肾透明细胞癌中X染色体生殖同源盆基因F1(Rhox F1)表达及其甲基化状态研究

Rhox F1 expression and its methylation in renal clear cell carcinoma

目的:探讨人肾透明细胞癌组织中X染色体生殖同源盒基因F1(Rhox F1)启动子CpG岛甲基化及其mRNA表达情况,分析MTHFR C677T不同基因型Rhox F1甲基化及mRNA的表达情况。方法:收集我院手术切除的38例肾透明细胞癌组织(肾癌组)及其配对的癌旁正常肾脏组织区(正常组)标本。采用实时PCR和甲基化特异PCR(MSP)技术分别检测肾癌组和正常组的Rhox F1 mRNA的表达和甲基化状态。采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)技术分别检测MTHFR基因677(C→T)多态性,分析MTHFR C677T不同基因型Rhox F1启动子去甲基化率及mRNA表达情况。结果:正常组中Rhox F1基因mRNA阳性表达率36.8%明显低于肾癌组的89.5%,差异有统计学意义(χ~2=22.619,P<0.001)。正常组中Rhox F1基因启动子甲基化率为78.9%,显著高于肾癌组的18.4%(χ~2=27.861,P<0.001)。34例Rhox F1 mRNA阳性表达的肾癌组织[MTHFR 677(C→T)]中TT基因型患者25例,CT基因型患者5例,CC基因型患者4例,Rhox F1 mRNA表达水平差异具有统计学意义(F=80.201,P<0.001),且TT基因Rhox F1 mRNA表达水平(0.91±0.01)显著高于CT基因型(0.33±0.03)和CC基因型(0.26±0.03),差异均有统计学意义(均P<0.001)。TT基因型中有24例Rhox F1启动子去甲基化(96.0%);CT基因型中有2例(40.0%);CC基因型中有1例(25.0%)。TT、CT、CC三组基因Rhox F1启动子去甲基化率差异具有统计学意义(P<0.001),其中TT基因Rhox F1启动子去甲基化率显著高于CT基因型和CC基因型(P=0.009,P=0.004)。结论:Rhox F1 mRNA在肾癌组织中高表达,Rhox F1启动子去甲基化率在肾癌组织中较高。MTHFR基因677(C→T)中TT基因型肾癌患者Rhox F1 mRNA表达水平和启动子去甲基化阳性率高于CT和CC基因型。

Objective To investigate the methylation and mRNA expression of CpG island in the promoter of X-chromosome homeobox gene F1(Rhox F1) in human renal clear cell carcinoma, and to analyze the methylation and mRNA expression of Rhox F1 in different MTHFR C677 T genotypes.Method A total of 38 cases of renal clear cell carcinoma(renal carcinoma group) and adjacent normal kidney tissue specimens(normal group) were collected in the Affiliated Hospital of Xuzhou Medical College after nephrectomy. The expression and methylation status of Rhox F1 gene in renal carcinoma group and normal group were detected by real-time PCR and methylation specific PCR(MSP). The MTHFR gene 677(C→T) polymorphism was detected by restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP), and the demethylation rate and mRNA expression of Rhox F1 promoter of different MTHFR C677 T genotypes were analyzed.Results The positive expression rate of Rhox F1 mRNA in the normal group(36.8%) were significantly lower than that in the renal cancer group(89.5%),the difference was statistically significant(χ2=22.619,P<0.001). Total methylation rate of Rhox F1 MRNA promoter in the normal group(78.9%) were higher than that in the renal cancer group(18.4%),the difference was statistically significant(χ2=27.861,P<0.001). There were 25 cases of TT genotype, 5 cases of CT genotype and 4 cases of CC genotype in 34 cases of renal cell carcinoma with Rhox F1 positive expression [MTHFR 677(C→T)]. The difference of Rhox F1 mRNA expression levels between TT, CT and CC genes was statistically significant(F=80.201,P<0.001) and the TT gene Rhox F1 mRNA expression level(0.91±0.01) was significantly higher than that in CT genotype(0.33±0.03) and CC genotype(0.26±0.03), and the differences were statistically significant(all P<0.001). There were 24(96.0%),2(40.0%) and 1(25.0%) Rhox F1 promoters demethylated in the TT genotype, in the CT genotype and in the CC genotype respectively. The demethylation rate of Rhx F1 promoter in TT, CT and CC groups was statistically significant(P<0.001). The demethylation rate of TT gene Rhox F1 promoter was significantly higher than CT genotype and CC genotype(P=0.009, P=0.004).Conclusion Rhox F1 mRNA is highly expressed in renal cell carcinoma, and the rate of demethylation of Rhox F1 promoter is higher in renal cell carcinoma. Among MTHFR C677 T genotypes, the expression level of Rhox F1 mRNA and the rate of Rhox F1 promoter demethylation of renal cancer patients with TT genotype were higher than those of others.