Effects of Leukaemia Inhibitory Factor Receptor on the Early Stage of Osteogenic Differentiation of Human Bone Marrow Mesenchymal Cells

Abstract

Leukaemia inhibitory factor (LIF) has a wide variety of biological activities. While recent studies have focused on the role of LIF in osteoblast differentiation, the exact role of LIFR during the early stage of osteogenic differentiation remains unclear. We observed that LIFR expression gradually decreased during the early stage of osteogenic differentiation of hMSCs. To evaluate how LIFR regulates osteogenic differentiation in greater depth, we transfected hMSCs with LIFR overexpression and siRNA lentiviral plasmids. Cells were divided into four groups: a negative overexpression control group, a LIFR overexpression group, a negative siRNA control group, and a LIFR siRNA group. On different days (0, 3, and 6) of the osteogenic differentiation of hMSCs, alkaline phosphatase (ALP) activity was assayed with an ALP staining and activity assay kit. Cells were harvested to assess the mRNA and protein expression of LIF, LIFR, and osteogenesis-related factors (ALP; RUNX2; osteonectin) by qRT-PCR and western blot analyses, respectively. In addition, culture supernatants were tested for the LIF content by ELISA. Our results showed that overexpression of LIFR significantly suppressed the osteoblast differentiation of hMSCs. In contrast, LIFR siRNA markedly improved this osteoblast differentiation as determined by ALP staining and activity measurements. Moreover, RUNX2, ALP, and ONN expression was also significantly changed by altering LIFR expression. We further analysed the expression of LIF and LIFR, revealing consistent LIF and LIFR trends during the osteogenic differentiation of hMSCs. Together, these results suggested that LIFR may be a novel negative regulator during the early stage of hMSC osteogenic differentiation.