Regulatory effect of CCCTC binding factor on Bcl-2 gene in pterygium and its mechanism

Objective

To investigate the regulation of transcription factor CCCTC binding factor (CTCF) on the expression of B-cell lymphoma 2 (Bcl-2) gene in pterygium and its molecular mechanism.

Methods

Pterygium tissue samples from 22 primary pterygium patients who underwent pterygium excision combined with autologous limbal stem cell transplantation in The First Hospital of Changsha from June 2017 to February 2019 were collected during the operation as pterygium group.Normal conjunctival tissue from 20 patients with ocular trauma due to conjunctiva rupture, eyeball rupture or eyeball perforation in the same period were collected during the repair of ocular trauma as control group.Real-time PCR and Western blot were used to detect the expression levels of CTCF and Bcl-2 in the two groups.The DNA methylation level of the Bcl-2 promoter in the samples of the two groups was detected by bisulfite sequencing PCR (BSP). Pterygium fibroblasts were isolated and cultured.Fibroblasts were identified by immunohistochemistry using vimentin antibody.The cultured pterygium fibroblasts were divided into a CTCF interference group transfected with CTCF interference plasmid, and a control group transfected with control plasmid.The expression levels of CTCF and Bcl-2 in pterygium fibroblasts in CTCF interference and control groups were detected by real-time PCR and Western blot.The cell vitality was detected with cell counting kit-8 at 12, 24, and 48 hours after transfection.The DNA methylation level of the Bcl-2 promoter in the cells of the CTCF interference and control groups after transfection was determined by BSP.Differences of the indexes among groups were analyzed.Correlation between Bcl-2 mRNA and Bcl-2 gene promoter methylation level of CTCF protein in pterygium tissue was analyzed by Pearson linear correlation analysis.This study protocol was approved by the Ethics Committee of The First Hospital of Changsha (No.KL-2017021). Written informed consent was obtained from the patients from whom the specimens were collected.

Results

The relative expression levels of CTCF mRNA and protein in pterygium group were 7.23±3.34 and 0.92±0.21, respectively, which were significantly higher than 1.10±0.44 and 0.28±0.07 in normal conjunctiva group (t=-8.136, -13.025; both at P<0.01). The relative expression levels of Bcl-2 mRNA and protein in pterygium group were 10.27±4.64 and 0.95±0.27, which were higher than 1.10±0.41 and 0.32±0.14 in normal conjunctiva group, showing statistically significant differences (t=-8.789, -10.782; both at P<0.01). The CTCF protein expression was significantly positively correlated with the Bcl-2 mRNA expression in pterygium group (r=0.746, P<0.01). The DNA methylation level of the Bcl-2 promoter in pterygium group was 0.65±0.09, which was lower than 0.83±0.06 in normal conjunctiva group, with a statistically significant difference (t=7.408, P<0.01). The DNA methylation level was significantly negatively correlated with the Bcl-2 mRNA expression in pterygium group (r=-0.635, P<0.01). After the interference of CTCF expression in pterygium fibroblasts, the relative expression levels of CTCF and Bcl-2 mRNA in CTCF interference group were 0.37±0.03 and 0.53±0.06, which were significantly lower than 1.02±0.06 and 0.99±0.07 in control group (t=20.035, 9.029; both at P<0.01). The relative expression levels of CTCF and Bcl-2 proteins in CTCF interference group were 0.23±0.06 and 0.56±0.07, which were lower than 0.52±0.05 and 0.92±0.12 in control group, showing statistically significant differences (t=6.914, 4.719; both at P<0.01). The cell viability of pterygium fibroblasts in CTCF interference group was 0.10±0.01, 0.17±0.01, 0.38±0.04 at 12, 24, and 48 hours after interference, respectively, which were lower than 0.12±0.01, 0.29±0.01 and 0.85±0.06 in control group, and the differences were statistically significant (t=3.718, 18.350, 15.621; all at P<0.01). The DNA methylation level of Bcl-2 promoter in CTCF interference group was 0.75±0.04, which was significantly higher than 0.61±0.03 in control group (t=-4.472, P<0.05).

Conclusions

CTCF is excessively expressed in pterygium, which may mediate the overexpression of Bcl-2 through down-regulating DNA methylation level.

Pterygium; Bcl-2; CCCTC-binding factor; DNA methylation