Quantitative assessment of AKAP12 promoter methylation in colorectal cancer using methylation-sensitive high resolution melting: Correlation with Duke's stage.
BACKGROUND
The expression of AKAP12 (A Kinase anchoring protein 12) is markedly reduced in a variety of cancers. The purpose of this study was to establish a methylation-sensitive high resolution melting (MS-HRM) assay for the quantitative detection of AKAP12 promoter methylation and expression and the association with clinicopathological variables in human colorectal cancer. We also assessed the effect of AKAP12 re-expression on cell growth and colony formation.


RESULTS
Downregulation or loss of AKAP12 mRNA expression was detected in 31 of 45 tissue samples (68.9%). No significant correlation was observed between the reduced expression levels and patient age, gender, Duke's stage or tumor differentiation. Methylation (>1%) of the AKAP12 promoter region was present in 35 of 45 (77.8%) carcinoma tissue samples and 6 of 45 (13.3%) adjacent tissue samples. AKAP12 methylation was significantly higher in the colorectal cancer tissues exhibiting advanced Duke's stages. Treatment of the three colorectal carcinoma cell lines (LoVo, COLO320 and SW480) with completely methylated AKAP12 with inhibitors of DNA methyltransferase (5-Aza-2'-deoxycytidine) markedly increased expression of AKAP12 and decreased methylation levels. Ectopic expression of AKAP12 in the LoVo cell line suppressed cell growth and inhibited colony formation.


METHODS
The AKAP12 gene was examined by quantitative RT-PCR, MS-HRM analysis and bisulfite sequencing in 45 paired tissue samples obtained from primary colorectal carcinomas and the corresponding adjacent tissues. In addition, five colorectal carcinoma cell lines (LoVo, COLO205, SW480, LS174T and COLO320) were investigated and western blot analysis was used to investigate changes in protein expression. A proliferation assay and soft agar assay were performed after overexpression of AKAP12.


CONCLUSIONS
Our study demonstrated that MS-HRM is a robust, fast and sensitive method for AKAP12 methylation analysis. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of this cancer.
Liu
Weiwei
W
Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
Guan
Ming
M
Su
Bing
B
Ye
Chuanzhong
C
Li
Ji
J
Zhang
Xinju
X
Liu
Chunfang
C
Li
Min
M
Lin
Yong
Y
Lu
Yuan
Y
D054758:A Kinase Anchor Proteins / Q000235:genetics* / Q000378:metabolism; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
D054758:A Kinase Anchor Proteins; C491162:AKAP12 protein, human; D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine