The role of vimentin as a methylation biomarker for early diagnosis of cervical cancer

Abstract

Multiple cytosine guanine dinucleotides (CpG island) are found in the VIM promoter region. The levels of VIM promoter methylation and VIM gene expression were investigated in 7 cervical cancer cell lines and 50 human tissue samples with a distinctive degree of malignant trans-formation. While multiple CpG sites in the VIM promoter were highly methylated in CIN III and invasive carcinoma cells, they were rarely methylated in normal cells. Our result shows that methylation in the VIM promoter appears to start from CIN I and CIN II, relatively early stages of multistep carcinogenesis. This epigenetic alteration in VIM promoter suggests the availability as a biomarker for the early diagnosis and prevention of cervical cancer. We also show that hypermethylation in the VIM promoter is responsible for transcriptional silencing of the VIM gene in cervical cancer cells. In addition, our result shows that exogenous overexpression of the VIM gene in SiHa cervical cancer cells slightly activated cell proliferation and migration as shown in soft agar colony formation and migration assays.

Fig. 1.. Methylation status of the VIM promoter in 7 cervical cancer cell lines. (A) Predicted CpG island in the genomic DNA containing the VIM gene. CpG dinucleotides in the VIM promoter are shown as vertical lines. Number, +1, indicates the translation start site of the VIM gene. Arrows indicate the position of primers used in the MSP or BSP assays (Table 2). The oligonucleotides used in this work are named as indicated. Abbreviations F and R denote forward and reverse, respectively. (B) MSP analysis of the VIM promoter in 7 cervical cancer cell lines. Methylation status is presented by the presence or absence of bands. UM or M represents PCR products amplified by oligonucleotide primers specific for unmethylated or methylated DNA, respectively. (C) Bisulfite sequencing of the CpG sites in the VIM promoters of 7 cervical cancer cell lines. Methylated or unmethylated cytosines are represented as closed or open circles, respectively. Their spacing reflects the CpG density of the region. Each row represents an individual cloned allele that was sequenced following sodium bisulfite DNA modification.

Fig. 2.. VIM gene expression in 7 cervical cancer cell lines. (A) Genomic DNA for the VIM gene is represented as exons (black boxes) and introns (thin lines linking the black boxes). The putative minimal promoter of the VIM gene is indicated by a horizontal thick line. Numbers indicate the position of exons coding the VIM gene. (B) The full-length transcript of the VIM gene is shown, where exons are indicated as boxes and numbers. Arrows indicate the positions of pRT-VIM-F and -R primers used in the RT-PCR (See Table 2). Abbreviations F and R denote forward and reverse, respectively. Nucleotide sequences were obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ BLAST/). (C) The transcriptional level of the VIM gene was measured in 7 cervical cancer cell lines. β-actin served as an internal control for the integrity of the cDNA.

Fig. 3.. RT-PCR analysis demonstrating reactivation of VIM gene expression in SNU-1299 cell line treated with demethylase 5′-Aza-dC and/or TSA. (A) RT-PCR analysis. RT-PCR was carried out using cDNA from cells subjected to different concentrations of the drug (See “Methods and Materials”). The β-actin served as an internal control for the integrity of the cDNA. (B) MSP analysis of the VIM promoter after treatment of 5′-Aza-dC and/or TSA. Methylation levels are presented by the thickness of bands. UM or M represents PCR products amplified by oligonucleotide primers specific for unmethylated or methylated DNA, respectively.

Fig. 4.. Schematic representation of correlation between VIM promoter hypermethylation and cervical cancer development. (A) MSP analysis of VIM promoter in normal, CIN I, CIN II, CIN III, and invasive cervical carcinoma tissues. The symbols of U or M denote PCR products amplified by oligonucleotide primers specific for unmethylated or methylated DNA, respectively. (B) Bisulfite sequencing of CpG sites in the VIM promoters of normal and carcinoma tissue samples. Each row represents an individual cloned allele that was sequenced. Circles represent CpG sites and their spacing reflects the CpG density of the region. Unmethylated or methylated cytosines are represented as open or closed circles, respectively.

Fig. 5.. Functional study of VIM in SiHa cervical cancer cells. (A) Soft agar colony-forming assays. SiHa cervical calls were transfected with pcDNA3 or pcDNA3-VIM vectors and the transfected cells were plated with 50 cell numbers per 6-well plate for 22 days. (B) Cervical cancer colony formation by the pcDNA3-VIM expression vector. (C) Western blotting verifying the overexpression of the VIM gene. (D) Phase micrographs of SiHa cells at various times after monolayer wounding. The pcDNA3 or pcDNA3-VIM-transfected SiHa cells were scratched and the closure of the scratch was photographed at the indicated times. This experiment was repeated 3 times and the closest result average is presented.