Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

Patient and control groups

The study patient group was formed of patients diagnosed with eyelid tumors in the Ophthalmology Department of Sivas Cumhuriyet University (SCU), Faculty of Medicine, Research and Application Hospital. As there was no similar study in literature regarding the determination of the number of patients, the sample size was established with the support of the Department of Biostatistics. Approval for the study was granted by the Local Ethics Committee (decision no: 2014-05/18) and all procedures were applied in compliance with the principles of the Declaration of Helsinki and the Good Clinical Practice guidelines. Of patients who underwent surgery within 1 year of the date of ethics committee approval, 20 suitable cases were identified. Of these 20 patients, 12 had BCC and the other 8 had different types such as small cell malignant tumor, trichoepithelioma, malignant fibrous histiocystoma, squamous cell carcinoma, melanoma, chronic calcified granuloma, and sebaceous gland carcinoma. Therefore, to obtain meaningful results, only the 12 patients with BCC were evaluated in this study. No restrictions were placed in terms of gender, age, or the histopathological type and degree of cancer, but there were no patients younger than 50 years old in the study. Informed consent for participation in the study was provided by all the patients before surgery. Patients were included in the study after determination of the histopathological type of the tumor in the pathology report of the sample excised during surgery. The study was conducted on the tumor tissue taken from all patients during the surgery and control samples [19], [20], [21] obtained from the normal tissue around the tumor in accordance with the recommendation of the pathologist (Table 1). The patients included in the study were those who had not received any preoperative radiotherapy or chemotherapy as that can significantly affect the expression of biomarkers. Blood samples were taken from these patients into citrated tubes for ELISA analysis of the protein levels. A control group was formed of 12 age and gender-matched healthy individuals with no history of cancer, from among those who presented at SCU, Faculty of Medicine, Research and Application Hospital, Department of Physical Therapy and Rehabilitation. The experimental stage of this study was conducted in the Biochemistry Department and the Histology Department of the Faculty of Medicine.

Gene expression analysis with the real-time PCR

RNA isolation from control and tumor tissue was performed in accordance with the protocol recommended by the manufacturer, using the RNeasy Fibrous Tissue (Qiagen) catalogue number 74704 and RNeasy (Qiagen) mini-isolation kit with catalogue number 74104, respectively.

The RT2 First Strand cDNA synthesis kit (Qiagen, cat. no: 330404) was used for the synthesis of cDNA. The concentrations of RNA and cDNA samples throughout the experiments were quantitatively measured by nanodrop (Maestro Nanodrop [Green Bioresearch, USA]).

RT-PCR analysis was performed using the RT² SYBR Green qPCR Mastermix kit (Qiagen). For precipitate that might occur in Mastermix tubes, it was kept at 42 °C for 1 min and then vortexed. A 25 μL qPCR mixture was prepared so that it contained 1 μL of diluted cDNA. In the experiments, all cDNA samples were examined 3 times under the same conditions, and the average of these three measurements was used in the analyses. The housekeeping gene (GAPDH) was used as an internal control to determine expression level differences between the control and study groups. The BAP1 (Qiagen, cat. no: PPH02153C), OGT (Qiagen, cat. no: PPH19166A), YY1 (Qiagen, cat. no: PPH00440F), and GAPDH (Qiagen, cat. no: PPH00150F) primers used in the study were optimized by the manufacturer, which minimized all the negativities in terms of creating a specific product because of the presence of guaranteed primers.

All the RT-PCR analysis results of the gene expression experiments were quantified using the Rotor-gene 6000 Series Software Version 1.7. Statistical analysis of the data was performed using the ΔΔCT method in “RT2 profiler RT-PCR Array Data Analysis version” software (https://geneglobe.qiagen.com/de/analyze/).

Protein levels analysis with ELISA

From the patients diagnosed with eyelid tumor and the control group, 4 mL blood samples were withdrawn into sterile citrate tubes. Plasma was obtained from the collected blood samples (1,000×g, 15 min) and stored at −80 °C in Eppendorf tubes.

The BAP1, OGT, and YY1 protein levels were determined using the ELISA method. The detectable ranges of human BAP1 (Sunred Biological Technology, cat. no: 201–12–3062), OGT (Sunred Biological Technology, cat. no: 201–12–7461), and YY1 (Sunred Biological Technology, cat. no: 201–12–7436) were 20–6,000 mg/L, 12.5–3,200 ng/L, and 0.05–15 ng/mL, respectively. The human BAP1, OGT, and YY1 ELISA kits had a sensitivity of 18.663 mg/L, 12.314 ng/L, and 0.045 ng/mL respectively. An automatic washer Bio-Tek ELX50 (BioTek Instruments, USA) device was used to wash the plates, and the Microplate Reader device (BioTek, Epoch, USA) was used for absorbance readings.

Protein expressions analysis with immunohistochemistry

The study group tissues were fixed in 10% buffered neutral formalin for 30–36 h. The tissues were stored at +4 °C after the dehydration, making them transparent, and then embedded in paraffin blocks. Sections of 5 µm were cut with a microtome and placed on a poly-L-lysine-coated slide, then fixed at room temperature and stored at +4 °C until staining. In the sections taken, BAP1 (cat. no: EPP10790), OGT (cat. no: EAP0752), and YY1 (cat. no: EPM12542) protein expressions were determined using immunochemistry markers. In summary, following deparaffinization in xylene and rehydration with graded series of ethanol, the sections were washed in distilled water. The endogenous peroxidase activity was blocked using 3% hydrogen peroxide in methanol for 15 min at RT. After washing in phosphate-buffered saline with Tween-20 (PBS-Tween-20), antigen retrieval was performed on the sections by boiling twice for 10 min in 10 mM sodium citrate buffer (pH 6). To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C. After washing, these sections were incubated with HRP‐streptavidin labeled, a biotinylated secondary antibody (TP-015-HA; Thermo Scientific, USA) and visualized with AEC and counterstained with hematoxylin. All the steps of the immunohistochemistry staining processes were applied to the negative control sections without the primary antibody incubation.

The intensity of the proteins was determined at ×10, ×20 and ×40 magnification with semi-quantitative scoring. For each patient, three randomly selected fields in non-consecutive sections (five for each tissue) were analyzed. Dye intensity was scored as weak (1+), medium (2+), and strong (3+) immunolabelling. Weak staining (1+) was used for preparation only selectable of primary antibody under ×40 microscope magnification, moderate staining (2+) was used for preparation only selectable of primary antibody under ×20 microscope magnification, and strong staining (3+) was used for preparation only selectable of primary antibody under ×40 microscope magnification [22]. Staining intensities 2 and 3 were considered as positive staining of the expression of BAP1, OGT, and YY1. Representative fields from each section were digitally photographed under light microscope (Olympus BX 51, Japan). All images were captured using the same light filter settings. While cell nuclear staining in the anterior stroma adjacent to the invasive tumor and within the tumor center, was regarded as positive for BAP1 staining, cytoplasmic positivity was evaluated as a non-specific reaction. The staining scoring of YY1 and OGT, cytoplasmic and nuclear expressions was based on positive staining in the nucleus and cytoplasm or both. For each patient, at least 500 cells in 5–10 representative high power fields were evaluated as previously described [23]. BAP1, OGT, and YY1 immunostaining were evaluated both in stroma adjacent to the invasive BCC tumor anterior and within the BCC tumor center. The percentage of positively stained cells was calculated.

Statistical analysis

Data obtained in the study were analyzed statistically using SPSS vn. 22.0 software (IBM Corp., Armonk, NY, USA). Data showing normal distribution were evaluated using analysis of variance, Tukey’s test, and the RT-PCR data were evaluated with the significance test of the difference between two means, the Student’s t-test. The error level was taken as 0.05. Data not conforming to normal distribution were evaluated with the Kruskal-Wallis, Mann-Whitney U, and X ² tests. The error level was taken as 0.05. The immunochemistry data were analyzed using the GraphPad Prism 7 (GraphPad Software, CA, USA) program. Conformity of the data to normal distribution was assessed with the D’Agostino-Pearson omnibus normality test. Results were presented as mean ± standard deviation (SD) values.

Patient information

Evaluation was made of 12 patients diagnosed with eyelid tumor, with no restriction in terms of gender, age, or histopathological type and grade of cancer. The histopathological type of the tumor was added to the data based on the pathology report of the tissue sample taken from the patients during surgery (Table 1). The characteristics of the patients with eyelid tumors, such as gender, age range, mean age, family history of cancer, and sun exposure, are presented in Table 1. The patients comprised 8 (66.7%) males and 4 (33.3%) females with a mean age of 65.30 ± 13.31 years (range, 50–88 years). Family history of cancer was determined in 6 (50.0%) patients and long-term exposure to the sun because of occupation was determined in 6 (50.0%) patients (Table 1).

Analysis of BAP1, OGT, and YY1 genes expression results

The expression levels of the BAP1, OGT, and YY1 genes in the eyelid tumor and control tissues were analyzed using the RT-PCR method. GAPDH was used as a housekeeping gene. The expression levels of working genes were normalized with GAPDH. The statistical analysis of the RT-PCR results was performed using the RT2 profiler RT-PCR Array Data Analysis version 3.5 software, Rotor-Gene 6000 software. The data obtained from the experiment were analyzed based on the ΔΔCt method. The RT-PCR data were evaluated using the Student’s t-test as the significance test of the difference between the two means.

A 1.84-fold increase was observed in the BAP1 gene expression levels in the tumor tissue group, which was statistically significant (p = 0.005). A 2.85-fold increase was observed in the OGT gene expression levels in the tumor tissue group, which was statistically significant (p = 0.002). There was a 3.06-fold increase in the YY1 gene expression levels in the tumor tissue compared to the control group, which was statistically significant (p = 0.001). All the differences in the expression levels of all the genes evaluated in this study were statistically significant (Figure 1).

Figure 1:

Comparison of the expression levels of BAP1, OGT and YY1 genes in the tumor group and the control group.

Analysis of BAP1, OGT, and YY1 plasma protein levels results

The characteristics of patients with eyelid tumors and the control group, such as gender, age range, and mean age, are presented in Table 2. The patient group comprised 8 (66.7%) males and 4 (33.3%) females with a mean age of 65.30 ± 13.31 years (range, 50–88 years). The control group comprised 8 (66.7%) males and 4 (33.3%) females with a mean age of 65.66 ± 12.17 years (range, 51–87 years) (Table 2).

The BAP1, OGT, and YY1 plasma levels in the patient and control groups were determined. The BAP1 levels in the patient group were found to be statistically significantly higher than those in the control group (p = 0.001). The OGT plasma levels were found to be similar in the patient and control groups (p = 0.319). The YY1 levels were statistically significantly higher in the patient group than in the control group (p = 0.008) (Table 3).

Pearson correlation analysis was applied to find relationships between variables. The correlation coefficients (correlations) between BAP1, OGT and YY1 in the control group were found to be very small and statistically insignificant (p > 0.05) (Table 4). In the patient group, there was determined to be a statistically significant positive relationship between BAP1 and YY1 (r = 0.850, p < 0.05). A statistically significant positive correlation was determined between BAP1 and OGT (r = 0.811, p < 0.05). A statistically significant positive correlation was determined between OGT and YY1 (r = 0.755, p < 0.05). The correlation coefficients were high in the correlation measures for all three proteins.

Using ROC curve analysis, the area under the curve (AUC), sensitivity and specificity values were calculated for cut-off points of BAP1, OGT, and YY1 protein levels. The cut-off point for BAP1 was 206.00 mg/L (95% confidence interval (CI): 0.746–1.000), OGT was 821.51 ng/L (95% CI: 0.393–0.857) and YY1 was 2.40 ng/mL (95% CI: 0.632–0.993). The cut-off points, and the sensitivity, specificity, and AUC values of those cut-off points are shown in Table 5. The ROC curves of the levels of BAP1, OGT, and YY1 in patients with eyelid tumors are shown in Figure 2.

Figure 2:

ROC curves of the levels of BAP1, OGT and YY1 in patients with eyelid tumors.

Analysis results of BAP1, OGT, and YY1 proteins expression

Immunohistochemical evaluation was performed on the 12 tissue samples of the patient group diagnosed with BCC. In immunohistochemical evaluation, it is known that the staining patterns of tissues taken from different patients may be different. Therefore, BAP1, OGT, and YY1 protein expressions in the tissue samples diagnosed with BCC in this study, were comparatively evaluated with the control tissues. In tumor areas with BCC, BAP1 immunolocalization was observed to be positive at different intensities in tumor cells (Figure 3A–B arrow). OGT immunolocalization in BCC was observed in D–E and F control samples (Figure 3). The immunoreactivity of OGT protein in the BCC samples was expressed in tumor cells but the staining intensity was weak. YY1 (G–H) immunolocalization in BCC patient tissues was observed to be positive at different staining intensities in tumor cells (Figure 3: G–H arrowhead).

Figure 3:

BAP1(A–B), OGT (D–E), and YY1 (G–H) immunolocalization in BCC. Negative control (C–F–I). White arrow: positive cells in tumor area and black arrow: positive cells in stromal cells. Chromogen; AEC, contrast dye; Hematoxylin. The intensity of the proteins was determined at ×10, ×20 and ×40 magnification with semi-quantitative scoring.

The staining intensity of BAP1, OGT and YY1 protein expression

The staining intensity of protein expression in BCC and control tissues was determined. The BAP1 protein staining score in the BCC tumor areas was high (Figure 4, p = 0.0001). The OGT protein was observed to be expressed at a much lower rate compared to BAP1 protein in BCC tumor areas (p = 0.182). The YY1 protein was observed to be expressed similarly to BAP1 in BCC tumor areas (Figure 4, p = 0.0001).

Figure 4:

BAP1, OGT and YY1 staining scores in BCC tumor and control areas.

Limitations

The main limitation of this study was the small sample size. According to the most recent Turkish Ministry of Health statistics, 454 cases of eye tumor were reported between 2013 and 2017. The eye tumor incidence in Turkey is much lower than the incidence of other types of tumors. Eyelid tumors constitute a small proportion of the 454 cases [66] (https://hsgm.saglik.gov.tr/depo/birimler/kanserdb/istatistik/Turkiye_Kanser_Istatistikleri_2017.pdf). Therefore, it is to be expected that the number of patients was low since this study was conducted only in the Sivas region. The study was conducted on a total of 12 patients; 8 (66.7%) male and 4 (33.3%) female, who were diagnosed with eyelid tumors within a one-year period (Table 1). Within the scope of the study, BAP1, OGT and YY1 protein levels were determined using the ELISA method. Protein expression was determined with the IHC method. However, Western blot analysis could not be performed due to the limited budget of this study project.