The Mechanism Of DNA Methylation Regulating The Expression Of CLDN6and Effect Tight Junction Function In Human Breast Cancer Cell MCF-7

Backgrounds:Breast cancer is one of the common malignant tumors in the female, and itsincidence is increasing year by year. The main reason for patient's death is tumormetastasis. Several research reported that abnormal expression of tight junction(TJ)protein may play an important role in malignant tumor.TJs are including occludin, Claudins (CLDNs), junctional adhesion molecules(JAM)and periphery plasmosin zonula occludens (ZO-l, ZO-2and ZO-3). Tightjunctions (TJ) are located at the extreme apical region of junction-associatedcomplexes in epithelial and endothelial cells, where they play important roles inmaintaining the epithelial permeability barrier and cell polarity. The barrier function isa physical barrier around the cells, which can close the gap of epithelial cells, andselectivity through soluble substances; Fence function is the lipid composition andprotein composition that separated the top side of epithelial cells,which maintainedthe cell polarity and not affected the two components of membrane. The destructionof TJ function is performed as barriers to the loss of cell polarity and signalingpathways. After the destruction of cell polarity, growth factors are enter into cells inthe form of autocrine and paracrine to stimulate epithelial carcinogenesis, through P38pathway activation the expression PKC modulation CLDNs to affect cell migration.CLDNs are a multi-gene family contains27members and their expressionpattern is tissue specificity. CLDNs are the most important backbone protein thatmaintains the structure and function of TJ. Molecular weight of CLDN6is23kDaand located on chromosome16p13.3. It is important for maintaining permeabilitybarriers and transepithelial resistance of epidermal cells. In previous work, we foundthat the expression of CLDN6was undetectable or down-regulated in human and rat mammary cancer cell lines. These results suggest that CLDN6may play a negativeregulatory role as a tumor suppressor gene in breast cancer development.Currently available studies suggest that tumor suppressor gene silencingassociated with epigenetic modification. Epigenetics is defined relative to the genetics,refers to the gene transcription before, do not change the sequence of chromatinstructural adjustment, influence gene expression, including DNA methylation, histonemodifications and chromatinremodeling, etc. DNA methylation is the process ofS-adenosylmethionine methyl transferred to5carbon atoms of cytosine, which iscatalyzed by DNA methyltransferase (DNA methyltransferase, DNMTs). There arethree mechanisms about DNA methylation leads to transcriptional repression:1.Preventing transcription factor binding by block Activation transcription factorrecognition sites;2. mediating transcription of tumor suppressor genes bycombination of Specific methylation bing protein and DNA methylation sequence,such as the MBD family;3. DNA methylation could also repress gene transcription byinduce histone modifications through DNA methylation binding protein collectedhistone modifying enzymes, such as histone deacetylases and histonemethyltransferase, which Causing chromatin conformational change. All of thesemechanisms interacted together to complete the regulation of tumor suppressor gene.Objects:This study sought to determine the mechanism of DNA methylation regulatingthe expression of CLDN6and the impact of DNA methylation on TJ function inMCF-7cell.Methods:1. Expression of CLDN6in breast cancer cell: RT-PCR, western blotting,immunofluorescent staining were used to determine the expression of CLDN6inHBL-100, MCF-7and MDA-MB-231cells.2. Expression of CLDN6and DNA methylation:RT-PCR, western blotting,immunofluorescent staining were used to determine the expression of CLDN6inMCF-7cells without or with5-Aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A(TSA) treated; Bisulfite Sequencing PCR (BSP) was used to determine methylation status at the CLDN6gene; Immunofluorescent staining was used to determine theexpression of CLDN6and DNMT1in breast invasive ductal carcinoma tissue, andanalysis their correlation.3. The mechanism DNA methylation regulating the expression of CLDN6:RT-PCR and western blotting were used to determine the expression of methy-CpGbinding domains (MBDs), DNA methyltransferase1(DNMT1), SUV39H1, H3-lysine9-demethylation (H3K9me2), H3-lysine9-trimethylation(H3K9me3),ChIP was usedto determine the combination of CLDN6with MBD1,2,3, DNMT1, H3K9me2,H3K9me3and SUV39H1; Nuclease accessibility assays was performed to obtaininformation about the impact of DNA methylation on chromatin conformation.4. The effects DNA methylation on TJ function of MCF-7: Treating MCF-7cellswith5-aza-Dc and TSA separately or simultaneously, then using transmission electronmicroscopy assay to determine TJ ultrastructure in these cells. Transepithelialelectrical resistance assay, paracellular tracer flux assay and transepithelial potentialassay were used to TJ function. RNAi was used to determine the knockdown CLDN6in MCF-7cells.Results:1. Expression of CLDN6in breast cancer cell: The expression of CLDN6inMCF-7and MDA-MB-231cells was lower than HBL-100. The Expression waslocated at cell membrane and cytoplasm.2. Expression of CLDN6and DNA methylation: MCF-7cells CLDN6genepromoter region (+1bp to+210bp) was methylated;5-aza-dC and TSA could inducethe expression of CLDN6separately or simultaneously. The expression of CLDN6inbreast invasive ductal carcinoma tissue was lower than control group, and lymphodemetastasis group was lower than control group; The expression of DNMT1in breastinvasive ductal carcinoma tissue was higher than control group; Expression ofCLDN6and DNMT1correlation analysis showed that no obvious correlation, butthere had the opposite trend.3. The mechanism DNA methylation regulating the expression of CLDN6:5-aza-dC and TSA can inhibit the combination of CLDN6with MBD1, DNMT1, H3K9me2and H3K9me3, TSA can promote the combination of CLDN6withSUV39H1; chromatin structure of CLDN6gene more closely between-300bp to+300bp, after treated with5-aza-dC and TSA separately or simultaneously, thechromatin structure becomes loose.4. The effects DNA methylation on TJ function of MCF-7: Treating MCF-7cellswith5-aza-dC and TSA separately or simultaneously could increase TJ number andtransepithelial electrical resistance, decrease FITC-dextran transmission, but therewere no effect on transepithelial potential and PNa/PCl; Slience expression ofCLDN6in the5-aza-dC and TSA separately or simultaneously treated group wouldenhance the migration and metastatic ability, but there were no effect ontransepithelial electrical resistance, transepithelial potential and PNa/PCl.Conclusions:1. CLDN6expression was related to DNA methylation in MCF-7cells anddemethylation could upregulate the expression of CLDN6.2. DNA methylation mediated down-regulation CLDN6was related with thebinding between the promoter of CLDN6and the proteins of MBD1, DNMT1,H3K9me2and H3K9me3, and was related with alteration of the chromatin structure.3. DNA methylation down-regulated CLDN6expression inhibited TJ function ofMCF-7cells.