Mapping And Analysis Of The Methylation Variable Positions Profiles Of HMLH1 Promoter CpG Islands In Human Sporadic Colorectal Carcinoma

BackgroundsCpG islands(CGIs) of human genome,located mainly in the promoter as well as the first extron regions,are GC rich short stretches(100-1000bp) and they link with～60%of human coding genes.Methylation of CpG sites in the promoter regions of genes can lead to decreased expression and silencing of the tumor suppressor genes and contribute to oncogenesis.However,the transcriptional silencing of affected genes can occur only when the abnormal methylation occurs in specific CpG sites.MVPs(Methylation variable positions) mean the distribution and incidence of abnormal methylation CpG sites in different diseases.It is a precise analysis for quantitative evaluation of global CGIs methylation status and patterns at genomic levels.Methylation of CpG sites in the promoter regions of hMLH1 gene can decrease its mRNA level or protein expression,affect its normal repairing function,lead to delayed correction of cancer related genes mutation and contribute to colorectal cancer.Previous researchers have studied methylation of the hMLH1 promoter using MSP method,but this method can only analyze CpG sites in the PCR primers.To characterize precisely the regions involved in the epigenetic silencing of hMLH1,we collected colonic samples from sporadic colorectal carcinoma patients,conducted the NaHSO₃ treatment-sequencing method for methylation analysis of hMLH1 promoter,and explored the correlation between our findings and clinical indicators.Objectives1.To construct an effective and stable method for quantifying all the CpG sites of target sequence by bisulfite sequencing.2.Map the complete methylation status of the hMLH1 promoter-a region that contains 46 CpG sites in 30 sporadic colorectal carcinomas patients. 3.Detect the expression of hMLH1 by immunohistochemistry.4.Analysis on the relationship between MVPs of hMLH1 promoter and the expression of hMLH1 together with clinical significance.Methods1.Construction of method by bisulfite sequencing1.1 Detection on methylation of the p16 promoter 5'—CpG of healthy peopleThe p16 promoter region from19556 to 20355(GenBank ID:AF 527803)was selected as the target sequence.Primers were designed by Methprimer software.The primers were completely complement to the bisulfite modification sequence and did not contain any CpG sites.Primers used were 5'-GTAGGTGGGGAGGAGTTTAGTTT-3'(sense) and 5'-AACTCCTC ATTCCTCTTCCTTAACT-3'(anti- sense),which were used to amplify a 712 bp product.Total genomic DNA was isolated from anticoagulated whole blood of healthy people, and modified by sodium Bisulfite.And then,the modified DNA was amplified by PCR, followed by Exonâ… and SAP purification.Subsequently,the PCR products were analyzed by automated DNA sequencing(ABI).1.2 Detection of methylation of the p16 promoter 5'-CpG of tumor cell line-SW480The tumor cell line-SW480 was cultured in 5A-medium supplemented with 10%CS at 37℃in 5%CO₂ Total genomic DNA was isolated from the SW480 cells.And then, methylation of p16 promoter 5'-CpG was detected as described previously.2.Mapping the complete methylation status of hMLH1 promoterAccording to Gene database of NCBI,some refereces and NCBI OMIM database,we obtain the reference sequence(GenBank ID:NM-000249)and present it to DBTSS database to find transcription initiation site and determine promoter region and take this region as our target fragment.Then the hMLH fragment was presented to Methprimer to prove it containing two CGIs was selected as the target sequence.Two pairs of primers were designed by Methprimer software. Primers were:5'-AGGATTTTTTGTTTTGTGATATTTG-3'(sense) and 5'-TTAA CCCTACTCTTATAACCTCCC-3'(anti-sense);5'-GGGAGGTTATAAGAGTAGGGT TAA-3' (sense) and 5'-AAAATACCTTCAACCAATCACC-3'(anti-sense),which could amplify 493 bp and 423 bp products,respectively.Thirty sporadic colorectal carcino-mas were obtained from surgical patients and this set of samples has been character-rized previously for clinicopathological parameters. 2.1 Detection of methylation of hMLH1 promoter in normal colorectal tissueAll of these 30 patients corresponding non-cancer colorectal tissue was obtained from a tumour-free location,which was at least 2 cm far away from the tumor and which was confirmed to be without any tumor cell infiltration by histology.Total genomic DNA was isolated from the 30 samples.And then,Genomic DNA of all samples was analyzed by the bisulfite genomic sequencing technique after bisulfite conversion as reported previously.2.2 Detection of methylation of hMLH1 promoter of cancerous samples2.2.1 Treatment of cancerous samplesIsolate cancerous cells under a light microscope followed hematoxylin staining,wash several times in phosphate-buffered saline(PBS) to remove cell debris.Extract Genomic DNA of these cancerous cells and treat them with sodium bisulfite.And then,the modified DNA was amplified by PCR.2.2.2 Cloning and sequencingThe PCR products were purified by alcohol and cloned into TA vector.Then insertpositive clones were selected by PCR.Sense primers used were:5'-AGGATTTTTTGTTTTG TGATATTTG-3' and 5'-GGGAGGTTATAAGAGTAGGGTTAA-3'.Anti-sense primer used was:5'-CAGGAAACAGCTATGAC-3'.They were taken to amplify 539 bp and 469 bp products,respectively.Ten clones per samples were sequenced using the ABI sequencing system.3.Detecting the expression of hMLH1 proteinParaffinized serial sections were cut at 5μm for immunohistochemistry and HE staining.Immunostaining was performed with antibodies directly against the C-terminal region of human MLH1 protein.Results1.Method of bisulfite sequencing was constructed successfully.2.The hypermethylation of hMLH1 gene was not detected in normal tissues.But it was detected in tumor tissues:the hypermethylation of CGIs 1 was in 20%(6/30) patients and CGIs 2 was 13.33%(4/30).3.The hMLH1 protein was detected in 50%tumor tissues.There was a highly significant correlation between the hypermethylation of CGIs 1 and loss of hMLH1 expression.In CGIs 1,CpG positions from 1 to 28 were hypermethylated. 4.There was no significant correlation between the hypermethylation of CGIs 1 and Patients age,gender,tumor location,invasive depth,lymphnode metastasis,and Dukes stage.But,there was a highly significant correlation between the hypermethylation of CGIs 1 and poorly differentiated carcinoma.Conclusions1.The construction of bisulfite sequencing should be helpful to further analysis on global CGIs methylation status of tumor suppressor genes.2.In CGIs 1,CpG positions from 1 to 28 are the critical region which could influence expression of hMLH1.3.MVPs of hMLH1 promoter could be used as a promising prognostic indicator of human sporadic colorectal carcinoma.