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Theranostics 2020; 10(10):4334-4348. doi:10.7150/thno.42982 This issue Cite

Research Paper

miR-128 plays a critical role in murine osteoclastogenesis and estrogen deficiency-induced bone loss

Gengyang Shen^1,2,3*, Hui Ren^1,3*, Qi Shang^2,3*, Zhida Zhang^2,3, Wenhua Zhao^2,3, Xiang Yu^2,3, Jingjing Tang^1,3, Zhidong Yang^1,3, De Liang^1,3, Xiaobing Jiang^1,3✉

1. The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China
2. Guangzhou University of Chinese Medicine, Guangzhou 510405, China
3. Lingnan Medical Research Center of Guangzhou University of Chinese Medicine, Guangzhou 510405, China
*These authors contributed equally to this work.

✉ Corresponding author: Xiaobing Jiang, Department of Spinal Surgery, The First Affiliated Hospital of Guangzhou University of Chinese Medicine. E-mail: spinedrjxbedu.cn.

Abstract

Postmenopausal osteoporosis (PMOP) is a severe health issue faced by postmenopausal women. microRNA-128 (miR-128) is associated with aging, inflammatory signaling, and inflammatory diseases, such as PMOP. It has also been reported to modulate in vitro osteogenic/adipogenic differentiation. However, its function in osteoclast formation is unknown.

Methods: First, the expression of miR-128 and nuclear factor of activated T cells 1 (Nfatc1, bone resorption master marker) was investigated in bone tissues derived from PMOP patients, while their correlation to each other was also investigated. The levels of miR-128 and Nfatc1 in bone specimens and bone marrow-derived macrophages (BMMs) from mice subjected to ovariectomy (OVX) were also assayed. Next, we employed mice BMMs modified for overexpression and inhibition of miR-128 levels to determine its effect on osteoclast differentiation. Moreover, we generated osteoclastic miR-128 conditional knockout (miR-128^Oc-/-) mice and isolated miR-128 deletion-BMMs to observe its biological function on bone phenotype and osteoclastogenesis in vivo, respectively. The miR-128^Oc-/- BMMs were used to explore the downstream regulatory mechanisms using pull-down, luciferase reporter, and western-blotting assays. Finally, the impact of miR-128 deficiency on OVX-induced bone loss in mice was evaluated.

Results: The miR-128 level was found to be positively correlated with the increase in Nfatc1 level in mouse/human bone specimens and mouse primary BMMs. In vitro experiments demonstrated miR-128 levels that were dependent on activity of osteoclast differentiation and miR-128 overexpression or inhibition in BMMs significantly increased or decreased osteoclastogenesis, respectively. In vivo, we revealed that osteoclastic miR-128 deletion remarkedly increased bone mass through the inhibition of osteoclastogenesis. Mechanistically, we identified sirtuin 1 (SIRT1) as the direct target of miR-128 at the post-transcriptional level during osteoclast differentiation. Increased levels of SIRT1 reduced nuclear factor κB (NF-κB) activity by decreasing the level of acetylation of Lysine 310, as well as inhibiting tumor necrosis factor-α (Tnf-α) and interleukin 1 (IL-1) expressions. Lastly, osteoclastic deletion of miR-128 significantly suppressed OVX-triggered osteoclastogenesis and exerted a protective effect against bone loss in mice.

Conclusions: Our findings reveal a critical mechanism for osteoclastogenesis that is mediated by the miR-128/SIRT1/NF-κB signaling axis, highlighting a possible avenue for the further exploration of diagnostic and therapeutic target molecules in PMOP.

Keywords: miR-128, osteoclastogenesis, PMOP, ovariectomy, aging, inflammation

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