Association Analysis Of FOXE-1 Methylation And Expression And Its Correlation With Colorectal Cancer

Objective:FOXE1(Folkhead box E1) belongs to the forkhead family of transcription factors, which is characterized by a distinct forkhead domain. This gene plays a crucial role in thyroid morphogenesis. Studies show the homozygous mutation of FOXE1can cause the thyroid dysgenesis such as hypothyroidism and cleft palate. FOXE1also has a close relationship with malignant tumor such as thyroid adenocarcinoma and squamous cell carcinoma in skin, lung and esophagus due to the polymorphism and mutation. It has been reported the promoter the FOXE1has the hypermethylation in pancreatic cancer and breast cancer but no relative reports in colorectal cancer. This study aims to detect the level of CpG island in promoter of FOXE1between the colorectal cancer tissue and paired normal mucosa, then combines with the research of the mRNA transcription and protein expression level and reveals the relationship between the regulation of the CpG island in promoter of FOXE1and the carcinogenesis in colorectal cancer.Materials and Methods:Cell lines:Three colorectal cell lines (HCT116, RKO, LS147) which were obtained from Cellular compartment of Chinese Academy of Sciences.Tissue specimens:A collection of83colorectal tissues were obtained from Cancer Hospital of Fudan University in Shanghai including paired normal tissues which at least5cm from the cancer tissue from the patients who underwent surgery from December2008to March2010. No patients received neoadjuvant radiotherapy or chemotherapy. All patients underwent RO resection and got pathological diagnosis after the operation. Tissues were obtained from patients less than30min after the resection and frozen in-196℃liquid nitrogen and then were frozen in RNALater solution which were used to extract Total RNA. Informed consents were signed and this study was approved by Ethics Committee of Fudan University. Tumor specimens were confirmed by routine histopathological examination based on Cancer Staging Manual Seventh Edition from American Joint Committee on Cancer (AJCC).Preliminary screeningGuoxiang Cai, M. D. used the genome methylation chip (Illumina Human Methylation27) to screen the methylated genes related to colorectal cancer. The results show that FOXE1is one of the hypermethylation genes.Method:1. Methylation-specific PCR (MSP) was used to detect the methylation status of of FOXE1in cell lines.The MSP-positive cell line was treated with demethylation drug5-aza-2-CdR. Quantitative Methylation Specific Real-time PCR (qMSP) was used to detect the methylation level of FOXE1; Taqman Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect FOXE1expression before and after treated with5-aza-2-CdR.2. DNA was extracted from the83tumor tissues and paired normal tissues and modified with bisulfate. qMSP was performed to detect the methylation level of FOXE1. RNA was extracted from the40tumor tissues and paired normal tissues frozen in RNA Later solution. qRT-PCR was used to detect the expression of FOXE1.Data analysisPercentage of methylated reference (PMR) was calculated as below:PMR=100×(target gene/reference gene) samples/(target gene/reference gene)MSssI-treated Human DNA. Methylation status is defined as negative when PMR<4while positive when PMR≥4. Delta CT Value of samples=CT Value of target gene-CT Value of reference.2-△Ct was defined as relative expression of miR-615. Correlation between methylation status and clinicopathological features were assessed by Pearson Chi-square test. The division of methylation state between83paired tumor and normal tissues was analyzed by paired Chi-square test. The expressional difference of foxel in40paired samples was assessed by paired t-test. Correlation between miRNA expression and methylation state was evaluated using independent sample t-test in40CRCs. Kaplan-Meier and Log-rank test were used to analyze the impact of methylation state on disease-free survival and overall survival. P-Value <0.05was considered as statistically significant and all p-Value was obtained by two-sides tests. All the analysis were performed using SPSS19.0software package.ResultThe MSP status of FOXE1is positive in RKO while negative in HCT116and LS147. Demethylation treatment can up-regulate the expression of FOXE1. The percentage of methylation-positive cases was significantly higher in83CRCs compared with paired normal tissues (p<0.001). In40paired samples, the expression level of FOXE1in tumor tissues is significantly lower than paired normal tissues (P=0.046). An inverse correlation was observed between the methylation level and expression of FOXE1 in40tumor tissues (p=0.048). No significant correlation were observed between methylation-negative and methylation-positive patients in DFS (p=0.618) and OS (p=0.500).Conclusion:The expression of FOXE1in colorectal cancer may be regulated by methylation, hypermethylation can down-regulate the expression of FOXE1. Aberrant hypermethylation of FOXE1can be highly oncogenic;the level of FOXE1methylation on cancer tissue is significantly higher than its pared normal tissue.