Cdk2-ap1 And Colorectal Cancer With Microsatellite Instability

Colorectal cancer (CRC) is one of the most common malignancies worldwide. In China, about 1.3 million people die from cancer each year. And CRC is the third most common cancer and the fourth most common cause of cancer-related deaths nationwide. With the changes of life style and diet structure, the incidence and mortality of CRC is increasing in recent decades. Therefore, it will be of great significance to find out CRC-related genes and interpret their exact roles, based on which the effective treatment strategies could be established.Microsatellites are repeated nucleotide sequences with high polymorphism distributed throughout the genome. MSI is caused by a failure of the DNA mismatch repair (MMR) system to repair errors that occur during the replication of DNA and is characterized by the accelerated accumulation of single nucleotide mutations that occur ubiquitously throughout the genome. And in terms of tumor, MSI is a phenomenon of appearing new allele due to any changes of length of a microsatellite resulted from either insertion or deletion of repeating units. MSI was first discovered and also most studied in CRC. Cyclin-dependent kinase 2-associated protein 1 (CDK2-AP1) is a highly conserved gene located in chromosome 12q24, which acts as a growth suppressor through negatively regulating the activity of cyclin dependent kinase 2 (CDK2) CDK2-AP1 was characterized by loss of heterozygosity and decreased expression of its translation product. CDK2-AP1 is continuously expressed in normal human tissues and has been shown to interact with multiple cell growth regulatory elements. The absence of this gene was found in many human tumors, and is associated with the increase of tumor invasion, lymph node metastasis, and bad prognosis.Previous studies have demonstrated the association between MSI and CDK2-AP1 expression in human CRC cell lines. Compared with MSS CRC cell lines, the reduced expression of CDK2-AP1, increased cell proliferation and less apoptosis were observed in MSI CRC cell lines, which could be reversed by transfecting CDK2-AP1 into MSI CRC cell lines. Further researches detected a novel somatic alteration del T in a poly (T) 8 track within the3'-UTR of the CDK2-APlgene in 21%(3/14) of MSI CRC cell lines and 13%(4/31) of MSI CRC tumor tissues in Caucasian. The presence of this mutation correlated significantly with decreased CDK2-AP1 expression in human CRC cell lines and tumors. So the del T in a poly (T) g track within the 3'-UTR of the CDK2-APlgene is perhaps a novel and meaningful somatic alteration correlated with MSI-H CRC.In this study, we aim to detect the distribution of the del T mutation in Chinese CRC patients and further identify its impact on the expression of CDK2-AP1. The study of the role of CDK2-AP1 in human CRC may lead to an improved understanding of the genetic elements and pathways that contribute specifically to MSI CRC natural history.In the beginning, we modified the traditional method of DNA phenol-chloroform extraction from formalin-fixed tissues, and successfully extracted eligible DNA samples of the CRC patients. And therefore we established the CRC tissue bank in our laboratory.Next, we used the fluorescent multiplex polymerase chain reaction (PCR)-capillary electrophoresis (FM-CE) to detect the five primary microsatellite loci recommended by the National Cancer Institute-sponsored conference:BAT25, BAT26, D2S123, D5S346, and D17S250. Finaly we found 73 MSI-H CRCs (two or more of the five microsatellite loci which showed instability) in 506 samples.At the same time, we detected the expression of CDK2-AP1 gene in both of the MSI CRC cell line RKO and the MSS cell line SW480 by real-time reversed transcription-PCR (RT-PCR). The result revealed that the expression of CDK2-AP1 in RKO was significantly decreased compared with SW480 (P< 0.05)We detected the expression of CDK2-AP1 by real-time-RT PCR in 7 MSI-H CRC samples and 10 MSS/MSI-L CRC samples, but there was no significant difference between the MSI-H and MSS/MSI-L.We still used the fluorescent PCR-capillary electrophoresis to detect the del T mutation of the CDK2-AP1 gene in all 73 MSI-H CRC samples and 60 MSS/MSI-L CRC samples. However, in all these samples, no del T modulation was found. And the data of sequencing assay supported this result.We also used the fluorescent PCR-capillary electrophoresis to detect the del T mutation of the CDK2-AP1 gene in RKO, CW2, Lovo, SW480, SW620, HT29 and CaCo2 CRC cell lines, but still no del T modulation was found, except (T) 7, (T) g and (T) 9 in the CW2 cell line. And the data of sequencing assay supported this result.No significant correlation was demonstrated between the MSI status and patient's gender, age, tumor size, lymph node metastasis, metastasis and stage. Interestingly, more MSI-H CRC tumors occurred in colon than in rectum (P= 0.04< 0.05), and the MSI-H CRC tumors were also found in early stage (P= 0.047< 0.05) In summary, we proved that FM-CE was an advisable method for large sample size MSI analysis. The finding that del T mutation might be detected in Caucasian populations did not exist in Chinese CRC samples, suggesting that the CRC may occur through different pathways in the two different population.