Analysis of microRNAs in senescent human periodontal ligament cells

Objectives: The accumulated cellular stress induced by senescence can trigger DNA damage and condemn to epigenetic change on the genome in aged periodontal tissue. Recently, it has been reported that senescent cells produce various secretory factors, referred to as the senescence-associated secretory phenotype (SASP) and involve exosomes including microRNAs (miRNAs). In this study, we investigated the pathophysiological role of miRNAs and SASP in senescent human periodontal ligament (HPDL) cells.
Methods: Replicative senescence was induced to primary HPDL cells. The expression of SASP-related genes, such as IL-6, IL-8, and MMPs, was examined by RT-qPCR. IL-6 and IL-8 concentrations in the culture supernatants of HPDL cells were analyzed by ELISA. Global expression profile of microRNAs (miRNAs) in HPDL cells was examined by miRNA microarray analysis and confirmed by RT-qPCR. NF-κB reporter activity was examined with luciferase assay.
Results: The replicative senescence model of HPDL cells in vitro was established. The senescent HPDL cells showed higher NF-κB activity with robust production of IL-6, IL-8, MMP1 and MMP9. Microarray analysis of miRNAs revealed the change of over 300 miRNA expressions during the progression of senescence. Moreover, we dissected the different expression of miRNA species such as miR-34a, miR-148b and miR-146a in senescenct HPDL cells.
Conclusions: Accumulation of DNA damage may elicit the cell cycle arrest and the production of inflammatory cytokines in a NF-κB dependent manner in the senescent HPDL cells. Moreover, various miRNAs might be involved in the expression of longevity genes and inflammatory cytokine in the HPDL cells. The miRNA dependent production of SASP factors in the senescent HPDL cells could exacerbate the inflammation and destruction of the aged periodontal tissues.