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. 2022 Oct 14;17(10):e0276076. doi: 10.1371/journal.pone.0276076

Fig 3. ZFAS1–PRC2 complex interaction regulating miR-9 expression in vitro in HCMEC.

Fig 3

Treatment of HCMECs with methylation inhibitors (DZNep) reduced HG induced mRNA upregulation of (A, B, C) EZH2, EED and SUZ12. In conjunction, DZNep also corrected HG induced decreased (D) miR-9 levels. Interestingly, transfection of HCMECs with ZFAS1 siRNA reduced HG induced (E, F) upregulation of EZH2 and EED mRNA, whereas(G) SUZ12 mRNA remained unchanged. (H) RIP assay using anti-EZH2 antibody performed in HCMECs showed glucose-induced increased binding of ZFAS1 with EZH2 (anti-IgG as negative control). (I) ChIP assay demonstrated increased binding of EZH2 on miR-9 promoter as measured by RT-PCR. (NG = 5 mM glucose; HG = 25 mM glucose; RIP = RNA immunoprecipitation; ChIP = chromatin immunoprecipitation RNA; D = DZNep; data expressed as a ratio to β-actin, mean ± SEM; *P < 0.05 compared to NG; ξ < 0.05 compared to HG; n = 6/group).