Department of Experimental Biology, The Faculty of Biology and Animal Science, University of Environmental and Life Sciences, 50-375 Wroclaw, Poland.
Laboratory of Gene Expression, Institute of Biotechnology CAS, Biocev, 25250 Vestec, Czech Republic.
Cells. 2020 Feb 19;9(2):479. doi: 10.3390/cells9020479.
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 (MC3T3) modulated expression of genes encoding osteogenic markers including collagen type I (), osteocalcin (), osteopontin (), and runt-related transcription factor 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 MC3T3 cells was characterized by lowered expression of several markers associated with osteoclasts' differentiation, foremost tartrate-resistant acid phosphatase () but also receptor activator of nuclear factor-κB ligand (), cathepsin K (), carbonic anhydrase II (), and matrix metalloproteinase (). 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.
miR-21 逐渐被认为是调节骨组织内稳态的分子。然而，由于 miR-21 对成骨细胞和破骨细胞的双重作用，其功能尚未完全了解。在这项研究中，我们使用小鼠前成骨细胞（MC3T3）和前破骨细胞（4B12）细胞系的间接共培养模型，研究了 miR-21 抑制对前成骨细胞分化和向破骨前细胞旁分泌信号的影响。miR-21 在 MC3T3 细胞（MC3T3）中的抑制调节了编码成骨标志物的基因的表达，包括胶原 I ()、骨钙素 ()、骨桥蛋白 ()和 runt 相关转录因子 2 ()。miR-21 在 MC3T3 成骨培养物中的抑制也影响了 OPN 蛋白的合成，OPN 蛋白对于细胞外基质（ECM）的适当矿化和破骨细胞附着在骨骼上是必不可少的。此外，研究表明，在成骨细胞中，miR-21 调节对前破骨细胞存活至关重要的因子的表达，如核因子 κB 受体激活剂配体（RANKL）。与破骨细胞分化相关的几种标志物在前破骨细胞中的表达降低，主要是抗酒石酸酸性磷酸酶 ()，但也有核因子-κB 受体激活剂配体 ()、组织蛋白酶 K ()、碳酸酐酶 II ()和基质金属蛋白酶 ()。总之，我们的数据表明，MC3T3 细胞中 miR-21 的抑制会损害分化和 ECM 矿化，并影响旁分泌信号，从而降低前破骨细胞的活力。
