| Objective: Colon cancer is one of the most common malignant tumors of digestivetract. It has been shown that the occurrence of colon cancer is associated with manykinds of factors. For the past few years, by the research of its pathogenesis, the silencingof antioncogene caused by methylation of DNA plays an important role in colon cancer.And the methylation of apoptosis associated speck-like protein containing CARD(ASC), an apoptotic gene, has caused extensive concern of scholars. As an antioncogene,which contain a pyrin (PYD) and a caspase recruitment domain (CARD), plays animportant role in regulating apoptosis and immunological reaction.By the study of the mathylation of ASC, at the same time, another multidomainprotein NACHT leucine-rich-repeat protein1(NALP1), which also contain PYD andCARD, raised our interest. NALP1can form immune complex at the PYD domain byinteracting with ASC which associates with apoptosis and immunological reaction. Inthe previous study, we explored the expression of ASC and NALP1in colon tissues andfound that the expressions of ASC and NALP1in normal colon tissues are higher thanthat in colon tumors. By this, we consider that, the expression of NALP1in coloncancer is influenced on the expression of ASC. In order to make further study of theassociativity between the state of the methylation of ASC and the expression of NALP1in human colon cancer, we treated three kinds of colon cancer cell lines with5-aza-2’-deoxycytidine (DAC) to restore ASC expression, and detected the expressionof NALP1before and after drug treatment. Our data was planed to prove thatmethylation of ASC influences the expression of NALP1and inhibits apoptosis inhuman colon cancer.Methods:1.To explore whether the two genes can occurring methylation, weanalyzed the condition of CpG islands in both ASC and NALP1by applied biosystems(Methyl Primer Experss Software v1.0).2. To restore the expression of ASC gene in three colon cancer cell lines by using5-aza-2’-deoxycytidine.3. To detect the state ofthe methylation of ASC in cancer cell lines by using methylation specific PCR (MSP).4.To analyze the expression of ASC before and after drug treatment in human coloncancer cell lines by using RT-PCR and Western blot.5. To analyze the expression ofNALP1before and after ASC restoration in human colon cancer cell lines by usingReal-time PCR and Western blot.6. To compare with three kinds of cancer cell linestreated whit DAC with each outer, we analyzed the effect of apoptosis caused by ASCmethylation by using Flow Cytometry.Results:1. Through the detection by software, we confirmed that there was a CpGisland in ASC gene but there was no CpG island in NALP1.2. MSP showed that ASCwas completely methylated in LS174T and HCT-116cell lines, but partly methylated inLoVo cell line.3. After drug treatment, the result showed that there was a higher or arestoration of ASC expression in human cancer cell lines by both RT-PCR and Westernblot.4. Both Real-time PCR and Western Blot showed that there was an increasedexpression of NALP1in all the cancer lines when ASC re-expressed.5. Treating threekinds of colon cancer cell lines with DAC to restore ASC expression，we found that theratio of apoptosis was higher than the control by Flow Cytometry in vitro expriment．Conclusion: It was indicated that ASC was methylated in three kinds of coloncancer lines, but NALP1can not be methylated because there was no CpG island in itsgene. The low expression of NALP1in colon cancer was not a direct effect ofmethylation but regulated by ASC. Restoration the expression of ASC can enhance cellsapoptosis. So our data supports that methylation of ASC can influence the expression ofNALP1, inhibit apoptosis and induce the occurence of colon cancer. |
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