The Research Of HMLH1 Gene Mutation Of Colorental Cancer In Guangxi

Background and ObjectiveColorectal cancer is the most common human malignancies,and the incidence rate of colorectal cancer is rising,in Shanghai colorectal cancer has been the second highest malignant tumor.Many studies showed that a large number of colorectal cancers were caused by gene mutations,gene mutations may be caused by carcinogenic agents,and can also be generated during DNA metabolism mismatch caused by the base.In order to ensure the integrity and stability of genetic material,there were many systems to prevent cell mutation, including excision repair,direct repair,recombinational repair and mismatch repair.MMR system was not only to rectify base mispairing in DNA recombination and replication for remaining genomic stabilitym,but to induce apoptosis of DNA damaging cell for eliminating canceration of the mutant cell.Human mismatch repair genes include hMSH2,hMLH1,hMSH3,hPMH1,hPMH2 etc,Which play a major role are hMSH2 and hMLH1 gene.The technique of Single strand conformation polymorphism analysis of polymerase chain reaction products(PCR-SSCP) is a new technology for mutation screening,which was created by Orita in 1989.The technique has been widely used in the research of tumor because it was a method of a simple,rapid and economic means of the point mutation screening.The purpose of this study is the detection of sporadic colorectal cancer(SCC) in patients with base mismatch repair(hMLH1) of mutation through the use of PCR-SSCP combined with DNA sequencing,then to investigate the role of mutated mismatch repair gene hMLH1 in the carcinogenesis and development of sporadic colorectal carcinoma.MethodsTumor DNA and DNA from corresponding normal neoplastictissue were extracted by using hydroxybenzene phenol-chlorofrom method.We analyzed the normal and tumor genomic DNA for hMLH1 mutation in exon 8,12,14,15and16 by using PCR-SSCP and silver staining.PCR was performed using 2.5μl of genomic DNA,10×buffer 5.0μl(25mmol/EMgcl₂),20pmol of each primer,2.5 mmol/L dNTPs 4.0μl,5U/μl Taq DNA polymerase 0.5μl and double distilled water 36.0μl,the total volume was 50.0μl.The Cycle conditions:preheatering at 94℃for 3 minutes,denaturing at 94℃for30 seconds,annealing at 55-63℃for 30 seconds, and extension at 72℃for 45 seconds,a total of 30 cycles PCR amplification,and with extension at 72℃for 5 minutes.Through PCR-single-strand conformation polymorphism analysis combined with silver staining method to identify abnormal staining bands.After purifying abnormal bands of PCR products,and the product of PCR which showed abnormal strand was sequenced directly.Results Among the 44 cases of colorectal cancer specimens,there was an abnormal SSCP electrophoresis band,and the mutation rate was 2.3%through DNA sequencing,nd mutation type was missense mutation,codon581(CCG→CTG,Pro→Leu).ConclusionThe study of this subject showed that there was hMLH1 gene mutation in GuangXi sporadic colorectal cancer,but it was just a sporadic case during the occurrence and development of colorectal carcinoma.