Role of E2F1, taking account for the transcriptional activity of lncRNA HOTAIR, in vascular calcification via epigenetic silencing of KLF16/Klotho

Background: Vascular calcification is a significant complication in patients with chronic kidney disease (CKD), which is closely related to hyperphosphatemia. However, its mechanism has not been fully elucidated yet. We mainly investigated the novel role and mechanism of transcription factor E2F transcription factor 1 (E2F1) by targeting lncRNA HOX transcript antisense intergenic RNA (HOTAIR) in high phosphate (Pi)-induced vascular calcification. Methods: Differential expressions of several candidates in E2F transcription factor family were assessed by RT-qPCR and Western blot analysis in both in-vitro and in-vivo models. Calcification of human aortic smooth muscle cells (HASMCs) was evaluated through Alizarin Red S staining, measurement of Ca 2+ content, and assessment of alkaline phosphatase (ALP) activity. Subcellular localization of HOTAIR was detected utilizing subcellular fractionation and fluorescent in-situ hybridization (FISH). Chromatin immunoprecipitation (ChIP), luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to validate the role of E2F1/HOTAIR in Krüppel-like factor 16 (KLF16)/Klotho axis. Concentrations of serum creatinine (Scr) and urine nitrogen (BUN) were measured to assess renal function in mice, followed by the evaluation of vascular calcification in mouse aorta ring. Results: E2F1, identified as significantly downregulated in HASMCs following Pi treatment, was found to be a transcriptional activator of HOTAIR. E2F1 overexpression attenuated Pi-induced HASMCs calcification, which was reversed by HOTAIR silencing. HOTAIR acted as a scaffold for lysine-specific demethylase 1A (KDM1A) and enhancer of zeste homolog 2 (EZH2), contributing to the epigenetic suppression of KLF16. KLF16, in turn, acted as a transcriptional inhibitor for Klotho, thereby suppressing Pi-triggered HASMCs calcification. Additionally, E2F1 overexpression alleviated calcium deposition in thoracic aorta of Pi-induced mice, which was overturned after HOTAIR silencing. Conclusion: HOTAIR, transcriptionally activated by E2F1, exerts a protective effect against vascular calcification by modulating KLF16/Klotho axis. This protective mechanism involves recruitment of KDM1A and EZH2. These findings provide potential new therapeutic targets for CKD treatment.