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首页> 外文学位 >Mechanistic Study of the Effect of CDH1 Promoter Hypermethylation on Drug Resistance and Related Gene Expression in Multidrug Resistant Human Hepatocellular Carcinoma R-HepG2 Cells.
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Mechanistic Study of the Effect of CDH1 Promoter Hypermethylation on Drug Resistance and Related Gene Expression in Multidrug Resistant Human Hepatocellular Carcinoma R-HepG2 Cells.

机译：CDH1启动子甲基化对多药耐药人肝细胞癌R-HepG2细胞耐药性及相关基因表达影响的机理研究。

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摘要

"Epigenetic" refers to a heritable change in the gene expression pattern that is not mediated by any alterations in the primary nucleotide sequence of a gene in the genome. This change involves methylation of DNA in the gene promoter regions, modification of histone residues and chromatin remodeling. Among them, methylation of DNA promoter region is an essential step in epigenetic gene silencing and is known to be closely related to carcinogenesis and cancer progression.;The aim of this study was to explore whether any methylation of DNA promoters mechanism is involved in drug resistance of a doxorubicin-induced human multidrug resistant hepatocellular carcinoma sub-linage R-HepG2 which was established from the doxorubicin sensitive HepG2 cell line in our laboratory. In this project, it was observed that the DNA promoter methylations of ESR1, Rassf2A, CDH1 and MDR1 in R-HepG2 were higher than those in HepG2 cells respectively by methylation specific polymerase chain reaction method. Bisulfite sequencing showed that the total 32 CpGs of CDH1 promoter region in R-HepG2 cells were hypermethylated while they were hypomethylated in HepG2 cells. CDH1 is the encoding gene of E-cadherin. The promoter hypermethylation induced CDH1 silencing in R-HepG2 cells was confirmed by reverse transcription polymerase chain reaction and Western blotting that CDH1 transcription and E-cadherin expression were maintained in HepG2 cells but both were lost in R-HepG2 cells. RT-PCR of 10 multidrug resistant related genes revealed that transcription of MDR1 was obviously increased in R-HepG2 cells, transcription of MRP1 and MRP5 were slightly increased in R-HepG2 cells, transcription of MRP6 and BCRP were slightly decreased in R-HepG2 cells comparing to those in the parental HepG2 cells. This result suggests that up-regulation of P-glycoprotein expression which is the protein product of MDR1 may be one of the major causes of multidrug resistance in R-HepG2 cells. Transient transfection of CDH1 cDNA increased the CDH1 transcription and E-cadherin expression in R-HepG2 cells. I also found that the CDH1 transfected R-HepG2-CDH1 cells showed increased amount of doxorubicin uptake, increased apoptotic population of cells exposed to doxorubicin, suppressed cell migration, and decreased P-glycoprotein expression comparing to those in R-HepG2 cells. It was also found that the transcription levels of SNAI2, TWIST1, ASNA1 and FYN were obviously higher in R-HepG2 cells than those in HepG2 cells. The transcription of FYN and TWIST1 were obviously decreased in CDH1 cDNA transfected R-HepG2-CDH1 cells which displayed a negative correlation with the transcription level of CDH1 and these results imply a suppressive role of CDH1 in regulating these genes which were involved in cancer metastasis and multidrug resistance.;Our preliminary study on effect of treatments of some potential anti-cancer drug candidates, namely Pheophorbide A (Pa), Pa combining with photodynamic therapy, Polyphyllin D (designated as HK-18), and its derivative designated as HK-27 on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that the promoter methylation of CDH1 was decreased in response to treatments of Pa, HK-18, and HK-27 in MDA-MB-231 cells.
机译：“表观遗传的”是指基因表达模式的可遗传改变，其不受基因组中基因的一级核苷酸序列的任何改变的介导。这种变化涉及基因启动子区域中DNA的甲基化，组蛋白残基的修饰和染色质重塑。其中，DNA启动子区域的甲基化是表观遗传基因沉默的重要步骤，并且已知与癌变和癌症进展密切相关。这项研究的目的是探讨DNA启动子机制的任何甲基化是否与药物抗性有关。由我们实验室中对阿霉素敏感的HepG2细胞系建立的阿霉素诱导的人多药耐药性肝细胞癌亚型R-HepG2的研究。在该项目中，通过甲基化特异性聚合酶链反应法观察到，R-HepG2中ESR1，Rassf2A，CDH1和MDR1的DNA启动子甲基化分别高于HepG2细胞中的甲基化。亚硫酸氢盐测序显示，R-HepG2细胞中CDH1启动子区域的总共32个CpGs被高甲基化，而在HepG2细胞中被低甲基化。 CDH1是E-钙粘蛋白的编码基因。通过逆转录聚合酶链反应和蛋白质印迹证实了启动子高甲基化诱导的R-HepG2细胞CDH1沉默，CDH1转录和E-钙粘蛋白表达在HepG2细胞中得以维持，但在R-HepG2细胞中均丢失。 10个耐多药相关基因的RT-PCR显示，R-HepG2细胞中MDR1的转录明显增加，R-HepG2细胞中MRP1和MRP5的转录略有增加，R-HepG2细胞中MRP6和BCRP的转录略有减少与亲本HepG2细胞相比。该结果表明，作为MDR1的蛋白质产物的P-糖蛋白表达的上调可能是R-HepG2细胞中多药耐药性的主要原因之一。 CDH1 cDNA的瞬时转染增加了R-HepG2细胞中CDH1转录和E-cadherin表达。我还发现，与R-HepG2细胞相比，经CDH1转染的R-HepG2-CDH1细胞显示出增加的阿霉素摄取量，暴露于阿霉素的细胞凋亡群体增加，细胞迁移受到抑制以及P-糖蛋白表达降低。还发现R-HepG2细胞中SNAI2，TWIST1，ASNA1和FYN的转录水平明显高于HepG2细胞。在转染了CDH1 cDNA的R-HepG2-CDH1细胞中，FYN和TWIST1的转录明显降低，这与CDH1的转录水平呈负相关，这些结果暗示CDH1在调节这些与癌症转移和转移有关的基因中具有抑制作用。我们对某些潜在的抗癌药物，例如Phophophidebide A（Pa），Pa结合光动力疗法，Polyphyllin D（命名为HK-18）及其衍生物HK-的治疗效果的初步研究。在人乳腺癌细胞系MCF-7和MDA-MB-231上的27显示，在MDA-MB-231细胞中，响应于Pa，HK-18和HK-27的处理，CDH1的启动子甲基化降低。

著录项

  • 作者

    Jiang, Lei.;

  • 作者单位

    The Chinese University of Hong Kong (Hong Kong).;

  • 授予单位 The Chinese University of Hong Kong (Hong Kong).;
  • 学科 Oncology.;Cellular biology.;Biochemistry.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 171 p.
  • 总页数 171
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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