1、 青海地区藏族、汉族HIF2a基因单核苷酸多态性与胃癌易感性的相关性研究 马金华 马金兰 王丽娟摘要 目的 探討青海地区藏、汉族HIF-2基因单核苷酸多态性与胃癌易感的相关性。 方法 收集青海省藏医院、青海大学附属肿瘤医院、青海省肿瘤医院就诊胃癌患者(长期居住于青海地区)藏族51例、汉族60例。使用外周血提取DNA。采用聚合酶链式反应(PCR)扩增,并使用测序法检测HIF-2基因的3个位点,分别为rs6715787、13419896、7598371,同时行相关性分析。 结果 rs6715787、rs7598371的单核苷酸多态性(SNP)基因型分别为C/G、C/C、G/G,rs13419896
2、的SNP基因型为A/G、G/G、A/A。两组基因型分布检验后符合Hardy-Weinberg平衡规律(P 0.05)。藏、汉族胃癌组rs13419896的A/G、G/G、A/A基因型频率分别为27.45%、29.41%,43.13%、45.00%,43.33%、11.67%,基因型频率分布两组差异有高度统计学意义(2 = 14.195,P 0.01),其等位基因差异有高度统计学意义(2 = 11.495,P 0.01)。藏族胃癌组等位基因A频率分布高于汉族胃癌组。藏、汉族胃癌组rs7598371的C/G、C/C、G/G基因型频率分别为29.42%、37.25%、33.33%,41.67%、5.
3、00%、53.33%,两组的基因型频率分布差异有统计学意义(2 = 18.118,P 0.01),其等位基因差异有高度统计学意义(2 = 16.002,P 0.01),藏族胃癌组等位基因C频率分布高于汉族胃癌组。藏、汉族胃癌组rs6715787的C/G、C/C、G/G基因型频率分别为35.29%、41.18%,23.52%、51.67%,8.33%、40.00%。两组基因型频率分布差异有高度统计学意义(2 = 16.675,P 0.01),其等位基因差异有高度统计学意义(2 = 13.518,P 0.01),藏族胃癌组等位基因C频率分布明显高于汉族胃癌组。基因位点藏汉族胃癌患者表达不同,其中青
4、海地区藏族人群胃癌患者携带C/C型的HIF-2基因的rs6715787高表达,藏族人群胃癌患者携带C/C型的HIF-2基因rs7598371高表达,汉族人群胃癌患者携带C/G型的HIF-2基因rs6715787高表达,汉族人群胃癌患者携带G/G型的HIF-2基因rs7598371高表达。 结论 HIF-2基因rs7598371、rs6715787、rs13419896与青海地区藏汉族胃癌可能无相关性,但青海地区藏族胃癌、汉族胃癌HIF-2基因单核苷酸多态性存在差异,基因位点藏汉族对照组、藏汉族胃癌患者表达不同,提示青海地区藏族胃癌、汉族胃癌患者的基因型不同,遗传基础可能不同。关键词 汉族;藏族
5、;胃癌;HIF-2基因;单核苷酸多态性 R735.2 A 1673-7210(2019)04(c)-0009-05The relationship between single nucleotide polymorphism of HIF-2 gene and susceptibility to gastric cancer of Qinghai area Tibetan, Han nationalityMA Jinhua1 MA Jinlan2 WANG Lijuan1 LI Jinzhang1 SHEN Cunfang1 MA Jinxiang31.Department of Medica
6、l Oncology, Qinghai University Affiliated Hospital, Qinghai Province, Xining 810001, China;2.Department of Nephrology, Qinghai University Affiliated Hospital, Qinghai Province, Xining 810001, China; 3.Medical College, Qinghai University, Qinghai Province, Xining 810001, ChinaAbstract Objective To ex
7、plore relationship between single nucleotide polymorphism of HIF-2 gene and susceptibility to gastric cancer of Qinghai area Tibetan, Han nationality. Methods A total of 51 Tibetan and 60 Han nationality patients (long lived in Qinghai area) with gastric cancer were collected. DNA was extracted from
8、 peripheral blood. Polymerase chain reaction (PCR) was used to detect 3 sites of HIF-2 gene, namely rs6715787, 13419896 and 7598371, and correlation analysis was performed. Results The SNP genotypes of rs6715787 and rs7598371 were C/G, C/C and G/G, respectively, and the SNP genotypes of rs13419896 w
9、ere A/G, G/G and A/A. The genotype frequencies of A/G, G/G and A/A in rs13419896 of Tibetan and Han nationality groups were 27.45%, 29.41%, 43.13% and 45.00%, 43.33%, 11.67%, respectively. The difference in genotype frequency distribution between the two groups was highly statistically significant (
10、2 = 13.518, P 0.01), and the difference in allele was highly statistically significant (2 = 14.195, P 0.01). The allele difference was statistically significant (2 = 11.495, P 0.01). The frequency distribution of allele A in Tibetan gastric cancer group was higher than that in Han nationality gastri
11、c cancer group. The C/G, C/C, G/G genotype frequencies of rs7598371 in the Tibetan and Han nationality groups were 29.42%, 37.25%, 33.33% and 41.67%, 5.00%, 53.33%, respectively. The difference of genotype frequency distribution between the two groups was highly statistically significant (2 = 18.118
12、, P 0.01), and the allele differences were highly statistically significant (2 = 16.002, P 0.01). The frequency distribution of allele C in Tibetan nationality gastric cancer group was higher than that in Han nationality gastric cancer group. The genotype distribution of the two groups was consisten
13、t with Hardy-Weinberg equilibrium law (P 0.05). The C/G, C/C, G/G genotype frequencies of rs6715787 in the Tibetan and Han nationality group were 35.29%, 41.18%, 23.52% and 51.67%, 8.33%, 40.00%, respectively. The frequency distribution of allele C in Tibetan nationality gastric cancer group was hig
14、hly significantly Higher nationality than that in han gastric cancer group (2 = 16.675, P 0.05),具有可比性。1.2 主要仪器与试剂聚合酶链式反应(PCR)扩增仪(Verity 96well,美国ABI),凝胶成像仪(Gene Genius,英国Syngene),3730XL测序仪(美国ABI),DNA marker(加拿大BBI公司),基因组DNA提取试剂盒(上海生工生物工程有限公司,生产批号:E604KA8640),PCR扩增试剂盒(上海生工生物工程有限公司)。1.3 方法1.3.1 血液采集 受
15、试者晨起空腹采集外周静脉血2 mL,采用EDTA抗凝处理。1.3.2 基因组DNA提取 取上述血样品,采用DNA试剂盒提取,电泳检测其完整度,分光光度计测其含量、纯度,-20冰箱保存备用。1.3.3 引物设计与合成 以美国国家生物信息中心的GenBank获取HIF-2a基因序列及相应位点的单核苷酸多态性(SNP)信息。使用PrimerExpress 2.0软件辅助设计引物。见表1。1.3.4 PCR反应 基因组DNA为模板,设计将HIF-2基因的rs13419896、rs7598371、rs6715787引物建立PCR反应体系25 L:DNA模板1 L,Taq buffer为2.5 L,dNT
16、P、上下游引物各0.5 L,5 U/L Taq DNA聚合酶0.25 L,ddH2O 14.8 L。PCR的条件为:95预变性3 min,94变性30 s,58退火30 s,72延伸45 s,进行35个循环;72后延伸10 min。5 L PCR扩增产物经1%琼脂糖凝胶上100 V电压下电泳40 min,溴化乙锭染色,凝胶成像系统照相、保存。扩增产物纯化后送测序、进行SNP技术分型,以测序结果确定基因频率、基因型并比较。巢式PCR优化体系的建立:采用巢式PCR扩增法即经2次PCR放大,可将单拷贝的目的DNA片段充分的扩增,以达检出的目的。1.4 统计学方法采用SPSS 22.0软件进行数据统计
17、分析,进行Hardy-Weinberg遗传平衡定律检验。计数资料采用百分率表示,组间比较采用2检验。以P 0.05为差异有统计学意义。2 結果2.1 DNA电泳检测结果藏汉族胃癌组结果见图1。2.2 PCR产物检测结果以上述DNA为模板进行PCR扩增,1%琼脂糖凝胶电泳,结果见图2。2.3 基因测序结果HIF-2基因rs6715787、rs13419896、rs7598371的SNP基因型分别是C/G、C/C、G/G型,A/G、G/G、A/A型,C/G、C/C、G/G型。2.4 基因型、基因频率分布结果2.4.1 Hardy-Weinberg平衡检验结果 rs6715787、rs1341989
18、6、rs7598371的SNP基因型分别为C/G、C/C、G/G,A/G、G/G、A/A,C/G、C/C、G/G。对照组检验基因型分布符合Hardy-Weinberg平衡规律(rs13419896 SNP:2 = 10.23,P 0.05;rs7598371SNP:2 = 13.35,P 0.05;rs6715787SNP:2 = 7.47,P 0.05)。2.4.2 rs13419896的SNP基因型及等位基因频率分布 两组基因型频率分布差异有高度统计学意义(2 = 14.195,P 0.01),其等位基因差异也存在高度统计学意义(2 = 11.495,P 0.01),藏族胃癌组等位基因A频
19、率分布明显高于汉族胃癌组。见表1。2.4.3 rs7598371的SNP基因型及等位基因频率分布 两组基因型频率分布差异有高度统计学意义(2 = 18.118,P 0.01),在汉族胃癌组中频率最高基因型分别为C/C型和G/G型。rs7598371等位基因差异有高度统计学意义(2 = 16.002,P 0.01),藏族胃癌组等位基因C频率分布明显高于汉族胃癌组。见表2。2.4.4 rs6715787的SNP基因型及等位基因频率分布 两组基因型频率分布差异有高度统计学意义(2 = 16.675,P 0.01),在汉族胃癌组中最高者分别为C/C型和G/G型,等位基因差异有高度统计学意义(2 = 13.518,P 0.01),藏族胃癌组等位基因C频率分布明显高于汉族胃癌组。见表3。 -全文完-