Study On Colorectal Cancer And Mismatch Repair Genes

There is an ample reason to study colorectal cancer (CRC): it is the fourth most common incident cancer and the second most common cause of cancer death in the world, and 875,000 people are diagnosed with CRC annually. Colorectal cancer is thus causally related to both genes and environment. Environmental risk factors for colorectal neoplasia include a positive family history, meat consumption, smoking, and alcohol consumption. Important inverse associations exist with vegetables, nonsteroidal anti-inflammatory drugs (NSAIDs), hormone replacement therapy, and physical activity.Genetics has a key role in predisposition to CRC and in its initiation and progression. There are several molecular pathways to colorectal cancer, especially the APC–β-catenin–Tcf (T-cell factor; a transcriptional activator) pathway and the pathway involving abnormalities of DNA mismatch repair. DNA mismatch repair (MMR) genes are one of the most important genes which are associated with colorectal cancer (CRC). They are involved in the repair of DNA base-base mispairs and strand misalignments that occur during DNA replication, and responsible for the genomic stability. There have been 6 MMR genes cloned from human cells—hMSH2,hMLH1,hMSH3,hMSH6,hPMS1 and hPMS2. It involved in both inherited syndromes (hereditary nonpolyposis colorectal cancer [HNPCC]) and approximately 15% of sporadic colorectal cancer.Identifying the genes conferring susceptibility or resistance to common human diseases should become increasingly feasible with improved methods for finding DNA sequence variants on a genome-wide scale. About 90% of sequence variants in humans are differences in single bases of DNA, called single nucleotide polymorphisms (SNPs). Single-nucleotide polymorphisms are generally believed to provide important information in the wide spectrum of life sciences. Because many studies have confirmed that a few SNPs have significant association with colorectal cancer, such as APC, HRAS1 and so on. Furthermore, MMR genes play the key role in genomic stability, and they have been identified as the pathogenic genes of HNPCC. Thus we can consider that the SNPs of MMR genes should have some phenotypic significance in sporadic colorectal cancer.There wasn't particular report about the association between SNPs of mismatch repair genes and Chinese sporadic colorectal cancer. Moreover, hMLH1and hMSH2 are two main genes of MMR genes, and their mutation can account 80% of all mutations of MMR genes which were occurred in CRC. So we chose 29 reported single-nucleotide variants (17 SNPs of hMLH1; 12 of hMSH2) that are rarely verified in a population-based study. All the selected SNPs are found in coding region of these two genes, and they can cause missense mutations that lead to amino acid change. We extracted the genomic DNA from peripheral blood of patients and control individuals, and sequencing was used to determine these SNPs. We identified SNPs and the genotype–phenotype association in Chinese populations of 150 healthy individuals and 160 sporadic colorectal cancer patients. Three SNPs (hMLH1 394G→C, 655A→G, 1151T→A) occurred with a frequency of 8.8-11.2% in the Chinese population. And these SNPs formed a serial of special combination and a series with combined effects. The haplotype hMLH1 655A-394C-1151A and the haplotype hMLH1 394G-655G-1151A occurred exclusively in sporadic colorectal cancers. None of the other 26 variants,including all the 12 SNPs of hMSH2, were detected or with very low frequence in the Chinese populationMismatch-repair-genes-deficient colorectal adenocarcinomas display distinctive molecular, pathological and clinical features, such as microsatellite instability (MSI), proximal location, poor differentiation, frequent mucinous and medullary phenotype, and marked peritumoral and intratumoral lymphocytic infiltration. MMR-deficient colorectal carcinomas have a more favorable clinical outcome than MMR-positive tumors, and the survival advantage conferred by the MSI phenotype is independent of tumor stage and other clinical and pathological variables.Although differences in mRNA expression between MMR-defective and MMR-proficient CRCs that represent the transcriptional level have been extensively discussed, we are interested in the post-transcriptional differences between MMR-deficient and MMR-positive CRCs. MicroRNAs (miRNAs) are 19- to 25-nucleotide-long non-coding RNAs that regulate gene expression at the post-transcriptional level via specific complementary sites at the 3'-UTR of target mRNAs, causing translational repression or degradation.Because the relationship at the post-transcription level is still obscure, we chose a genome-wide miRNA microarray and quantitative real-time PCR to identify differentially expressed miRNAs. We characterized the post-transcriptional profile of mismatch repair-deficient colon cancer and reveal the pathophysiological role of aberrant miRNA. Eight miRNAs (five up-regulated: miR-21, let-7a, miR-200b, miR-200c, miR-145; three down-regulated: miR-127, miR-155, miR-133b) were identified to be de-regulated in MMR-deficient CRC cells and tissues. miR-21 was up-regulated and had a noticeably high score during cluster analysis in all MMR-deficient cell lines and tissues. Oncogene PLAG1 was then experimentally validated as the functional target of miR-21. Meanwhile, the expression of IGF2 was determined to be coincident with its regulator PLAG1 in all of the above samples. Most importantly, by utilizing both CRC cells and nude mice models, cells treated with precursor miR-21 showed stimulated proliferation in vitro and in vivo. The results were consistent with PLAG1 knock-down, which increased cell apoptosis. In addition, the in vivo study using nude mice showed that up-regulated miR-21 made the MMR-positive CRCs display conspicuous lymphocyte infiltration, which was an important character of MMR-deficient CRCs. miR-21 can inhibit tumor growth of MMR-deficient CRC by modulating the expression of PLAG1 and IGF2. Collectively, our data demonstrate that miR-21 post-transcriptionally targets oncogene PLAG1 and inhibits cell proliferation in colon cancer for the first time. It may serve as a novel therapeutic target in colorectal cancer.