HTPAP单体型与基因表达及肝细胞癌(HCC)预后的关系

The association of HTPAP haplotype in hepatocellular carcinoma (HCC) with gene expression and prognosis

目的拟通过分析不同HTPAP单体型与基因表达及肝细胞癌(hepatocellular carcinoma,HCC)预后的关系,探讨HTPAP单体型对HCC术后复发及预后的影响。方法随机选取377例HCC样本,提取其中以AGCTAC、GCGGGT、AGCTGC和GCGGAT等4种主要HTPAP单体型组成的HCC RNA。绝对定量PCR检测HTPAP基因表达水平,免疫组化染色、RT-PCR和Western blot检测HTPAP蛋白表达水平,分析单体型与基因表达的关系。随机选取665例HCC样本并提取DNA,焦磷酸测序方法对6个单核苷酸多态性(singlenucleotide polymorphisms,SNPs)进行检测。单体型构建后,分析HTPAP单体型与HCC预后的关系。结果 377例HCC中有327例由上述4种主要单体型构成。基因表达分析提示,HCC中HTPAP在GCGGGT纯合子组和AGCTAC/GCGGGT杂合子组的表达低于AGCTAC/GCGGAT杂合子组、AGCTAC/AGCTGC杂合子组及AGCTAC纯合子组,但差异无统计学意义(P=0.062)。免疫组化染色发现GCGGGT纯/杂合子组HCC较其他单体型HCC的HTPAP表达显著降低(P=0.035)。Kaplan-Meier分析提示,GCGGGT纯/杂合子组HCC较其他单体型HCC术后易复发且预后差(P<0.001)。Cox风险比例模型发现GCGGGT单体型是HCC术后复发及预后差的独立相关因子。结论 HCC的不同HTPAP单体型在mRNA水平的表达差异无统计学意义,但在蛋白水平存在显著差异。GCGGGT单体型可作为HCC术后复发和预后差的预测指标。

Objective To investigate the association of HTPAP haplotype with gene expression and prognosis of hepatocellular carcinoma （HCC）, so as to evaluate whether HTPAP haplotype could affect recurrence and overall survival （OS） of HCC. Methods We evaluated the relationship between HTPAP expression and haplotypes in randomly selected 377 HCC samples composed of 4 kinds of HTPAP haplotype （AGCTAC, GCGGGT, AGCTGC and GCGGAT） by quantitive real-time PCR, immunohistochemical and Western blot analysis. We further employed pyrosequencing （PSQ96） todetect and reconstruct genotypes in another independent 665 HCC samples in order to determine whether the haplotypes to be associated with time to recurrence （TTR） or OS of HCC. Results Totally 327 of 377 HCC samples were composed of the above HTPAP haplotypes. The gene expression levels in groups of GCGGGT homozygote and AGCTAC/GCGGGT heterozygotes were lower than those in groups of CATCGA homozygote, AGCTAC/AGCTGC heterozygotes and AGCTAC/GCGGAT heterozygotes, but no significance was found between them （P = 0. 062）. Immunohistochemical analysis revealed that the protein expression levels in groups of GCGGGT homozygote and heterozygotes were significantly lower than those in other haplotypes groups （P = 0. 035）. Kaplan-Meier analysis showed that patients with GCGGGT haplotype exhibited shorter TTR and shorter postoperative OS （all P~ 0. 001） compared with those without GCGGGT haplotype. Cox multivariate regression analysis indicated that GCGGGT haplotype was an independent predicator for HCC postoperative recurrenc. Conclusions Different HTPAP haplotypes of HCC have no statistical difference in mRNA expression level, but significant differences in protein expression level. GCGGGT haplotype was an independent predicator for postoperative recurrence and poor prognosis in HCC.