Post‐menopausal oestrogen deficiency induces osteoblast apoptosis via regulating HOTAIR/miRNA‐138 signalling and suppressing TIMP1 expression

In this study, we aimed to explore the molecular mechanisms underlying the development of osteoporosis in post‐menopausal females. Real‐time PCR was conducted to measure the expression of potential lncRNAs involved in the osteoporosis of post‐menopausal females. In addition, Western blot and IHC assays were used to study the possible correlation among HOTAIR, miR‐138 and TIMP1, while a computational analysis was carried out to predict the ‘seed sequence’ responsible for the binding between miR‐138 and HOTAIR/TIMP1. Furthermore, luciferase reporter assays were conducted to validate the negative regulatory relationship between miR‐138 and TIMP1/HOTAIR. To evaluate the effect of oestrogen on the function of HOATIR and its downstream effectors, luciferase activity was measured in cells cotransfected with different vectors or treated with different doses of oestrogen. The results of the luciferase assay were further validated by real‐time PCR, Western blot, MTT assay and flow cytometry. Among the candidate lncRNAs, HOTAIR was the only lncRNA down‐regulated in post‐menopausal females. HOTAIR bound to miR‐138 and negatively regulated its expression. Meanwhile, miR‐138 could also bind to TIMP1 mRNA and reduce its expression. Furthermore, a dose‐dependent up‐regulation of HOTAIR was observed in cells treated with oestrogen, and the elevated HOTAIR increased the level of TIMP1 by targeting miR‐138. In addition, oestrogen promoted cell viability and suppressed cell apoptosis, and effects of oestrogen were blocked by the silencing of HOTAIR. Therefore, it can be concluded that oestrogen deficiency could induce the apoptosis of osteoblasts and lead to osteoporosis in post‐menopausal females via modulation of the HOTAIR/miR‐138/TIMP1 signalling axis.