Significance Of Secreted Frizzled-Related Protein In The Process Of Tumorigenesis In Human Sporadic Colorectal Carcinomas

Objective:(1) To investigate the functions of promoter hypermethylation of secreted frizzled-related proteins(SFRPs) in colorectal cancer(CRC).(2) To investigate the expression of SFRPs in colorectal cancer tissues.(3) To study the change of gene expression profile in human colorectal cell line HCT116 transfected with SFRP4-expressing plasmid and provide important information for the further research on the protential functional mechanisms of SFRP4 in colorectal tumorigenesis.Methods:(1) The promoter hypermethylation of SFRPs in 20 sporadic CRC tissues and adjacent normal mucosa was detected by methylation-specific PCR.The amplified DNA was subcloned into the T-A cloning vector and sequenced.Two CRC cell lines,HCT116 and SW480,were treated by 5-aza-2'deoxycytidine for demethylation.The promoter hypermethylation and protein expression of SFRPs in CRC cell lines were detected by methylation-specific PCR and western blot respectively.(2) The mRNA expression of SFRPs was analyzed in 20 paired CRC and corresponding adjacent non-cancerous tissues by quantitative real time RT-PCR and the protein expression of SFRP1 and SFRP4 was verified by Western blot in these samples and further measured by immunohistochemistry(IHC) in 206 colorectal tissues.(3) pcDNA3.1-SFRP4 was transfected into HCT116 cells,and the empty vector-transfected HCT116 cells were used as the control.Differentially expressed genes were screened with Affymetrix array.The results of cDNA microarray were confirmed by RT-PCR.Results:(1) Hypermethylation of SFRP1,2,4,5 was detected in 95%(19/20),85% (17/20),15%(3/20),65%(13/20),and 60%(12/20),40%(8/12),5%(1/20),35% (7/20) of the colorectal cancer tissues and adjacent normal mucosa,respectively. SFRP1,2,5 methylation was more frequently found in CRC than in adjacent normal mucosa(P＜0.05).SFRP1,2,4,5 were methylated in HCT116,and SFRP1,2 were methylated in SW480.The SFRPs protein expression was absent when their promotors were methylated in colorectal cancer cell lines.5-aza-2'deoxycytidine treatment re-expressed the silenced SFRP protein expressions effectively.(2) The mRNA levels of SFRP1 and SFRP5 were significantly downregulated in 85%and 80%of CRC,but SFRP4 was overexpressed in 16 of 20 CRC samples.These findings were concordant with those obtained from the Western blotting in SFRP1 and SFRP4. Moreover,IHC analysis demonstrated a significant difference among CRC,HIN and adenoma in SFRP1 and SFRP4 expression.(3) SFRP4 transfection induced extensive change of the gene expression profile of HCT116 cells.The differentially expressed genes were involved mainly in p38MAPK pathway,TNF pathway and cell apoptosis.Conclusion:(1) Methylation of SFRP1,2,5 is associated with the evolution of colorectal cancer.Methylation of SFRPs is closely related to the expression silencing of SFRPs.(2) The differences in expression suggest SFRP1 and SFRP4 appear to be more suitable candidate markers for colorectal lesions.Unlike SFRP1 as a negative regulator,SFRP4 may have quite different biological role.(3) Due to molecular changes in some important pathways influenced by SFRP4 transfection,SFRP4 may take part in the regulation of these pathways.