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孕烷X受体(pregnane X receptor,PXR)可通过调节细胞色素P450同工酶3A4—CYP3A4的表达而影响肿瘤细胞对化疗的敏感性,而其表达水平则会受到自身基因甲基化的影响.本文研究了结肠癌组织中pxr基因甲基化的分布情况及其对pxr,cyp3a4表达的影响,并在多种结肠癌细胞系中分析了pxr基因甲基化是否与5-氟尿嘧啶(5-FU)耐药性相关.收集结肠癌病灶区、癌旁区及正常结肠组织样本,分别提取基因组DNA及RNA.PCR-限制性酶切分析检测pxr基因外显子3甲基化;real-time PCR检测pxr及cyp3a4基因的表达.鉴定LOVO、LS180、LS174T、HT29、HCT116等5种结肠癌细胞中pxr外显子3甲基化与pxr,cyp3a4表达的相关性并分别筛选出PXR高/低表达的细胞株进行5-FU耐药性分析.结果显示,结肠癌病灶组织中pxr外显子3甲基化频率显著增加,伴有pxr,cyp3a4表达的增强.在结肠组织及结肠癌细胞系中,pxr与cyp3a4的表达均密切相关,且均与pxr甲基化程度相关.PXR高表达细胞株LS180对5-FU的耐药性显著升高,以siRNA分别下调pxr及cyp3a4的表达,均可增加LS180对5-FU的敏感性.结果提示,pxr基因外显子3区甲基化与PXR及CYP3A4的高表达密切相关,并与结肠癌细胞对5-FU的抗药性相关.
Pregnane X receptor (PXR) can affect the chemosensitivity of tumor cells by regulating the expression of cytochrome P450 isoenzyme 3A4-CYP3A4, and its expression level will be affected by its own gene methylation In this paper, we investigated the distribution of pxr methylation and its effect on the expression of pxr and cyp3a4 in colon cancer tissues and analyzed whether pxr methylation was associated with 5-fluorouracil (5- FU) resistance.Collect colon cancer, paracancer and normal colon tissue samples were extracted genomic DNA and RNA.PCR-restriction analysis of pxr gene exon 3 methylation; real-time PCR was used to detect the expression of pxr and cyp3a4 genes and the correlation between pxr exon 3 methylation and pxr and cyp3a4 expression in 5 colon cancer cell lines including LOVO, LS180, LS174T, HT29 and HCT116, and the high / low PXR The results showed that the methylation frequency of exon 3 of pxr in colorectal cancer tissues was significantly increased accompanied with the increase of pxr and cyp3a4 expression in colon and colon cancer cell lines , Pxr and cyp3a4 expression are closely related, and both pxr methylation.PXR high expression cell line LS180 resistance to 5-FU was significantly increased, respectively, siRNA downregulation of pxr and cyp3a4, can increase LS180 on 5-FU sensitivity.The results suggest that, The methylation of exon 3 of pxr gene is closely related to the high expression of PXR and CYP3A4, and is related to the drug resistance of colon cancer cells to 5-FU.