| Background and ObjectiveColorectal cancer (Colorectal cancer, CRC) is the general term for colon and rectal cancer, which is one of the most common gastrointestinal malignancy. Occurrence and development of the disease involve in the changes of multiple genes, these genes include three type genes:oncogenes, tumor suppressor genes and DNA repair genes. More and more research evidences suggest that methylation of many tumor suppressor gene promoter 5'CpG island lead to inactivation of gene expression. Recent studies have also found that detection of tumor suppressor gene promoter methylation in human fecal specimens could be used as a viable colorectal cancer screening epigenetic biomarker. Therefore, in-depth study of epigenetic modification of tumor suppressor genes, to elucidate the molecular mechanism of colorectal cancer, diagnosis, treatment and prognosis, is important, and may become the future tumor target of treatment. Intercellular adhesion molecule 1/lung cancer inhibitor-1 (CADM1/TSLC1) is tumor suppressor gene found in the study of lung cancer, belongs to immunoglobulin superfamily and is homology with neural cell adhesion molecule, the gene can inhibit cancer cell growth, in a calcium and magnesium ions independent manner affinity reaction to mediate cell-cell adhesion and cell signal transduction. CADM1/TSLC1 also induces immune cells to recognize and kill tumor cells, induce tumor cell apoptosis and inhibit cell proliferation. Expressions of CADM1/TSLC1 have been shown in many normal human tissues, and reduce in many cancer tissues; an important mechanism of the gene promoter methylation leads to decreased expression. Differential expression of lung molecular-1 (DAL-1/4.1B/ EPB41L3) is one kind of membrane skeleton protein, contains a conservative 4.1 protein/Ades protein/root protein/membrane spike protein (FERM) region interacte with CADM1/TSLC1 cytoplasmic domain of actin anchor connection. DAL-1/4.1B/EPB41L3 exists in the basolateral membrane of the mouse small intestinal epithelial cells, and the immunohistochemistry results showed that the gene significantly associated with shift and proliferation of intestinal epithelial cells. Loss of DAL-1/4.1B/EPB41L3 expression will lead to decrease of cell adhesion; re-expression of DAL-1/4.1 B/EPB41L3 protein can increase cell adhesion and induce cell apoptosis. In human non-small cell lung cancer, DAL-1/4.1B/EPB41L3 expression compared with the corresponding adjacent tissues was decreased significantly in cancer tissues, deletion of the gene expression is also present in human meningiomas, ependymomas and breast cancer, which means that in the tumors DAL-1/4.1B/EPB41L3 play an important role in pathogenesis. In lung cancer, renal cell carcinoma, breast cancer research, reduction of DAL-1/4.1 B/EPB41L3 gene expression was mainly caused by abnormal methylation of the promoter. Homologous Drosophila tumor suppressor molecule-3 (MPP3) belong to membrane-associated guanylate kinase molecules (MAGUKs) family of cytoplasmic proteins, contains a carboxyl-terminal (PSD-95/Dlg/ZO-1) PDZ, which can combinate with CADM1/TSLC1 to regulate cell polarity and the role of cell adhesion. MPP3 gene may lead to inactivation of tumor suppressor CADM1/TSLC1 pathway. Including non-small cell lung cancer, in a variety of human cancers, there is often genetic heterozygosity in MPP3 gene chromosome, which is one of the mechanisms of MPP3 inactivation. Therefore, in the epithelial cells CADM1/TSLC1 DAL-1/4.1B/EPB41L3 and MPP3 genes constitute the cascade to play a role in tumor suppressor pathways. CADM1/TSLC1 PDZ binding subunit, DAL-1/4.1B/EPB41L3 protein core FERM interconnected region and MPP3 trimer formation play a pivotal role in the maintenance of normal tissue structure, cell adhesion, cell polarity and morphology, inactivation of the pathway may lead to tumor incidence and development, and regulation of the pathway genes promoter methylation may be an important molecular mechanism.Materials and Methods1) Colorectal cancer cell culture and collection of tissue samples of colorectal cancer:colorectal cancer cell lines (SW480, HT29, COLO205, SW1116, SW620, LOVO, HCT116, HCT8) were cultured in 10% FBS RPMI-1640 medium; After collection of 54 cases of colorectal cancer and adjacent non-cancer tissue, samples were pretreated by both methods:1) into the tissue preservation fluid,-80℃low temperature refrigerator; 2) regular 10% neutral formalin fixed;2)RT-PCR detection of gene mRNA expression:extraction of all cell lines and tissue samples total RNA, synthesis of cDNA, PCR reactions, agarose gel electrophoresis, semi-quantitative analysis of CADM1/TSLC1, DAL-1/4.1B/EPB41 L3 and MPP3 mRNA level of gene expression;3)Western Blotting detection of gene protein expression:extraction of the cell lines and tissues total protein, determination of protein concentration, denatured protein, protein electrophoresis on the sample, transfer film, closed, with a combination of resistance and the second antibody reaction respectively, the final chemical ECL Detection of light and semi-quantitative analysis CADM1/TSLC1, DAL-1/4.1B/EPB41L3 and MPP3 gene protein expression;4)BSP and MSP detection of gene promoter methylation status:extraction of cell lines and tissue samples genomic DNA, bisulfite modified, BSP and MSP amplification of DNA fragments containing CpG islands, detection CADM1/TSLC1, DAL-1/4.1B/EPB41L3 and MPP3 gene promoter methylation status of sites; BSP and MSP assay cells gene promoter methylation, MSP assay tissue gene promoter methylation;5)Immunohistochemical detection of tissue CADM1/TSLC1 protein expression:tissue dehydration, transparent, dipping wax, embedded, producer, dewaxing, hydration, antigen retrieval, respectively, with a combination of resistance and the second antibody reaction, DAB was color, hematoxylin, dehydrated slices, transparent and Fengpian;6)5-aza-dC treatment of colorectal cancers:first 105/ml colorectal cancer cell line inoculated in six well plate, prepared 10μM5-aza-dC in the role of 2 and 5 days in the cell, the blank control group PBS instead of 5-aza-dC, in the first 8 days cells were collected. Using RT-PCR method and Western Blotting Detection of the cell lines CADM1/TSLC1, DAL-1/4.1B/EPB41L3 and MPP3mRNA and protein expression changes.7)Statistical analysis:using One Way ANOVA test and independent sample T test compared the gene expression level. Kendall's tau-b used method of testing gene expression and promoter methylation status of the relationship. Were tested using Chi-Square, Fisher's exact test and independent sample T test analysis of promoter methylation and clinical features.Results 1)CADM1/TSLC1 relationship with colorectal cancer:CADM1/TSLC1 mRNA in three colorectal cancer cell lines (SW480, HT29 and COLO205) expression was deleted; in four cell lines (SW620, LoVo, HCT116 and HCT8) in CADM1/TSLC1 mRNA expression decreased. Loss of CADM1/TSLC1 was in 18/54 (33%) colorectal carcinoma, CADM1/TSLC1 was significantly reduced in 21/54 (39%) colorectal cancer. Immunohistochemical results showed CADM1/TSLC1 protein were expressed in the cell membrane and cytoplasm, expression can be seen in non-cancerous tissue membrane, but in cancer tissue was mainly in cytoplasm.2/8 colorectal cancer cell lines (SW480, HT29) were detected promoter hypermethylation, and 4/8 colorectal cancer cell lines (HCT116, HCT8, SW620, LoVo) were detected partially methylation, and CADM1/TSLC1 expression of these cell lines was decreased. After treatment of 5-aza-dC, SW480 and HT29 cell line appeared CADM1/TSLC1 expression. CADM1/TSLC1 gene promoter methylation was detected in 29/39 (74%) colorectal cancer patients, whose expression of CADM1/TSLC1 mRNA reduced or missing. In 26/32 (81%) patients of the progress Tumor (T3 and T4 period) were detected CADM1/TSLC1 gene promoter methylation, whereas in 6/32 (19%) cases of early cancer (T1 and T2 period) were detected CADM1/TSLC1 gene promoter methylation, the difference between the two groups was significant (X2=4.459, P=0.035). In addition, Dukes'C and Dukes'D of patients with colorectal cancer CADM1/TSLC1 gene promoter methylation frequency was significantly higher than Dukes'A period and Dukes'B of patients (X2=6.480, P= 0.011);2)DAL-1/4.1B/EPB41L3 relationship with colorectal cancer:DAL-1/4.1B/E PB41L3mRNA in six colon cancer cell line (SW480, HT29, COLO205, SW1116, SW620, and LOVO) was expressed with no significant difference between the expressions. DAL-1/4.1B/EPB41L3 promoter CpG sites are unmethylated.5-aza-dC role in six colorectal cancer cell lines and found that there was no significant change DAL-1/4.1B/EPB41L3mRNA. DAL-1/4.1B/EPB41L3 mRNA 5/21 (24%) cases of colorectal cancer DAL-1/4.1B/EPB41L3 mRNA expression loss.2/21 (10%) cases of colorectal carcinoma DAL-1/4.1 B/EPB41L3 promoter methylation bands appear. The results show that the DAL-1/4.1B/EPB41L3 promoter methylation in colorectal cancer is a rare phenomenon; there are other silent molecular mechanisms of DAL-1/4.1B/EPB41L3 expression;3)MPP3 relationship with colorectal cancer:Loss of MPP3 expression was in two colorectal cancer cell lines (SW1116, LoVo), which were with MPP3 promoter methylation status. After treatment of 5-aza-dC, expression of MPP3 in these two colorectal cancer cell lines SW1116, LoVo were recovered. Loss of MPP3 expression was in 10/23 (43%) cases of colorectal carcinoma, MPP3 expression in colorectal cancer was significantly reduced.8/23 (35%) cases colorectal cancer tissues was detected MPP3 gene promoter methylation, and that eight cases of tissue loss MPP3mRNA expression. MPP3 promoter methylation in 8/14 (57%) cases advanced tumors (T3, and T4 period) to detect gene methylation, and in the 1/9 cases (11%) of early cancer (T1, and T2 period) to detect gene methylation, the difference was significant (P=0.040). In Dukes'C, D staging MPP3 and CADM1/TSLC1 promoter methylation correlated significantly (P=0.021). MPP3 and/or CADM1/TSLC1 promoter methylation were in 10/14 patients in advanced tumors (T3 and T4 period) reviewMeasured gene promoter methylation, and in the 1/9 cases of early cancer (T1 and T2) to detect gene methylation, the difference was significant (P=0.009).Conclusions1)CADM1/TSLC1 in colorectal cancer:in colorectal cancer, CADM1/TSLC1 decreased expression may be the important molecular mechanisms lead to the development of the disease. Promoter methylation is the main mechanism cause CADM1/TSLC1 gene silencing. CADM1/TSLC1 promoter methylation was more often in advanced colorectal cancer (T3, T4) than early colorectal cancer (Tis, T1, T2), suggesting that CADM1/TSLC1 may inhibit tumor invasion and growth; CADM1/TSLC1 promoter methylation at Dukes'C and D was more common than Dukes'A and B, suggesting that promoter methylation inducing of CADM1/TSLC1 silence corelated with the degree of malignancy of colorectal cancer. CADM1/TSLC1 promoter methylation is the role of demethylation as an effective target of drug targets.2)DAL-1/4.1B/EPB41L3 in colorectal cancer:We detected six cases of colorectal cancer cell line DAL-1/4.1B/EPB41L3mRNA, found that gene expression is not significantly reduced; but in 21 cases of colorectal cancer tissue samples, the gene expression of cancer in the 24% tissue loss, and the corresponding adjacent tissues were normal expression of the gene. The results raise two questions:1) In addition to the use of the six colon cancer cell line other than decreased expression may DAL-1/4.1B/EPB41L3mRNA other cell lines,Options for further study; 2) As the SW480, HT29, COLO205, SW620 and LOVO colorectal cancer cell lines, were detected in the expression of different levels of CADM1/TSLC1 reduced, suggesting CADM1/TSLC1 and DAL-1/4.1B/EPB41L3 number of molecules combine to form dimers decreased accordingly, which may cause DAL-1/4.1B/EPB41L3 protein expression is not significantly reduced in the case, but the protein membrane localization error and the loss of membrane proteins with each other play the role of tumor suppressor gene function, leading to changes in characteristics of normal cells to cancer cells, but this requires further study cell lines DAL-1/4.1B/EPB41L3 protein localizations. Colorectal cancer tissue samples decreased expression of the gene while the experimental results suggest DAL-1/4.1B/EPB41L3 in colorectal cancer may play an important role. Decreased expression in colorectal cancer DAL-1/4.1B/EPB41L3, may cause proliferation of colon cancer, tumor growth and invasion ability of anti-apoptotic capacity and distant metastasis were enhanced, resulting in increased tumor malignancy. In colorectal cancer, gene promoter methylation might not be the primary mechanism for decreased expression DAL-1/4.1B/EPB41L3.3)MPP3 in colorectal cancer:in colorectal cancer, MPP3 disease decreased expression may lead to the development of the important molecular mechanisms. Promoter methylation is a major cause MPP3 gene silencing mechanism. MPP3 promoter methylation in advanced tumors (T3 and T4 of) the incidence of tumors than in early (T1 and T2), suggesting MPP3 loss can lead to tumor cell invasion. In Dukes'C, D staging MPP3 and CADMl/TSLC1 promoter methylation is relevant, we speculated that the part of the development of colorectal cancer to Dukes'C, D period of time, MPP3 CADM1/TSLC1 gene promoter and gene promoter methylation may occur simultaneously, this may lead to MPP3 and CADM1/TSLC1 tumor suppressor protein function while the loss of further damage CADM1/TSLC1 cascade pathway suppressor function, so that further deterioration. In MPP3 and/or promoter methylation CADM1/TSLC1 incidence of advanced tumors (T3 and T4 period) than in early tumors (T1 and T2) high, the results suggest these two genes were detected promoter may forecast range of patients with colorectal tumor-infiltrating, the choice of treatment for clinical reference. In summary, the resultsCADM1/TSLC1 cascade pathway that tumor suppressor gene expression changes in colorectal cancer is an important event, so in the pathogenesis of colorectal cancer play an important role. In colorectal cancer can be detected MPP3 and CADM1/TSLC1 frequent promoter methylation, in particular T3 and T4 in patients with colorectal cancer, the clinical diagnosis of colorectal cancer invasion to provide a theoretical basis for demethylation Drug treatment of colorectal cancer the role of a new target goal, the use of drugs to restore MPP3 demethylation and CADM1/TSLC1 expression may be an effective treatment of colorectal cancer.
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