Study on Microsatellite Instability in Colorectal Cancer Patients with ...

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Cancer Research

[Abstract]

IntroductionColorectal cancer is a common malignant tumor in the world. The incidence of colorectal cancer shows a rising trendency. Accumulated evidence has established that carcinogenesis is associated with alterations in cellular oncogenes and in tumor suppressor genes necessary for malignant transformation. About twenty percent of colorectal cancer is characterized by a familial aggregation. Hereditar-y non ? polyposis colorectal cancer (HNPCC) is thought to be the most common form of inherited colorectal cancer. HNPCC is an autosomal dominant disorder which accounts for 5% ? 15% of colorectal cancer cases. Most cases of HNPCC and part of sporadic colorectal cancer are caused by a defect in the DNA mismatch repair gene ( MMR) but not in oncogenes or tumor suppressor genes. Tumors which have defective mismatch repair gene show a characteristic molecular phenotype, termed microsatellite instability ( MSI).Microsatellites are polymorphic, short, tandem repeat segments dispersed throughout the human genome. MSI is a form of genetic instability characterized by expansions and contractions of simple sequence repeats in DNA. It occurs because of a defect in DNA MMR. Nowadays, MSI has been paid more attention to because of its key role in the initiation and development of carcinoma. MSI is considered a new mechanism of colorectal cancer.In our experiment, polymerase chain reaction ? single strand conformation polymorphism (PCR ? SSCP) and sliver ? staining methods were performed to detect incidence of MSI in 29 cases of CRC with familial predisposition and in 30 sporadic CRC. The goal is to discuss the molecular mechanisms of CRC with familial predisposition and the possibility to discover disease in early stage, directclinical treatment and judge the prognosis.Materials and MethodsI. Materials1. Samples All samples used were obtained from paraffin ? embedded tissues of the surgical resected specimens of patients admitted to the 1 st Affiliated Hospital of China Medical University during 1997 -2003.2. Reagents Primer; BAT25, BAT26, D5S346, D17S250 and D2S123. (GIAGEN company); TaKaRa Taq?, Proteinase K, Agarose Regular, DNA Marker DL2000 ( TaKaRa Biotechnology company ) ; the other reagents made in China were all analytical pure.II. MethodsSerial five 8 ? jxm thick sections of selected tissue blocks were obtained. Corresponding samples of colorectal mucosa that were free of tumor and metaplasia , were obtained from each case as control tissue. Sections were dewaxed. Then genomic DNA was purified by proteinase K digestion and phenol ? chloroform extraction. The ratio of A260 and A280 was measured by ultraviolet spec-troscopic photometer. The concentration of DNA was calculated. PCRs were performed to amplify colorectal lesions and corresponding normal DNA. After amplification, PCR products were separated by 2% Agarose gels to detect DNA. Then PCR products were electrophoresed on non ? denaturing 8% ( 29 :1) poly-acrylamide gels, and bands were visualized by silver staining.Alterations in the microsatellites in the form of changes in the size of DNA strands were detected by comparing tumor DNA and normal DNA. It was judged negative if their strands have no difference. MSI was defined as a band shift in either of the two alleles or the appearance of a band with different sizes in the tumor or normal sample. Using a panel of five microsatellites, a specimen was defined as high ? level MSI ( MSI ? H) if two or more of the five markers displayed instability,as low -level (MSI ? L) if one marker displayed instability, and as microsatellite stable (MSS) if none of the markers exhibited MSI.III. Statistical analysisStatistical analysis was performed using the t ? test and Fishers exact test. P 0. 05 ).MSI incidence rates in different reports vary. We therefore postulate that MSI rates may be associated with markers, the criteria for definition of MSI, the number of samples, race and different areas.Our results demonstrate that MSI plays an important role in CRC with familial predisposition. MSI analysis is very useful in detecting tumors with mismatch repair genes as the first step. The verticle relatives of MSI carriers have a higher risk of HNPCC. We expect to discover HNPCC in early stage among those people by surveillance and have the possibility of early surgical intervention that could be curative.

Title: Study on Microsatellite Instability in Colorectal Cancer Patients with Familial Predisposition

Category: Cancer Letters

Filename: Study on Microsatellite Instability in Colorectal Cancer Patients with Familial Predisposition.pdf

Pages: 153

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