Hypermethylation Of EDNRB Promoter Contributes To The Risk Of Colorectal Cancer

Objective: Colorectal cancer (CRC) is one of the most common digestive malignancies in theworld. EDNRB is a new candidate tumor suppressor gene which is often down-regulated or evensilenced by promoter hypermethylation in various human cancers. However, the function ofEDNRB gene in CRC remains unknown. In this study, we examined the expression and DNAmethylation of EDNRB in CRC tissues, as well as its clinical significance.Methods:1. Tissue samples collection: Cancer and adjacent normal tissue samples werecollected at the time of surgery from42primary sporadic CRC patients in the Department ofGastrointestinal Surgery in XX Hospital between June2012and April2013. Adjacent normaltissues were collected at least5cm away from the tumor. Tissues were stored in liquid nitrogenimmediately after excision and then stored at-80℃.2. MSP: Spin column was used to extractDNA from42pairs of colorectal cancer tissues and normal tissues. DNA concentration and qualitywere determined with spectrophotometer. Eluted DNA was bisulphite-treated with ZYMO EZDNA Methylation-Gold Kit according to the manufacturer’s instruction. Then methylation statusof EDNRB promoter was determined by MSP. PCR products were subjected to2.0%agarose gelelectrophoresis at100V for20min, and visualized directly under UV illumination.3. qRT-PCR:Total RNA was extracted from fresh frozen CRC and normal tissues from the same patients withTRIzol reagent by the manufacturer. RNA concentration and quality was determined. The first-strand cDNA was synthesized according to the manufacturer’s instruction of M-MLV ReverseTranscriptase. The qRT-PCR reactions were conducted in a96-well plate. The values of Ctdisplayed by the instrument were recorded. Samples were confirmed whether there was a clearlyvisible band (86/194bp) on the gel with EDNRB or GAPDH primers.4. Statistics: Comparisons ofEDNRB promoter methylation were made by the correction formula of Chi-square test. The2–ΔCtmethod was used to analyze the result of qRT-PCR. Two groups of related data were analyzedusing paired t-test. The Mann-Whiteney U test was used to statistical analysis two groups ofindependent data which does not meet normality test. We considered p <0.05as statisticallysignificant.Results: The EDNRB methylation rate of42colorectal cancer tissues (positive rate of92.86%)was significantly higher than the EDNRB methylation rate of42normal tissues (positive rate of 59.52%, p=0.001). Consequently, significantly lower level of EDNRB mRNA in CRC tumorsamples was observed than that of the normal samples (0.31+0.91versus0.70+1.18, p=0.032).No significant relationship was observed between EDNRB expression status or methylation and theclinical features.Conclusions:1. The hypermethylation of EDNRB in CRC tissues was more frequent than in correspondingnormal tissues.2. There was no significant difference between hypermethylation of EDNRB in CRC tissuesand clinicopathological factors.3. Significantly lower EDNRB mRNA level in CRC tumor samples was observed than that innormal samples.4. There was no significant difference between EDNRB mRNA level in CRC tissues andclinicopathological factors.Our results suggested that hypermethylation of EDNRB promoter might be responsible for itsdownregulation in CRC, and play an important role in the development of CRC. EDNRB genepromoter methylation can be a marker of early diagnosis of colorectal cancer, and also may beused as a new target for biological treatment.