Regulatory Mechanism Of Aberrant Methylation Of IGFBP-rP1 In Colorectal Carcinoma

Colorectal cancer (CRC) is one of the prevalent malignant tumors that threaten our health. In spite of the development of radiotherapy, chemotherapy and bio-targeted therapy, the mortality declines indistinctively. In China, the CRC incidence grows rapidly, especially in ZheJiang, ShangHai and JiangSu provinces. The low early detection rate is one of the important factors affecting the cancer therapy, so the early detection will promote the cancer prognosis.In 1999, our laboratory built three cDNA libraries using SSH (suppression subtractive hybridization):adenoma VS normal mucosa(A-N), adenocarcinoma VS adenoma(T-A), adenocarcinoma VS normal mucosa(T-N), and screened a series of genes with differential expression. IGFBP-rP1 (insulin-like growth factor binding protein-related protein 1) was screened from adenocarcinoma VS normal mucosa (T-N) cDNA subtraction library and is overexpressed in the colorectal adenocarcinoma tissue.IGFBP-rP1 is also named as IGFBP7 (Insulin-like growth factor binding protein 7), mac25 (meningioma associated cDNA 25), PSF (prostacyclin-stimulating factor), AGM (angiomodulin) and TAF (tumor-derived adhesion factor). It belongs to the IGFBP family and is a member of insulin-like growth factor (IGF) axis. IGFs have multiple functions regulating cell differentiation, proliferation and apoptosis. IGFBPs can bind IGFs and modulate the bioavailability of IGFs in the circulation. IGFBP-rPl, as a secreted protein, is widely detected in different types of body fluids, such as serum, urine, amniotic fluid and cerebrospinal fluid, and in various types of organs, such as thymus, prostate, bladder, liver, lung, stomach and colon. The majority view is that IGFBP-rPl plays a potential tumor suppressor role against human carcinogenesis with complex control mechanism, because it is down-regulated in most human tumors, including prostate carcinoma, breast carcinoma, liver carcinoma and meningioma, and overexpression of IGFBP-rPl can induce the apoptosis, arrest cell cycle and inhibit the proliferation of immortalized or malignat human cells. However, it has been reported that there is an opposite IGFBP-rPl expression pattern in several cancers, including colorectal cancer. This paradoxical phenomenon implied a reversible regulatory mechanism in this gene. Lin et al. found DNA methylation is the main regulatory mechanism underlying aberrant IGFBP-rPl expression in colorectal cancer, and aberrant methylation of the exon 1 of IGFBP-rP1 is inversely correlated with the expression of IGFBP-rP1 in colorectal cancer.Gene expression is regulated by two main mechanisms, genetics and epigenetics. Alterations of gene sequence are the typical situation of genetic modification, including gene amplifications, gene deletions, point mutations, loss of heterozygosity, chromosomal rearrangements, and overall aneuploidy, However, Epigenetic changes occur at a higher frequency than genetic changes and can be reversible upon treatment with pharmacological agents. Epigenetic modification is defined as a heritable, reversible change in gene expression that does not result from DNA sequence alterations, such as DNA methylation and demethylation, histone acetylation and deacetylation, non-coding RNA, etc. Aberrant DNA methylation is the most common type of epigenetic mechanism. It has become clear that hypermethylation of the 5'-flanking CpG islands silences gene expression and many suppressor genes are regulated by this pathway, abnormal DNA methylation is considered an early event in human carcinogenesis.DNA methylation is an epigenetic event in which DNA methyltransferases (DNMTs) cause the addition of a methyl group connected to the fifth carbon position of a cytosine residue in CpG dinucleotides. The mammalian DNMTs family mainly encompasses DNMT1, DNMT3a and DNMT3b. In mammals, DNMT3a and-3b are mainly responsible for establishing methylation at previously unmethylated sites, whereas DNMT1 is the major maintenance methyltransferase, reproducing existing methylation patterns during cell division. Selective targeting of DNMTs can result in gene-special demethylation and reexpression of many suppressor genes.The PcG protein EZH2 (Enhancer of Zeste homolog 2) is a histone methyltransferase associated with transcriptional repression. The Polycomb group protein EZH2 controls CpG methylation through direct physical contact with DNA methyltransferases. EZH2, as part of the PRC2/3 complexes, can physically recruit DNMTs to certain target-genes and that this process is essential for silencing the genes.Recent reports have demonstrated that the transcription of the Polycomb group (PcG) gene EZH2 is controlled by growth factors through the pRB-E2F pathway, which affects its interaction with DNMT1, thus this exerts influence of methylation. Downregulation of the potential suppressor gene IGFBP-rP1 is associated with inactivation of the retinoblastoma protein in human breast cancer. Then what is the relationship between RB and the aberrant DNA methylation of IGFBP-rPl and what is the role of DNMTs in the regulatory mechanism of the aberrant DNA methylation of IGFBP-rP1? It is our task to make them clear.In this study, we found that DNMTs, EZH2 and RB all express in four colorectal cell lines(RKO, HT29, SW620 and SW480). In order to disclose the regulatory mechanism of aberrant methylation of IGFBP-rPl, RNA interference (RNAi) technology was chosen to delete the expression of DNMTs, EZH2 and RB in colorectal RKO cell line without the endogenous IGFBP-rP1 expression.In view of the important role of DNMTs on the methylation in human cancer cells, our research was started from the study whether DNMTs take part in the aberrant DNA methylation of IGFBP-rP1. The expression of DNMT1, DNMT3a and DNMT3b were deleted respectively in colorectal RKO cell line and stable transfection cell lines were built with low level of DNMT1, DNMT3a, DNMT3b, but the restoration of IGFBP-rPl expression were not observed, so co-transfection was used to delete two of them at the same time, and the weak re-expression of IGFBP-rP1 was detectd in the monoclone cell line with decrease of DNMT1 and DNMT3b simultaneously, and the methylation status of IGFBP-rPl exon 1 was detected by MSP and BSP. The results of ChIP(Chromatin Immunoprecipitation) illustrate that not DNMT3b but the protein of DNMT1 can bind the IGFBP-rP1 exon 1 directly. Based the above results, we made it clear that cooperation of DNMT1 and DNMT3b plays an important role in the aberrant DNA methylation of IGFBP-rP1 in colorectal RKO cell line.Because EZH2 and RB were reported to control DNMTs recent years, our concern is wether they also are involved in the regulatory of aberrant DNA methylation of IGFBP-rP1. EZH2 was deleted effectively in RKO, but we didn't observed the restoration of IGFBP-rP1 expression, while interference of RB gene in RKO induced the re-expression of IGFBP-rP1, The monoclone cell lines of selective deletion of RB were built to detect the methylation status of IGFBP-rP1 exon 1 by BSP. There is not interaction between RB and IGFBP-rP1 exon 1 in ChIP result, but we found the reduction of DNMT1 and DNMT3b in the RKO-RBi cell line.From the above results, we drew the following conclusions:1. Selective deletion of both DNMT1 and DNMT3b simultaneously in RKO cell can restore the IGFBP-rP1 expression, and DNMT1 binds the exon 1 of IGFBP-rP1 directly.2. Respective knockdowns of EZH2, DNMT1, DNMT3b and DNMT3a can not influence the expression of IGFBP-rP1.3. Decrease of RB in RKO cell can induce the hypomethylation of IGFBP-rP1 and restore the expression of IGFBP-rPl.4. Deletion of RB can result in the reduction of DNMT1 and DNMT3b, which maybe a pathway that RB regulates the methylation of the exon 1 of IGFBP-rP1.