Down-regulation of the expression of CCAAT/enhancer binding protein α gene in cervical squamous cell carcinoma

Clinical data and cervical squamous cell carcinoma samples were collected from patients at the First Affiliated Hospital and the Third Affiliated Hospital of the Medical College of Shihezi University, Xinjiang, China between January 2008 and April 2012. Cervical cancer samples were taken for every consecutive patient with different grades of pathology. None of these patients received chemotherapy or radiotherapy before the cervical tissue samples were obtained. All histological diagnoses were confirmed by experienced pathologists in the hospitals. Written informed consent was obtained from each patient; approval was obtained from the Ethics Committee of the Medical College of Shihezi University, China. Cervical squamous cell carcinoma tissues and adjacent normal cervical tissues were collected together from each patient. Tissue samples were snap-frozen immediately after removal and were stored at -80°C.

Representative formalin fixed paraffin-embedded tissue blocks were selected. Five μm sections were cut, deparaffinised and rehydrated through graded alcohols. Antigen retrieval was performed by heating the slides in citrate buffer at 98°C for 30 min in a water bath. Endogenous peroxidase was quenched for 10 min with peroxidase blocking reagent (Dako Corporation). Primary antibodies, anti-C/EBPα (1:200; sc-61, Santa Cruz Biotechnology) and anti-Ki-67 (1:100; BD Biosciences Pharmingen) were incubated for 60 min at room temperature. Antibody staining was visualized using the ChemMate Envision detection system (Dako Cytomation). Sections were counterstained. The counterstaining was performed with Meyer’s hematoxylin solution. Negative controls were run simultaneously with preimmune immunoglobulin. Rabbit polyclonal antibody (Ab-1) used was diluted 10- and 50-fold. The specificity of the immunohistochemical reactions was assessed by performing the assays in the presence of an excess of relevant versus irrelevant peptides. The peptide used for immunization completely suppressed staining whereas an irrelevant peptide at the same concentration had no effect. The C/EBPα protein and Ki-67 protein IHC signal was scored on the following scale taking into account both the proportion of cells stained and the intensity of staining in those cells as follows: score 0, no cells stained; score 1, weak or absent nuclear staining and <5% of cells containing C/EBPα and Ki-67 localized to the nuclei of cells; score 2, nuclear staining and between 5% and 25% of the cells containing C/EBPα and Ki-67 localized prominently to the nuclei of cells; score 3, nuclear staining and between 26% and 50% of the cells containing C/EBPα and Ki-67 localized prominently to the nuclei of cells; score 4, nuclear staining and more than 50% of the cells containing C/EBPα and Ki-67 localized prominently to the nuclei of cells. Two observers quantified independently.

Gene methylation was analyzed using MALDI-TOF MassARRAY method [12]. DNA from cervical tissue samples was extracted using QIAamp DNA Mini Kit (QIAGEN). Bisulfite treatment was performed using EZ DNA methylation kit (Sequenom, USA). PCR was performed in a total volume of 5 μl containing 0.5 U Hot Star Taq polymerase (Qiagen), 10 pmol of forward and reverse primers 0.5 μl, 10× PCR buffer (Mg²⁺ free) 0.5 μl, ddH₂O 0.5 μl, MgCl₂ 0.4 μl, 25mM dNTP 0.1 μl and 5 ng template. Cycling was performed using a Mastercycler (Eppendorf) under the following conditions: 15 min at 94°C; 45 cycles at 94°C for 20 sec, annealing at 62°C for 30 sec and extension 72°C for 1 min; and finally, extension at 72°C for 3 min. Shrimp alkaline phosphotase (SAP) mixture (2 μl) was then added to each reaction. The reactions were vortexed briefly, centrifuged at 3,000 rpm for 5 min, and incubated at 37°C for 20 min, and 85°C for 5 min.

RNA transcription was performed in a volume of 5 μl containing RNase Free-ddH₂O (3.15 μl), 5× T cleavage & Polymerase buffer (0.89 μl), T7 RNA & DNA polymerase (0.44 μl), T cleavage mix (0.24 μl), DTT (100 mM, 0.22 μl), and RNAase A (0.06 μl). After incubation at 37°C for 3 hr, 6 mg Clean Resin (Sequenom) was added to desalt RNA. MassARRAY MS (Bruker-Sequenom USA) was used to analyze the data.

cDNA of C/EBPα gene was cloned into pcDNA3.1(-) vector and confirmed by sequencing after cloning into the pcDNA3.1(-) expression vector. HeLa cells were initially obtained from ATCC (American Type Culture Collection, Manassas, VA) and maintained in our lab. Non-transfected HeLa cells and those transfected with pcDNA3.1(-) were used as controls. HeLa cells were plated at 1–2 × 10⁵ cells per well in a six-well cell culture plate 12–24 hr before the transfection. Two μg of C/EBPα constructs and control plasmids pcDNA3.1 were mixed with 6 μl of Lipofect AMINE 2000 (Invitrogen). The mixture was incubated at room temperature for 20 min. After washing the cells with 1× PBS, the DNA/ Lipofect AMINE 2000 mixtures were transferred to HeLa cells. Plasmids pcDNA3.1 were also transferred into separate HeLa cells as controls. Transfected HeLa cells were then incubated at 37°C in the 5% CO₂ for 24 hr. The effect of C/EBPα overexpression was determined by measuring immunofluorescence luciferase activity using an assay system according to the manufacturer’s protocol (Promega). Each experiment was repeated with multiple batches of cells.

The survival rate of HeLa cells transfected by C/EBPα pcDNA3.1 construct and pcDNA3.1, and non-transfected HeLa cells, was determined using the 3-(3,4-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The HeLa cells transfected with C/EBPα pcDNA3.1 constructs, transfected cells with pcDNA3.1, and non-transfected HeLa Cells were seeded in 96-well plates (Falcon; Becton Dickinson Labware) at 0.6 × 10⁴ cells per well in DMEM containing 10% FBS. The cells were incubated for 24 hr at 37°C; the number of cells was quantified with the MTT cell growth assay kit according to the manufacturer’s instruction. Briefly, 20 μl of MTT solution was added to each well, incubated for 4 hr, and then scanned at 570 nm by microplate reader MTP-300 (Corona); the quantification was repeated 10 times. The statistics were obtained from ten individual experiments by analysis of variance.

Cells were serum-starved overnight. The top chambers of 6.5 mm Corning Costar transwells (Corning, NY, New York, USA) were loaded with 0.2 ml of cells (5 × 10⁵ cells/ml) in serum-free media. DMEM media containing 20% FBS 0.6 ml was added to the bottom wells and the cells were then incubated at 37°C overnight. Cells on the top layer were removed and the images of the cells at the bottom of the membrane were captured using a canon camera microscope (×200). The statistics were obtained from five individual experiments by analysis of variance.

Statistical analysis was performed using SPSS 17.0 software. C/EBPα protein levels were compared between cervical carcinoma and chronic cervicitis using non-parametric test. C/EBPα mRNA level in cervical carcinoma tissues and the adjacent normal tissues was analyzed by Student ‘t’ test. The promoter of C/EBPα gene methylation by MALDI TOF MassARRAY method was analyzed with Wilcoxon Rank Sum Test.

C/EBPα protein level was strongly positive (score 4) in the nuclei of chronic cervicitis cells. There were 70 strong staining (score 4), 19 medium staining (score 3), 20 weak staining (score 2) and 16 negative staining (score 0–1) in chronic cervicitis tissues. There were, however, 44 strong staining (score 4), 59 medium staining (score 3), 34 weak staining (score 2) and 22 negative staining (score 0–1) in cervical carcinoma. C/EBPα protein level was the highest in well-differentiated cervical carcinoma, followed by moderately differentiated cervical cancer, and was the lowest in poorly differentiated cervical cancer (Figure 1). Thus, the difference between chronic cervicitis and cervical carcinoma in C/EBPα protein level was significant (P < 0.001) (Table 1). C/EBPα protein level in the well-differentiated cervical carcinoma was significantly higher than that in the moderately differentiated cervical carcinoma. Further, C/EBPα protein level in moderately differentiated cervical carcinoma was significantly higher than that in poorly differentiated cervical carcinoma (P < 0.001) (Table 2). Immunohistochemistry staining of serial sections of tissues showed that expression of C/EBPα protein was increased whereas expression of Ki-67 protein expression was reduced in chronic cervicitis. However, in CIN3 (carcinoma in situ) and squamous cell carcinoma, C/EBPα protein expression was decreased whereas Ki-67 protein expression was increased (Figure 2). Therefore, it is possible that C/EBPα protein could regulate cervical cell proliferation.

Fifteen cervical carcinoma tissues and their corresponding normal cervical tissues were examined for CEBPα gene expression by real time quantitative RT-PCR. The average CEBPα mRNA level was 3.07 ± 1.04 in cervical cancer tissues and 5.63 ± 2.98 in their corresponding normal cervical tissues (Figure 3A). The difference between cervical tissues and their corresponding normal cervical tissues was significant (t = -3.150, P < 0.01). Thus, the expression of CEBPα gene was reduced significantly in cervical carcinoma tissues.

Forty-seven cervical carcinoma tissues and 25 normal tissues were analyzed for methylation in the promoter of C/EBPα gene by MALDI TOF MassARRAY. We found that the rate of C/EBPα gene methylation in CpG 5, CpG-14.15, CpG-19.20 were significantly higher in cervical tissues than in normal cervical tissues (P < 0.05, P < 0.01, P < 0.05, respectively; Figure 3B). Methylation in the promoter of C/EBPα gene, thus, may have caused the reduction in the expression of this gene.

The proliferation of HeLa cells transfected by C/EBPα pcDNA3.1 construct was inhibited significantly compared to those transfected by pcDNA3.1 plasmid and non-transfected HeLa cells using MTT assay (P < 0.001) (Figure 4A). This study indicates that C/EBPα gene may inhibit HeLa cell proliferation. To assess whether overexpression of C/EBPα is sufficient to reduce cell migration, we measured the migration of HeLa cells using transwell migration assays. The number of HeLa cells migrated in C/EBPα pcDNA3.1 construct transfected group is significantly less than that in the two control groups, i.e. pcDNA3.1 plasmid transfected group (without C/EBPα gene) and the HeLa cells not subject to transfection (Figure 4B). Thus, the expression of C/EBPα protein in HeLa appears to inhibit cell migration. The numbers of migration cells are significantly different between C/EBPα pcDNA3.1 construct transfected group and the two control groups with mean ± SD (n = 5) (P < 0.001) (Figure 4C).