The Role of miR-21 in Osteoblasts–Osteoclasts Coupling In Vitro

Agnieszka Smieszek,Mateusz Sikora,Lukas Valihrach,Klaudia Marcinkowska,Ariadna Pielok,Krzysztof Marycz

doi:10.3390/cells9020479

Abstract

MiR-21 is being gradually more and more recognized as a molecule regulating bone tissue homeostasis. However, its function is not fully understood due to the dual role of miR-21 on bone-forming and bone-resorbing cells. In this study, we investigated the impact of miR-21 inhibition on pre-osteoblastic cells differentiation and paracrine signaling towards pre-osteoclasts using indirect co-culture model of mouse pre-osteoblast (MC3T3) and pre-osteoclast (4B12) cell lines. The inhibition of miR-21 in MC3T3 cells (MC3T3inh21) modulated expression of genes encoding osteogenic markers including collagen type I (Coll-1), osteocalcin (Ocl), osteopontin (Opn), and runt-related transcription factor 2 (Runx-2). Inhibition of miR-21 in osteogenic cultures of MC3T3 also inflected the synthesis of OPN protein which is essential for proper mineralization of extracellular matrix (ECM) and anchoring osteoclasts to the bones. Furthermore, it was shown that in osteoblasts miR-21 regulates expression of factors that are vital for survival of pre-osteoclast, such as receptor activator of nuclear factor κB ligand (RANKL). The pre-osteoclast cultured with MC3T3inh21 cells was characterized by lowered expression of several markers associated with osteoclasts’ differentiation, foremost tartrate-resistant acid phosphatase (Trap) but also receptor activator of nuclear factor-κB ligand (Rank), cathepsin K (Ctsk), carbonic anhydrase II (CaII), and matrix metalloproteinase (Mmp-9). Collectively, our data indicate that the inhibition of miR-21 in MC3T3 cells impairs the differentiation and ECM mineralization as well as influences paracrine signaling leading to decreased viability of pre-osteoclasts.

Highlights

MicroRNAs are a class of non-coding, single stranded RNAs with average length around 22 nucleotides
The higher mRNA level for collagen type 1 (Coll-1) was noted in MC3T3 inh21/ 4B12 cultures following 15 days of osteogenic differentiation, the observed differences were not statistically significant (Figure 1d,e)
In co-culture of MC3T3 with 4B12, the inhibition of miR-21 had no influence on Runx-2 levels after 3 and 15 days of culture (Figure 1j,l), lowered expression of miR-21 was correlated with increased mRNA level for Runx-2, noted after 7 days of osteogenic differentiation (Figure 1k)

Summary

Introduction

MicroRNAs (miRNAs) are a class of non-coding, single stranded RNAs with average length around 22 nucleotides. The molecules are highly conserved across species and have various biological functions that are connected with the regulation of gene expression by targeting 30 -untranslated region (30 -UTR) of messenger RNA [1,2]. MiRNAs regulate multiple processes crucial for homeostasis maintenance, such as cell proliferation, differentiation, survival and apoptosis. Mounting evidence indicates that miRNAs play a vital role during osteogenesis process by modulating expression of genes. Cells 2020, 9, 479 essential for recruitment and differentiation of various bone cells, including progenitor and mature cells [2,3]. MiRNAs are important molecules that control the process of extracellular matrix (ECM). MiR-29b-dependent suppression of collagen occurs at the late stage of differentiation, which facilitates the maturation of collagen fibrillar matrix for mineral deposition [4]

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