The Expression Of Dact1 And Dact3 Gene In Carcinogenesis Of Colorectal Tumor

BackgroundColorectal carcinoma is one of the most common tumor types in our country. In present, scientists focus their studies on cancer surppressor genes to find out the moleculor mechanisms of tumor oncogenesis. Wnt pathway,which plays an important role in the development of colorectal cancer,is a hot spot of research. On the while, Dact is a key regulator site in the Wnt pathway.The purpose of this study is to observe the methylation of promoter of gene Dact1(Dact3) and the expression of Dact1(Dact3) mRNA in colorectal tumor and to analyze its relationship between them with carcinogenesis of colorectal tumor.Materials and MethodsColorectal cancer tissues 50 pairs of cancerous and corresponding paracancerous were surgically obtained from colorectal cancer patients ,and 20 of normal colorectal tissues were surgically obtained from PPH patients, all of which in the Hangzhou Hospital of anhui Medical University between 2008.1 and 2009.7. All the cancerous tissues had clear pathological diagnosis. The paracancerous were far away about 5cm from cancerous mucoses, and also had clear pathological- diagnosis: normal mucoses cases. All patients were preoperatively end up receiving chemotherapy and other anti-cancer therapy. All the tissues were stored at -80℃for reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific polymerase chain reaction (MSP).RT-PCR:Total RNA was isolated from tissue samples using Trizol Reagent according to the manufacturer's protocol. The concentration and purity of total RNA were determined from the A260/A280 ratio using a UV spectrophotometer .One microgram of total RNA from each specimen was subjected to RT-PCR. Theβ-actin and Dact1(Dact3) PCR products were electrophoresed on a 2% agarose gel and visualized by staining with ethidum bromide. The integrated density values of the bands representing amplified products were acquired and analyzed by a image analysis system.MSP:DNA was extracted from the 50 colorectal cancers and paracancerous and 20 of normal colorectal tissues using phenol-chloroform extraction, than treated with bisulfite. briefly, DNA was denatured with NaOH and modified with sodium bisulfite. DNA samples were then purified using Wizard DNA purification resin, treated again with NaOH, precipitated with ethanol, and resuspended in water for template to PCR. l 0ul samples of each PCR reaction products were loaded directly onto 2.0% agarose gels, stained with 0.1% ethidium bromide, and visualized under UV illumination. Results determination: Only when PCR products were amplified with methylated primer, then the promoter of Dact1(Dact3) gene could be regarded as methylation.Statistical analysis: All the data were analyzed with SPSS11.5 statistics software. The statistical significance was defined as P<0.05.ResultsThe detection of colorectal cancer Dact1 mRNA positive expression (12%, 6 / 50) by Qualitative RT-PCR was lower than in the paracancerous (90%, 45/50) and normal colorectal tissues (95%, 19/20). And there were significantly different between colorectal tumor tissues and paracancerous, normal colorectal tissues respectively(P<0.05). In the same way, colorectal cancer Dact3 mRNA positive expression (10%,5/50) were lower than in paracancerous (86%,43/50) and normal colorectal tissues (90%,18/20). And there were significantly different between colorectal tumor tissues and paracancerous, normal colorectal tissues respectively(P<0.05). Bisulphite sequencing revealed that the methylation was unequally distributed within the Dact1 promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas tissues (46% , 23/50) . In contrast, normal colorectal tissues displayed lower hypermethylation (5%,1/20) as well as in paracancerous (12%,6/50). On the contrary, the hypermethylation of Dact3 have no significant difference between colorectal tumor tissues (46%,23/50) and paracancerous(10%,5/50), as well as normal colorectal tissues(10%,2/20)(P>0.05).ConclusionPromoter hypermethylation of Dact1 were presented in the vast majority of colorectal tumor, and only rarely in paracancerous and normal colorectal tissues, which was maybe correlated with Epigenetic silencing and down-regulation of the Dact1 gene in colorectal tumor .On the other hand, promoter hypermethylation of Dact3 had no significant difference between colorectal carcinomas tissues and paracancerous, as well as normal colorectal tissues.It maybe had no relevance in the loss (low expression) of Dact3 mRNA expression with the state of promoter methylation of Dact3.