生发展。全基因组关联分析将胃癌的一个易感基因定位于5p13.1（PRKAA1基因的所在区域）。通过进一步生物信息学分析，发现PRKAA1启动子区SNPrs461404、rs10035235分别位于转录因子NF-κB1、RFX3的结合区域，而第一内含子区SNP rs13361707正好处于转录因子GATA1的结合区域。荧光素酶报告基因实验也证实不同等位基因的序列所驱动的荧光素酶表达量存在着显著的差异，说明这些位点具备一定的功能。本课题拟在前期工作的基础上精确定位PRKAA1基因上的功能位点，分析其功能学机制，探讨其与肿瘤的易感发生、临床表型及预后之间的关系。本研究将建立功能性肿瘤相关基因多态性位点的分析流程，为今后胃癌的个体化治疗和预防提供重要理论依据。

前期我们实验小组证实PRKAA1基因的SNP rs461404、rs10035235显著增加胃癌危险性。提示PRKAA1基因为胃癌的易感基因。本课题旨在研究PRKAA1基因在胃癌发生发展过程所起作用及其功能机制。.首先临床样本验证PRKAA1的表达，结果表明PRKAA1在癌组织中水平较癌旁显著升高，其差异表达情况与TCGA数据库结果相一致。进一步Kaplan-Meier生存分析显示PRKAA1低表达的患者预后较PRKAA1高表达的患者预后好，生存期更长。随后，我们从PRKAA1基因的上下游调控通路研究PRKAA1参与胃癌发生发展的生物学机制。幽门螺杆菌（H. pylori）感染在胃癌发生发展过程中起到重要作用。实验表明，HP感染促进 NF-Bp50表达，调控PAKAA1基因的启动子转录活性，促进PRKAA1的表达，参与胃癌发生。通过细胞实验表明，PRKAA1在胃癌细胞株中表达升高，敲低PRKAA1可以抑制细胞增殖、细胞周期阻滞，促进细胞凋亡。动物实验表明，敲低PRKAA1可以抑制裸鼠种植瘤的成瘤性。进一步研究表明，敲低PRKAA1基因可抑制凋亡因子PCNA及Bcl-2的表达，并激活JNK1和Akt信号通路，调控胃癌细胞增值凋亡。另一方面，敲低PRKAA1可抑制胃癌细胞侵袭和转移，同时发现敲低PRKAA1可以抑制裸鼠转移瘤的成瘤性。不同细胞株中敲低PRKAA1基因可以抑制MMP2表达及NF-B信号通路。进一步挽救实验表明PRKAA1过表达可抑制由于NF-Bp50敲低后所诱导的胃癌细胞侵袭转移能力的下降。说明PRKAA1通过调控NF-B信号通路参与胃癌细胞侵袭转移。.在此青年基金的资助下，本人申请并主持了徐州市课题一项，获江苏省“六大人才高峰”高层次人才资助项目一项，荣获徐州市第七期“拔尖人才”称号，入选江苏省第五期“333工程”第三层次培养对象。发表专著1篇（第三副主编），SCI论文1篇，本研究形成的3篇SCI论文正在投稿中。在国内学术会议上进行论文投递。