Current eye research · 2023
EZH2 Promotes Corneal Endothelial Cell Apoptosis by Mediating H3K27me3 and Inhibiting HO-1 Transcription.
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Lin Ying, Su Huanjun, Zou Baoyi, Huang Minli
Аннотация
PURPOSE: This paper aims to explore the molecular mechanism of Enhancer of Zeste Homolog 2 (EZH2)-mediated H3K27me3 in human corneal endothelial cells (HCEC) apoptosis by inhibiting Heme oxygenase-1 (HO-1) transcription to provide a potential target for the treatment of corneal apoptosis.
METHODS: HCECs were cultured in vitro and transfected with si-EZH2, pcDNA3.1-EZH2, pcDNA3.1-HO-1, GSK-J4 (an effective H3K27me3 demethylase inhibitor), and corresponding controls. Western Blot assay was used to detect the levels of EZH2, HO-1, H3K27me3, and apoptosis-related proteins (Bcl-2, Bax, and Cleaved-caspase-3) in HCECs; CCK-8 assay was conducted to detect cell viability and flow cytometry to analyze the apoptosis. HO-1 mRNA levels were detected by RT-qPCR and changes in H3K27me3 levels on the HO-1 promoter were detected by chromatin immunoprecipitation.
RESULTS: HCECs transfected with si-EZH2 showed significantly lower EZH2 mRNA and protein levels, higher HCEC viability, lower apoptosis rates, higher antiapoptotic protein Bcl-2 expression, lower proapoptotic protein (Bax and Cleaved-caspase-3) levels, and significantly higher HO-1 expression. HCECs transfected with pcDNA3.1-EZH2 showed the opposite results. EZH2 repressed HO-1 transcription by mediating H3K27me3. H3K27me27 was enriched in the HO-1 promoter and overexpression of EZH2 increased H3K27me27 levels. Promotion of H3K27me3 partially reversed the mitigating effect of si-EZH2 on HCEC apoptosis. Overexpression of HO-1 partially reversed the apoptosis-promoting effects of EZH2 and H3K27me3 on HCECs.
CONCLUSIONS: EZH2 promotes HCE cell apoptosis by mediating H3K27me3 to inhibit HO-1 transcription.
METHODS: HCECs were cultured in vitro and transfected with si-EZH2, pcDNA3.1-EZH2, pcDNA3.1-HO-1, GSK-J4 (an effective H3K27me3 demethylase inhibitor), and corresponding controls. Western Blot assay was used to detect the levels of EZH2, HO-1, H3K27me3, and apoptosis-related proteins (Bcl-2, Bax, and Cleaved-caspase-3) in HCECs; CCK-8 assay was conducted to detect cell viability and flow cytometry to analyze the apoptosis. HO-1 mRNA levels were detected by RT-qPCR and changes in H3K27me3 levels on the HO-1 promoter were detected by chromatin immunoprecipitation.
RESULTS: HCECs transfected with si-EZH2 showed significantly lower EZH2 mRNA and protein levels, higher HCEC viability, lower apoptosis rates, higher antiapoptotic protein Bcl-2 expression, lower proapoptotic protein (Bax and Cleaved-caspase-3) levels, and significantly higher HO-1 expression. HCECs transfected with pcDNA3.1-EZH2 showed the opposite results. EZH2 repressed HO-1 transcription by mediating H3K27me3. H3K27me27 was enriched in the HO-1 promoter and overexpression of EZH2 increased H3K27me27 levels. Promotion of H3K27me3 partially reversed the mitigating effect of si-EZH2 on HCEC apoptosis. Overexpression of HO-1 partially reversed the apoptosis-promoting effects of EZH2 and H3K27me3 on HCECs.
CONCLUSIONS: EZH2 promotes HCE cell apoptosis by mediating H3K27me3 to inhibit HO-1 transcription.