Development of a Microsatellite Instability Reporter System in Human Colorectal Cancer

• Main Authors: Yi-ting Chen, 陳伊婷
• Other Authors: Christina Ling Chang
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/92161571421179939521

碩士 === 國立成功大學 === 分子醫學研究所 === 97 === Colorectal Cancer (CRC) is ranked as the 3rd leading cause of cancer mortality in Taiwan. Patients with inflammatory disease such as ulcerative colitis have a high risk of colon cancer. Germline mutations in the DNA mismatch repair (MMR) genes account for hereditary nonpolyposis colorectal cancer (HNPCC). On the other hand, hypermethylation of the hMLH1 promoter accounts for sporadic colon cancer. Both genetic and epigenetic events lead to MMR inactivation. Additionally, oxidative stress also inactivates the function of MMR. Inactivated MMR is unable to correct insertions or deletions of the repeat unit in microsatellites, which is manifested as microsatellite instability (MSI). MSI has been detected in patients with colon cancer and ulcerative colitis, and MSI-positive patients will develop drug resistance. To reduce CRC incidence and mortality, it is important to prevent MSI from development. Since CRC is caused by both genetic and epigenetic alterations, we hypothesize that the MSI phenotype serves as a nexus to simultaneously detect epigenetic (such as DNA methylation) and genetic (such as insertions/deletions) events. I constructed a dual fluorescence MSI reporter system consisting of (CG)5, (CG)8 and (CG)13 microsatellites. From MMR-deficient HCT116 human colorectal cancer cell line, I generated at least two stable clones harboring each (CG)n microsatellite , as well as a negative control harboring the (N)26 random sequence. H2O2 increased the MSI frequency in a dose-dependent manner in the stable clones harboring (CG)n. Compared to (CG)5, (CG)13 appears to be more sensitive to report the MSI phenotype. Furthermore, H2O2 also increased the MSI frequency in a time-dependent manner. Direct sequencing of MSI-positive (i.e., RFP+/GFP+) cells revealed that one (CG) unit deletion was a major event that restored the correct reading frame of RFP. In the future, the MSI reporter system will be used for screening anti-MSI compounds in order to reduce the incidence and mortality of colorectal cancer.