Zheng Hongjun, Ramnaraign David, Anderson Britta A, Tycksen Eric, Nunley Ryan, McAlinden Audrey
Department of Orthopaedic Surgery Washington University School of Medicine St Louis MO USA.
St Louis University School of Medicine St Louis MO USA.
JBMR Plus. 2018 Jul 18;3(2):e10071. doi: 10.1002/jbm4.10071. eCollection 2019 Feb.
MicroRNAs (miRNAs) are known to play critical roles in many cellular processes including those regulating skeletal development and homeostasis. A previous study from our group identified differentially expressed miRNAs in the developing human growth plate. Among those more highly expressed in hypertrophic chondrocytes compared to progenitor chondrocytes was miR-138, therefore suggesting a possible role for this miRNA in regulating chondrogenesis and/or endochondral ossification. The goal of this study was to determine the function of miR- 138 in regulating osteogenesis by using human osteoarthritic dedifferentiated chondrocytes (DDCs) as source of inducible cells. We show that over-expression of miR-138 inhibited osteogenic differentiation of DDCs in vitro. Moreover, cell shape was altered and cell proliferation and possibly migration was also suppressed by miR-138. Given alterations in cell shape, closer analysis revealed that F-actin polymerization was also inhibited by miR-138. Computational approaches showed that the small GTPase, RhoC, is a potential miR-138 target gene. We pursued RhoC further given its function in regulating cell proliferation and migration in cancer cells. Indeed, miR-138 over-expression in DDCs resulted in decreased RhoC protein levels. A series of rescue experiments showed that RhoC over-expression could attenuate the inhibitory actions of miR-138 on DDC proliferation, F-actin polymerization and osteogenic differentiation. Bone formation was also found to be enhanced within human demineralized bone scaffolds seeded with DDCs expressing both miR-138 and RhoC. In conclusion, we have discovered a new mechanism in DDCs whereby miR-138 functions to suppress RhoC which subsequently inhibits proliferation, F-actin polymerization and osteogenic differentiation. To date, there are no published reports on the importance of RhoC in regulating osteogenesis. This opens up new avenues of research involving miR-138 and RhoC pathways to better understand mechanisms regulating bone formation in addition to the potential use of DDCs as a cell source for bone tissue engineering. © 2018 The Authors. is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
已知微小RNA（miRNA）在许多细胞过程中发挥关键作用，包括那些调节骨骼发育和体内平衡的过程。我们团队之前的一项研究在发育中的人类生长板中鉴定出了差异表达的miRNA。与祖代软骨细胞相比，在肥大软骨细胞中表达更高的miRNA中有miR-138，因此表明该miRNA在调节软骨生成和/或软骨内骨化中可能发挥作用。本研究的目的是通过使用人类骨关节炎去分化软骨细胞（DDC）作为诱导细胞来源，确定miR-138在调节成骨过程中的功能。我们发现，miR-138的过表达在体外抑制了DDC的成骨分化。此外，miR-138改变了细胞形态，并抑制了细胞增殖以及可能的迁移。鉴于细胞形态的改变，进一步分析发现miR-138也抑制了F-肌动蛋白聚合。计算方法表明，小GTP酶RhoC是一个潜在的miR-138靶基因。鉴于其在调节癌细胞增殖和迁移中的作用，我们对RhoC进行了进一步研究。事实上，DDC中miR-138的过表达导致RhoC蛋白水平降低。一系列拯救实验表明，RhoC的过表达可以减弱miR-138对DDC增殖、F-肌动蛋白聚合和成骨分化的抑制作用。在接种了同时表达miR-138和RhoC的DDC的人脱矿骨支架中，也发现骨形成得到了增强。总之，我们在DDC中发现了一种新机制，即miR-138通过抑制RhoC发挥作用，进而抑制增殖、F-肌动蛋白聚合和成骨分化。迄今为止，尚无关于RhoC在调节成骨过程中重要性的公开报道。这开辟了新的研究途径，涉及miR-138和RhoC途径，以更好地理解调节骨形成的机制，此外还有将DDC用作骨组织工程细胞来源的潜在用途。© 2018作者。由Wiley Periodicals, Inc.代表美国骨与矿物质研究学会出版。
