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

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.

Small and Long Non-coding RNAs as Functional Regulators of Bone Homeostasis, Acting Alone or Cooperatively

“…Our recent data showed complex engagement of miR-21 in the process of osteoblast-osteoclast coupling. 25 We have confirmed previous studies showing that inhibition of miR-21 expression in MC3T3-E1 cells causes a decrease in mRNA expression of crucial osteogenic markers, such as osteocalcin (Ocl), osteopontin (Opn), collagen type I, and Runx2. Furthermore, we confirmed that MC3T3-E1 cells with lowered expression of miR-21 did not support the osteoclastogenesis process, which might be related to the fact that its targets, such as Opn or receptor activator of nuclear factor κB ligand (RANKL), are regulated in a dynamic manner in the process of osteogenesis.…”

“…The essential functions of miR-21 and miR-124 in bone tissue turnover were previously described in detail. 25 , 26 This pair of miRNAs works in an opposite way and takes part in bone-dependent disease progression. miR-21 is known from its engagement in the process of osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), as well as maintaining proper bone remodeling.…”

“…Furthermore, we confirmed that MC3T3-E1 cells with lowered expression of miR-21 did not support the osteoclastogenesis process, which might be related to the fact that its targets, such as Opn or receptor activator of nuclear factor κB ligand (RANKL), are regulated in a dynamic manner in the process of osteogenesis. 25 …”

“… 69 Moreover, miR-21 also elevates the RANKL/OPG ratio; however, it is also considered to be a crucial factor that positively affects osteoblast activity and enhances the mineralization of bone tissue. 25 …”

Hafnium (IV) oxide obtained by atomic layer deposition (ALD) technology promotes early osteogenesis via activation of Runx2-OPN-mir21A axis while inhibits osteoclasts activity

“…2 b). miR-21-5p is widely known osteogenesis stimulator, whereas miR-16 suppress expression of key osteogenic markers and mineral calcium deposition by mesenchymal stem cells during osteogenic differentiation [ 22 , 23 ]. Immunofluorescence staining revealed significant differences in distribution of RUNX2 and osteoprotegerin (OPG), as well as substantial alternation in the expression of OPN between experimental groups.…”