Genetic Or Epigenetic Changes Down-regulation Of Imprinted Gene SLC22A18 Expression In Human Breast Cancer

Breast cancer is the most common disease among women worldwide.Although China has the low morbidity of breast cancer,its yearly increasing rate is 1%-2% higher than the average rate in the world.Over the past ten years,the ever-increasing incidence has made breast cancer one of the most frequent malignancies among Chinese women.The precise molecular mechanism of breast cancer is not well understood.Many studies have provided evidence of genetic alterations in breast cancers such as point mutations,loss of heterozygosity(LOH),and homozygous deletions in many tumor suppressor genes,which are associated with carcinogenesis.More recently,it has also been reported that epigenetic plays an important role in the development and progression of human breast cancer.The field of epigenetic includes DNA methylation,genomic imprinting and chromatin remodelling.Genomic imprinting is a form of epigenetic inheritance that distinguishes maternal and patemal alleles. Genomic imprinting plays a critical role in fetal growth,cell proliferation and adult behavior.There are numerous reports of tumors showing imprinted genes also involved in carcinogenesis.It is now well established that DNA methylation is involved in regulating gene transcription repression.Evidences indicate that DNA methylation is the main mechanism of genomic imprinting.Recently,more and more study show the aberrant expression and the variation imprinting status may be a critical biomarker in carcinogenesis.SLC22A18(solute carrier family 22,member 18 ),a paternally imprinted gene is located in chromosome 11p15.5,also known as TSSC5,BWR1A,IMPT1 and ORCTL2,encodes an effiux transporter-like protein with ten transmembrane domains, whose regulations may contribute to drug sensitivity,cellular metabolism and growth. SLC22A18 mRNA expression is observed in the fetal kidney and liver,and also in multiple adult tissues such as the liver,kidney,prostate and colon.The protein is found on the apical membrane surfaces of the proximal tubules in human kidney.It has been speculated that it may be involved in resistance in export of genotoxic substances whose retention may increase the risk of tumor formation.More recently, TSSC5 has been shown to be a substrate for a conserved E3 ubiquitin ligase RING105, and the manipulation of RING 105 may assist in restoring TSSC5 function and normal growth control in cancer.Structural mutations,deletion and abnormal imprinting patterns of SLC22A18 have been identified in a number of sporadic tumors,including Wilm's tumor,hepatoblastomas,hepatocelluar carcinomas and breast cancer.These resluts indicated that SLC22A18 may play a role in tumorigenesis.The molecular mechanisms that lead to inactivation of the SLC22A18 gene remain poorly understood.Because approximately 80%of breast tumors were diagnosed as infiltrating ductal carcinomas(IDCs),we searched for genetic alterations,mutation and allelic loss,and the methylation status of SLC22A18 gene in patients with IDCs in China.Part 1 Expresstion of imprinted gene SLC22A18 in infiltrating ductal breast carcinomaPurpose To investigate the expression of imprinted gene SLC22A18 in breast cancer and breast benign diseases.Then the expression and its clinical relevance in breast caner were explored.Methods Real-time quantitative reverse transcriptase-polymerase chain reaction(Realtime RT-PCR) and immunohistochemistry(IHC) was applied on 56 cases of infiltrating ductal breast carcinoma(IDC),56 cases of corresponding adjacent non-cancerous tissues and 20 benign breast tissues in order to detect mRNA and protein expression of SLC22A18 gene.Statistical analysis was carried out to analyze the correlation between SLC22A18 gene expression and various clinical parameters in these breast cancer patients.Results SLC22A18 mRNA expression in 56 IDC tissues was lower than that in all corresponding adjacent non-cancerous tissues(z=-4.15,P＜0.01).SLC22A18 mRNA expression was lower in breast cancer cases,when compared with that in benign cases(z=-2.76,P＜0.01).SLC22A18 mRNA expression in 48 IDCs was lower than that in 8 dural carcinoma in situ(part of IDC)(z=-2.19,P＜0.05).There was a decreased or completely diminished SLC22A18 protein expression in breast cancer.A significant difference of SLC22A18 protein expression was also observed in IDC and benign groups(P＜0.01).Neither mRNA nor protein expression of SLC22A18 gene correlated with clinicopathologic parameters such as age of patients, size of tumor,clinical stage,pathologic subtype,histologic grade or lymph node metastasis(P＞0.05).Conclusions Our results indicate that SLC22A18 expression is markedly down-regulated in IDC.Decreased expression of SLC22A18 gene may play an important role in the carcinogenesis of IDC.PartⅡMutation,loss of heterozygosity and genomic imprinting of SLC22A18 in human breast cancerPurpose To clarify the role of SLC22A18 in breast cancer,we examined the gene for mutation,loss of heterozygosity,and genomic imprinting of SLC2218 in IDC,and explore the relationship between mutation,deletion,genomic imprinting and SLC22A18 expression.Methods Single strand conformation polymorphism analysis of polymerase chain reaction products(PCR-SSCP),polymerase chain reaction-restricted fragments length polymorphism(PCR-RELP) and DNA sequencing were used to detect the mutation,deletion,and genomic imprinting of SLC2218 in 56 paired IDC tissues. Then the molecular mechanism of breast cancer was explored.Results Of the 56 paired IDC,only 1 showed abnormal SSCP band shift of exon9.DNA sequencing showed C deletion at 1087 locus,resulting in a stop codon TGA.No band shifts of exon 7 were found in 56 IDCs and corresponding adjacent non-cancerous tissues.We screened 56 paired samples and identified 23 heterozygotes of SLC22A18 in corresponding adjacent non-cancerous tissues,and 19 heterozygotes in IDCs.So loss of heterozygosity(LOH) occurred in 4(17.3%) of 23 informative IDCs.SLC22A18 showed a gain of imprinting(GOI) in 5/19(26.3%) IDCs.GOI was also demonstrated in 2/19(10.5%) corresponding adjacent non-cancerous tissues.2 of 8 dutal carcinoma in situ(part of IDC) also gained imprinting.In the specimens with SLC22A18 DNA heterozygote,SLC22A18 mRNA expression in 4 LOH of IDCs was lower than that in 19 heteozygotes in IDCs(P＜0.05).SLC22A18 mRNA expression in GOI samples was lower than that in 14 no GOI samples(P＜0.05).LOH and GOI did not correlat to SLC22A18 protein expression.Conclusions The phenomena of mutation,LOH and GOI of SLC22A18 were occurred in IDCs.LOH and GOI were related to the transcriptional expression.LOH and GOI of SLC22A18 may be involved in breast carcinogenesis and prognosis.PartⅢSLC22A18 promoter aberrant methylation in breast cancerPurpose To study the methylation status of imprinted gene SLC22A18 in IDCs, and the correlation between methylation status and clinical characteristics in IDCs. Further,to investigate the expression and promoter methylation in IDCs.Methods The methylation status at the promoter regions of SLC22A18 gene was examined by methylation-specific polymerase chain reaction(MSP) in the specimens of IDC from 56 patients.Results By employing MSP we determined the frequency of aberrant methylation for SLC22A18 in 56 IDCs samples and their corresponding adjacent non-cancerous tissues.The percentages of methylation for SLC22A18 in 56 IDCs were 73.2%and in the corresponding adjacent non-cancerous tissues,39.3%, respectively.The frequency of methylation of SLC22A18 in 56 IDCs was higher than that in the corresponding adjacent non-cancerous tissues(73.2%versus 39.3%,P＜0.01).The mRNA and protein expression of SLC22A18 gene in methylation group of IDCs was significantly reduced when compared with that of the unmethylation group of IDCs(P＜0.01,P＜0.05).The frequency of methylation of SLC22A18 was not correlated with clinicopathologic parameters such as age of patients,size of tumor, clinical stage,pathologic subtype,histologic grade or lymph node metastasis(P＞0.05).Conclusions The reduced expression of SLC22A18 was associated with aberrant DNA methylation in the promoter region.Promoter methylation was related to status of imprinting.Detection of promoter methylation is potentially usedful as epigenetic marker in tumor progress.PartⅣEffects on the growth inhibition of human breast carcinoma cell and reversion of SLC22A18 methylation by 5-aza-2'-deoxycytidine and Trichostatin APurpose In order to further study whether DNA methylation is the direct cause of aberrant SLC22A18 expression,breast cancer cell line MDA-MB-231 was treated with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine(5-aza-dC) and histone deacetylase inhibitor Trichostatin A(TSA).Meanwhile,we observed the changes of cell growth and cell cycle after treatment.Methods Cell proliferation,cell cycle,the level of methylation and expression of SLC22A18 in breast cancer cell line MDA-MB-231 after treatment with 5-aza-dc and/or TSA was observed under WST-1 method,FCM,MSP,Realtime RT-PCR and Western blot.Results Treatment with 5-aza-dc alone or 5-aza-dc/TSA on breast cancer cell lineMDA-MB-231 could reverse aberrant hypermethylation of the SLC22A18 promoter and increase SLC22A18 mRNA and protein expression.Combination of 5-aza-dc and TSA could further reverse hypermethylation of the promoter and up-regulated the expression.The results suggesting that up-regulation of SLC22A18 expression was the direct effect of demethylation.We also found that the proliferation of breast cancer cells was suppressed and the cell cycle was arrested in S stage after treatment with 5-aza-dc and TSA.Conclusions SLC22A18 promoter aberrant methylation was associated with a decease its expression.Aberrant DNA methylation may be the main mechanism of down-regulation of SLC22A18 in IDC.The combination of 5-aza-dc and TSA could further reverse promoter methylation and increase gene expression.PartⅤThe relationship ofDNMT3b,HDAC1 and SLC22A18 promoter DNA by chromatin immunopreeipitationPurpose To establish effective chromatin immnuoprecipitation(CHIP) and identify the interaction between the chromatin DNA of MDA-MB-231 cells and DNMT3b,HDAC 1 protein by Chip analysis.Methods The MDA-MB-231 cells were treated by 5-aza-dc and/or TSA.Then the MDA-MB-231 cells were treated by formaldehyde so that a complex composed of protein and chromatin DNA could stably exit in vivo.The DNA-protein complex was precipitated with and an appropriate amount of anti-DNMT3b and HDAC1 antibody. The DNA-protein complex was eluted and purified to extract the specific DNA.The specific DNA was amplified using PCR.The PCR products were was loaded directly onto 1.5%agarose gels,stained with ethidium bromide and visualized under UV illumination.Results ChIP assay performed with specific DNMT3b and HDAC1 antibody showed that DNMT3b and HDAC1 were associated with the SLC22A18 promoter in MDA-MB-231 cells.Treatment with 5-aza-dc/TSA and 5-aza-dc,but not TSA, reduced the level of association of DNMT3b with SLC22A18 promoter.Thus, recruitment of DNMT3b to methylated SLC22A18 promoter deceases upon demethylation of the promoter.Chip assay performed with specific HDAC 1 antibody showed that HDAC1 was associated with the SLC22A18 promoter in MDA-MB-231 cells.The association was significantly reduced by TSA or TSA/5-aza-dc treatment and was unaffected by 5-aza-dc alone.The DNMT3b and HDAC1 protein expression of SLC22A18 was unaffected by 5-aza-dc and/or TSA treatment.Conclusions DNMT3b and HDAC1 forming a complex,which bound to methylated CpG sites and made chromatin structure from open to close,blocked transcriptional factors to bind to theirs specific binding sites and then suppress SLC22A18 expression.PartⅥThe proliferation of breast cancer cells after transfection of SLC22A18Purpose To elucidate the effect of SLC22A18 overexpression on the breast cancer cells.Methods Constructed vector plRES2-EGFP-SLC22A18,then the vector was transiently transfected into MDA-MB-231 cells.Cell proliferation,cell cycle and expression of SLC22A18 were observed under WST-1 method,FCM,Real-time RT-PCR and Western blot.Results The plasmid plRES2-EGFP- SLC22A18 was constructed successfully. After transiently transfected the vector into MDA-MB-231,the mRNA and protein expression of SLC22A18 were decreased.The ability of cell proliferation was down-regulated.The cell cycle changed,the percentage of S stage was lower than the control.Conclusions The proliferation of breast cancer cell line MDA-MB-231 was inhibited after transiently transfected the vector plRES2-EGFP-SLC22A18.Conclusions1.Decreased expression of SLC22A18 gene may play an important role in the carcinogenesis of IDC.2.The phenomena of mutation,LOH and GOI of SLC22A18 were occurred in IDCs. LOH and GOI were related to the transcriptional expression.LOH and GOI of SLC22A18 may be involved in breast carcinogenesis and prognosis.3.The reduced expression of SLC22A18 was associated with aberrant DNA methylation in the promoter region.Promoter methylation was related to status of imprinting.Detection of promoter methylation is potentially usedful as epigenetic marker in tumor progress.4.Aberrant DNA methylation may be the main mechanism of down-regulation of SLC22A18 in IDC.5.DNMT3b and HDAC1 were associated with the SLC22A18 promoter in MDA-MB-231 cells.DNMT3b and HDAC1 forming a complex,which bound to methylated CpG sites and made chromatin structure from open to close,blocked transcription factors to bind to theirs specific binding sites and then suppress SLC22A18 expression.6.The proliferation of breast cancer cell line MDA-MB-231 was inhibited after transiently transfected the vector plRES2-EGFP- SLC22A 18.Originalities of this workFor the first time,the genetic and epigenetic mechanisms involved in imprinting gene SLC22A18 down-regulation in breast cancer were studied.The reduced expression of SLC22A18 in IDC was associated with aberrant DNA methylation in the promoter region.DNA methylation and GOI of SLC22A18 may be involved in breast carcinogenesis and prognosis.ChIP assay was established.DNMT3b and HDAC1 forming a complex,which bound to methylated CpG sites and made chromatin structure from open to close,blocked transcription factors to bind to theirs specific binding sites and then suppress SLC22A18 expression.