Mechanical force inhibited hPDLSCs proliferation with the downregulation of MIR31HG via DNA methylation.

Abstract

This study aimed to investigate how mechanical force affects the proliferation of human periodontal ligament stem cells (hPDLSCs). CCK-8 assays and staining of ki67 were performed to evaluate hPDLSCs proliferation. qRT-PCR, ELISA, or Western blot analysis were used to measure the expression levels of interleukin (IL)-6, miR-31 host gene (MIR31HG), DNA methyltransferase 1 (DNMT1), and DNA methyltransferase 3B (DNMT3B). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays were conducted to determine whether MIR31HG was targeted by DNMT1 and DNMT3B. MassARRAY mass spectrometry was used to quantify DNA methylation levels of the MIR31HG promoter. Mechanical force inhibited hPDLSCs proliferation with the downregulation of MIR31HG and upregulation of IL-6, DNMT1 and DNMT3B. Knockdown of MIR31HG suppressed hPDLSCs proliferation, and knockdown of DNMT1 or DNMT3B reversed mechanical force-induced downregulation of MIR31HG. Dual-luciferase and ChIP assays revealed DNMT1 and DNMT3B bound MIR31HG promoter in the region 1,015bp upstream of the transcriptional start site. Treatment with 5'-aca-2'-deoxycytidine downregulated DNA methylation level in MIR31HG gene promoter, while mechanical force promoted the methylation of MIR31HG gene promoter. These findings elucidated how mechanical force affects proliferation via MIR31HG in hPDLSCs, providing clues for possible MIR31HG-based orthodontic therapeutic approaches.