The SiHa (HPV 16-positive) cell line was kindly provided by Dr.Silvio Gutkind (Moores Cancer Center, UCSD, USA) with proof that there was no contamination, and the C33a (HPV-negative) cell line was purchased from the American Type Culture Collection (HTB-31TM; lot no. 63596879). The cells were grown and maintained in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% antibiotic-antimycotic (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere of 5% CO₂.

The E6 recombinant plasmid was inserted into a PCDNA3 vector, which was provided by Assoc. Prof. Dr. Andrew Yeudal (Augusta University, USA). The E7 recombinant plasmid was inserted into PCDNA3.1 myc-his, which was constructed as per a previous study (13). Both recombinant plasmids were sent for sequencing to confirm that the sequences were correct.

E6 and E7 recombinant plasmids were transfected into C33a cells. C33a cells were seeded into 6-well plates at 3×10⁵ cells/ml and incubated overnight. Next, 2 µg E6 or E7 recombinant plasmid and pcDNA 3.1/myc-his (PC) empty plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After 72 h, transfected cells were collected to study E6- and E7-mediated CADM1 and DAPK1 promoter methylation, and CADM1 and DAPK1 mRNA expression. Moreover, cells transfected with E7 were harvested for chromatin immunoprecipitation. The transfection was performed in triplicate for all experiments.

SiHa, C33a and C33a cells transfected with E6 or E7 were subjected to DNA extraction. Briefly, cells were digested with lysis buffer II containing SDS (Sigma-Aldrich; Merck KGaA) and proteinase K (USB) at 50°C overnight. Phenol/chloroform extraction and ethanol precipitation were then carried out as previously described (14).

SiHa, C33a and C33a cells transfected with E6 or E7 and empty vector were subjected to RNA extraction. Total RNA was extracted using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, 5 µg total RNA from each sample was synthesized to cDNA using the RevertAid first-strand cDNA synthesis kit, according to the manufacturer's specifications (Thermo Fisher Scientific, Inc.).

A total of 750 ng DNAsample was subjected to bisulfite treatment using the EZ DNA Methylation-Gold kit (Zymo Research Corp.), according to the protocol provided by the manufacturer. The eluted DNA was subsequently used to carry out methylation-specific PCR using 0.3 µM methylated and unmethylated specific primers (Table I). The DNA was initially denatured for 15 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 60°C for CADM1 and 53°C for DAPK1, and 1 min at 72°C, and 72°C for 7 min. Then, 10 µl PCR products were observed by gel electrophoresis on 8% acrylamide gels and stained with SYBR (Lonza Group, Ltd.). The methylated and unmethylated band intensities of each sample were visualized and measured using Storm840 and ImageQuant Software (Amersham Biosciences; GE Healthcare). The experiment was performed in triplicate.

Quantitative PCR was performed to observe the DAPK1 and CADM1 mRNA expression in SiHa C33a cells and c33a transfected cells. Amplification was performed with 0.1 µM DAPK1 and 0.1 µM CADM1 primers (Table I), including primers for 0.1 µM GAPDH, which was used as a reference gene, and 19 Power SYBR Green PCR master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cDNA was amplified using a 7500-fast real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with initially denatured for 10 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C, and 72°C for 7 min. The fold changes in expression of DAPK1 and CADM1 between experimental and control cellswere then determined using the ^ΔΔCt method (18). The experiment was performed in triplicate.

Chromatin immunoprecipitation was carried out in E7 recombinant plasmid- and empty plasmid-transfected C33a cells, as previously described, with some modifications (19). To observe the binding of E7 protein (with or without the CR3 region) at the CADM1 promoter, the chromatin fragments were immunoprecipitated with 10 µg of the H3K4 (Abcam (Cambridge, UK), HPV16-E7 (Santa Cruz, CA, USA) and IgG (Santa Cruz, CA, USA) antibodies. Next, the precipitated DNA was further analyzed by PCR using 0.3 µM CADM1-ChIPF and 0.3 µM CADM1-ChIPR (Table I) with initially denatured for 10 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 62°C, and 1 min at 72°C, and 72°C for 7 min. Then, 10 µl PCR products were observed by gel electrophoresis using a 8% acrylamide and 1% agarose gel stained with SYBR (Lonza Group, Ltd.). The CADM1 bands were visualized using Storm840 and ImageQuanNT Software (Amersham Biosciences; GE Healthcare). The experiment was performed in triplicate.

To confirm the accuracy of the E6 and E7 recombinant plasmid sequences, E6 and E7 recombinant plasmids were transformed into competent Escherichia coli XL-1 blue cells for cloning, and then the plasmids were extracted using a GeneJET Plasmid Miniprep kit (Fermentas; Thermo Fisher Scientific, Inc.) for sequencing analysis immediately after transfection. After alignment between the sequence of recombinant plasmid and GENBANK (www.ncbi.nlm.nig.gov), the accuracy of the E6 and E7 sequences were confirmed.

To test the significance of the E6- or E7-mediated induction of CADM1 and DAPK1 promoter methylation and the decrease in gene expression, two sample t-tests between two groups of samples, E6 or E7 transfected cells and control cells, were used. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of HPV in mediating promoter methylation and decreasing gene expression, the SiHa cervical cancer cell line with HPV type 16 infection and the C33a cervical cancer cell line without HPV infection were used for the observation of methylation and the expression status of CADM1 and DAPK1. There was methylation at the CADM1 and DAPK1 promoters in SiHa cells, whereas there was no methylation at the CADM1 and DAPK1 promoters in C33a cells (Fig. 1A and B). There was no expression of CADM1 in SiHa cells, while the expression of this gene in C33a cells was detected. For DAPK1, significantly decreased expression was detected in SiHa cells compared to that in C33a cells; P=0.005. (Fig. 1C and D).

To investigate whether E6 or E7 of HPV induce de novo methylation of DAPK1 and CADM1, E6 or E7 recombinant plasmids and empty PCDNA3.1 (as a control) were successfully transfected into C33a cell (Fig. 2A and B). CADM1 and DAPK1 promoter methylation and their expression were observed. For methylation status in E6- or E7-transfected cells compared to control cells, significant CADM1 promoter methylation was observed in E7-transfected cells compared with control cells (P<0.0001), while the promoter methylation of this gene could not be detected in C33a cells transfected with E6. To observe the methylation of DAPK1 in C33a-transfected cells, the results showed that significantly increased DAPK1 promoter methylation was detected in E6-transfected cells compared to cells with the empty vector (P=0.0003), whereas there was no DAPK1 methylation in E7-transfected cells (Fig. 3A and B). For the expression study, after E7-transfected into C33a cells, there was a significantly decreased CADM1 expression in E7-transfected C33a cells (P=0.008) compared to cells with the empty vector. To investigate the expression of DAPK1, after E6 and empty vector transfection into C33a cells, there was significantly decreased expression of DAPK1 (P=0.001) in E6-transfected cells compared to cells with the empty vector (Fig. 3C and D).

It was hypothesized that E7 promotes CADM1 methylation by forming a complex with Dnmt1 at the CADM1 promoter, which is the same mechanism as that for CCNA1. To demonstrate that this hypothesis was viable, a chromatin immunoprecipitation assay was carried out in C33a cells transfected with E7 and the empty vector by precipitating with anti-HPV16 E7 and performing PCR to obtain a 229-bp CADM1 product. As shown in Fig. 4, the product band of the CADM1 promoter was detected in E7-overexpressing C33a cells, but not in the cells with the empty vector. These data suggested that E7 can bind at the CADM1 promoter.