miR-29b-3p regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically stimulated osteocytes

Zeng et al. Cellular & Molecular Biology Letters https://doi.org/10.1186/s11658-019-0136-2 (2019) 24:11 RESEARCH LETTERS Cellular & Molecular Biology Letters Open Access miR-29b-3p regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically stimulated osteocytes Qiangcheng Zeng1†, Yang Wang2,3†, Jie Gao1,4, Zhixiong Yan2, Zhenghua Li1, Xianqiong Zou2, Yanan Li2, Jiahui Wang2 and Yong Guo1,2* * Correspondence: guoyong74@163. com † Qiangcheng Zeng and Yang Wang contributed equally to this work. 1 key laboratory of Functional Bioresource Utilization in University of Shandong, Shandong Key Laboratory of Biophysics, Dezhou University, Dezhou 253023, China 2 Department of Biomedical Engineering, College of Biotechnology, Guilin Medical University, No. 1 Zhiyuan Road, Lingui District, Guilin City 541100, Guangxi, China Full list of author information is available at the end of the article Abstract Background: Mechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation. Methods: Mouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated. Results: The mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts’ miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation. Conclusions: In osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes. Keywords: Mechanical tensile strain, Osteocyte, Osteoblast differentiation, miRNA microarray © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zeng et al. Cellular & Molecular Biology Letters (2019) 24:11 Introduction Mechanical stimulation plays an essential role in the metabolic balance of bone. Physiological loading can induce bone formation, whereas a lack of loading or excessive loading leads to bone resorption [1–4]. As the dominant cells in bone tissue, osteocytes respond to mechanical stimulation, sense and integrate mechanical stimuli into biochemical signals to regulate both bone formation and resorption [5]. Previous studies mainly focused on osteocytes’ response to fluid shear stress which inhibits osteocytes apoptosis and promotes survival by modulating the Bcl-2/Bax expression ratio, enhances expression levels of NO and PGE2, and increases COX2 and the OPG/RANKL ratio, playing a dominant role in regulating the activities of both osteoblasts and osteoclasts [6–8], thus regulating bone reconstruction and remodeling. However, how osteocytes convert the mechanical stimulation into a biological signal and regulate bone formation (activity of osteoblasts) or resorption (activity of osteoclasts) remains not fully elucidated. MiRs are small non-coding, single-strand RNAs, which control gene expression by targeting to 3′ untranslated regions of mRNA resulting in translational repression or degradation [9]. It was previously found that miR plays a pivotal role in bone formation [10], and many miRs which regulate bone formation have been identified [10, 11]. Some mechanoresponsive miRs were recently identified, they played significant roles in bone formation. For example, miR-33-5p and miR-132 are responsive to mechanical loading and regulate osteogenesis via targeting Hmga2 and mTOR signaling pathway, respectively [12, 13]. Our previous study confirmed that a mechanical tensile strain of 2500 με at 0.5 Hz for 8 h promoted osteogenesis and mechanoresponsive miRs in osteoblasts were identified [14]. The study urged us to investigate osteocytes’ response to the mechanical tensile strain and to search for mechanoresponsive miRs of osteocytes. miR-29b regulated osteoblast differentiation (in MC3T3 osteoblasts, miR-29b overexpression promotes osteogenic differentiation) [15], and IGF-1 was confirmed to be a target gene of miR-29b [16, 17]. We speculated that miR-29b was responsive to mechanical strain applied to osteocytes and involved in osteoblast differentiation. However, the mechanism by miR-29b osteocytes convert a mechanical signal into a biological signal and regulate osteoblast differentiation has not been fully elucidated. In this study, the osteocytes’ biological response to a mechanical tensile strain of 2500 με at 0.5 Hz for 8 h was investigated, and some novel mechanosensitive miRs were selected. In addition, the involvement of miR-29b in osteocytes’ response to mechanical strain and osteoblast differentiation were studied. Methods Cell culture A mouse MLO-Y4 osteocyte cell line (provided by JENNIO Biological Technology, Guangzhou, China) was cultured in dishes with α-MEM medium (α-MEM, Invitrogen), containing 10% FBS and 1% penicillin. Then the cells were transferred to mechanica (...truncated)