ABSTRACT
PURPOSE: Age-related cataract (ARC) is the most common cause of visual impairment and blindness in older adults. However, the role of CUL4B in the ARC remains unclear. Therefore, we investigated CUL4B expression and its effects on apoptosis. MATERIALS AND METHODS: CUL4B expression levels were detected by a quantitative real-time polymerase chain reaction from the anterior lens capsules of patients with ARC and HLE-B3 cells treated with different concentrations of H2O2. CUL4B expression was silenced by siRNA transfection to evaluate apoptosis. CUL4B and apoptotic proteins B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), caspase-3, cleaved caspase-3, Bax, Bak, and Bid were assessed using western blot analysis. Apoptosis was monitored using the TUNEL assay. RESULTS: CUL4B expression was downregulated in the anterior lens capsules (P < 0.0001) and H2O2-treated HLE-B3 cells (P = 0.0405). CUL4B protein levels were significantly lower in 100 µmol/L (P = 0.0012) and 200 µmol/L (P = 0.0041) H2O2-treated HLE-B3 cells than in the untreated cells. CUL4B expression was significantly knocked down at the mRNA (P = 0.0043) and protein levels (P = 0.0002) in HLE-B3 cells. Bcl-2 (P = 0.0199), Mcl-1 (P = 0.0042), and caspase-3 (P = 0.0142) were significantly downregulated, whereas cleaved caspase-3 (P = 0.0089) and Bak (P = 0.009) were significantly upregulated in the knockdown group. The TUNEL assay showed a greater induction of apoptosis. CONCLUSIONS: CUL4B downregulation promotes the apoptosis of lens epithelial cells. Our study may help in understanding the role of CUL4B in ARC pathogenesis.
Subject(s)
Apoptosis , Cataract , Cullin Proteins , Humans , Cataract/metabolism , Cataract/genetics , Cataract/etiology , Cullin Proteins/genetics , Cullin Proteins/metabolism , Cullin Proteins/biosynthesis , Male , Female , Aged , Blotting, Western , Real-Time Polymerase Chain Reaction , Middle Aged , Aging , Gene Expression Regulation , Lens Capsule, Crystalline/metabolism , Lens Capsule, Crystalline/pathology , In Situ Nick-End LabelingABSTRACT
Recently, aberrant DNA methylation of the HIST1H4F gene (encodes Histone 4 protein) has been shown in many types of cancer, which may serve as a promising biomarker for early cancer diagnosis. However, the correlation between DNA methylation of the HIST1H4F gene and its role in gene expression is unclear in bladder cancer. Therefore, the first objective of this study is to explore the DNA methylation pattern of the HIST1H4F gene and then further elucidate its effects on HIST1H4F mRNA expression in bladder cancer. To this end, the methylation pattern of the HIST1H4F gene was analyzed by pyrosequencing and the effects of the methylation profiles of this gene on HIST1H4F mRNA expression in bladder cancer were examined by qRT-PCR. Sequencing analysis revealed significantly higher methylation frequencies of the HIST1H4F gene in bladder tumor samples compared to normal samples (p < 0,0001). However, when we evaluated the correlations between hypermethylation of HIST1H4F and the clinicopathological parameters (tumor stage, tumor grade, lymph node metastasis, muscle-invasion), no significant difference was found between the groups (p > 0.05). In addition, we examined the role of hypermethylation of the HIST1H4F gene on HIST1H4F mRNA expression. We found that hypermethylation of HIST1H4F in the exon have no effect HIST1H4F mRNA expression in bladder cancer (p > 0.05). We also confirmed our finding in cultured T24 cell line which HIST1H4F gene is hypermethylated. Our results suggest that hypermethylation of the HIST1H4F seems to be a promising early diagnostic biomarker in bladder cancer patients. However, further studies are needed to determine the role of HIST1H4F hypermethylation in tumorigenesis.
Subject(s)
Histones , Urinary Bladder Neoplasms , Humans , Histones/genetics , Histones/metabolism , Urinary Bladder Neoplasms/metabolism , DNA Methylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Gene Expression , Gene Expression Regulation, Neoplastic , Biomarkers, Tumor/metabolismABSTRACT
Background/aim: Age-related cataract is the most important visual impairment all over the world. Epigenetic modifications, especially overexpression of histone deacetylases, have become the focus of interest for cataract development in recent years. Sirtuin 1 (SIRT1), a class II histone deacetylase and a member of the sirtuin family, is one of the best-characterized histone deacetylases and has a pivotal role in age-related diseases. However, the association of SIRT1 with age-related cataracts has not yet been fully elucidated. Therefore, we aimed to determine the expression of SIRT1 in age-related cataract patients. Materials and methods: Expressions of SIRT1 were evaluated by quantitative polymerase chain reaction (qPCR) in patients and healthy controls. RNA samples were collected from the anterior capsule and peripheral blood samples of age-related cataract patients. Human lens epithelial cell line B3 and peripheral blood samples of healthy subjects were used as controls. Results: We determined that the expression of SIRT1 in blood and anterior capsule samples increased significantly compared to the control group (P < 0.05). Conclusion: The expression level of SIRT1 plays a vital role in the development of age-related cataract and it can be used as a biomarker. Thus, SIRT1 inhibitors can be used in the treatment of age-related cataract disease.
