Research progress of microRNA in prevention and treatment of osteonecrosis of the femoral head

Changsui Yu,Liguo Zhu*,Xiaofeng Zhang,ZhongbaoYu,Fengyuan Zhan,Shuren Wang,Xilin Xu,Zhengchun Wang,Jun Zhao,Xinsheng Xie

1Wangjing Hospital,China Academy of Chinese Medical SciencesGraduate School,Beijing,China.2Zhong-bao Yu of Chinese medicine clinic,Kuandian,Liaoning,China.3First Hospital Affiliated to Heilongjiang University,Haerbin,Heilongjiang,China. 4Department of Orthopedics,the Second Hospital Affiliated to Heilongjiang University,Haerbin,Heilongjiang,China.5Heilongjiang University of Chinese Medicine,Haerbin,Heilongjiang,China.

Introduction

Cells contain a variety of non-coding RNAs.Among them,microRNA(miRNA)is considered to widely present in human tissues or cells.In addition,abundant gene regulatory molecules occur in a variety of cell organisms that can affect the output of many protein coding genes.The miRNA gene produces a microtranscript of approximately 22 nucleotides that acts as an antisense factor for other RNAs[1,2].

Osteonecrosis of the femoral head(ONFH) is a common orthopedics disease,and if not treated in a timely manner,the femoral head would completely collapse in about 80%of the patients,which is rather challenging for the Department of Orthopedics[3].The pathogenesis of this disease includes increased intraosseous pressure, lipid metabolism disorder,intravascular coagulation, damage of microvascular endothelial cells, apoptosis of osteoblasts and osteocytes,and annihilation of the immune system[4].Previous studies showed that miRNAs can modulate the physiology and pathology of the body through target genes, including cell proliferation,differentiation,apoptosis,and tissue development[5-7].

Expression and function of miRNA in the femoral head

Lawrie et al.[8]first discovered that miRNA was a potential non-invasive marker for the diagnosis of ONFH; this characteristic established the clinical application of miRNA in the diagnosis of orthopedic diseases[8,9].Wang et al.[9]evaluated the serum and miRNA of patients for steroid-induced ONFH and found 27 types of miRNAs.The study showed that these miRNAs were directly involved in cell differentiation,apoptosis,proliferation,and regulation.The differential expression of miRNA could be used not only as the basis of its function but also as a marker for the diagnosis of various diseases[10-12].

Expression and function of miRNA in the serum of femoral head

Wang et al.[9-13]purified miRNA,performed highthroughput sequencing,compared the results with the differential expression of miRNA in the serum of patients with ONFH,and found that mir-3960 was significantly increased in the serum of patients with ONFH.A total of 207 miRNAs were screened from the serum of patients with ONFH.15 miRNAs,including miR-423-5p,mir-3960,mir-195-5p,mir-15b-3p,and mir-1304-3p, were significantly expressed, and 12 miRNAs,including mir-99a-5p,mir-532-5p,miR-140-5p,mir-10a-5p,mir-10b-5p,mir-181c-5p and miR-433,were also significantly expressed. These elevated expressions suggested that these miRNAs were directly involved in cell apoptosis, regulation,proliferation,and differentiation,while mir-195-5p and mir-15b-3p, the members of miR-15/16 families that played a crucial regulatory role in cell apoptosis.Xia et al.[14]found that miR-15b was significantly expressed during the inhibition of the induction of cell apoptosis and reduction of the expression of Bcl-2 protein. Thus, this study suggested that miR-15b and miR-16 were involved in the process of Bcl-2-targeted regulation and cell apoptosis.Another study showed that both mir-15b-3p and mir-195-3p had an upward trend in the serum of patients with steroid-induced ONFH,demonstrating that mir-15b-3p and mir-195-3p might also contribute to cell apoptosis[9].

Expression and function of miRNA in the cells of femoral head

Studies[15]showed that the miRNA expression contributed to cell proliferation and differentiation,while its up-regulated expression in the serum of patients with ONFH could effectively promote cell proliferation, differentiation, and vascularization.The main role of TGF-β[16](transforming growth factor-beta) promoted tissue regeneration, cell differentiation,and embryonic development.TGF-β inhibited Cbfa1(corebinding factor subunit alpha-1)and osteocalcin through Smad3 in mesenchymal cells. Furthermore, Runx2 (Runt-related transcription factor 2) [17] is a vital important regulatory transcription factor of cell function that can promote the differentiation of osteoblasts.Hjelmeland et al.[18]found that dermal fibroblasts regulated the activity of Smad3 and Runx2 and prevented the differentiation into osteoblast lineage.Therefore,Smad3 was a key protein regulating the osteogenic differentiation of mesenchymal stem cells(MSCs).Pais et al.[19]demonstrated that Smad3 inhibited miR-140,TGF-β,and Runx2 as a targeting pathway in order to promote the osteogenic differentiation of MSCs. Further experiments showed that the expression of miR-140 was decreased in patients with ONFH,indicating that miR-140 was inhibited to promote the osteogenic differentiation of MSCs.In addition,it promoted the osteoblast differentiation and bone morphogenetic protein 2 ossification by regulating Runx2,mir-3960,and mir-2861[13].Wang et al.showed that the expression of miR-3960 was significantly increased in patients with ONFH;this phenomenon promoted the osteogenic differentiation of MSCs, rendering it as a mechanism underlying the promotion of osteogenic differentiation[9].

Expression and function of miRNA in femoral head cartilage

Mature miRNA plays a major role in the development of femoral head cartilage.Wienholds et al.[20,21]used in-situ hybridization to analyze miRNAs and found a specific expression pattern in cartilage tissues.In order to identify the specific expression of miRNA in chondrocytes,Miyaki et al.[22]used the miRNA chip to compare the gene expression profiles of bone marrow MSCs from primary chondrocytes and human femoral head cartilage [23].Since miR-140 in the articular cartilage of the femoral head was inhibited by IL-1,6,8,12,the number of miR-140 molecules in the femoral head cartilage was significantly decreased.Several studies suggested that the decrease in the expression of miR-140 was related to the pathogenesis of ONFH[24,25].miR-140 played a key role in the femoral head cartilage,whereas mRNAs could regulate multiple targets, such as Adamts-5 and miRNAs.Recent studies showed that HDAC4 and IGFBP5 could downregulate miR-140,while miR-140 could inhibit the expression pathway of Adamts-5, thereby regulating the overall balance between synthesis and degradation of the cartilage matrix.According to the gene expression analysis, the cartilage-related catabolic factors, such as matrix-upregulated chondrocyte catabolic enzymes and cartilage matrix genes could downregulate the expression of miR-140[26,27].In conclusion,miR-140 played a key role in maintaining the stability and integrity of the femoral head cartilage by regulating the expression of Adamts-5[28,29].

miRNA in the prevention and treatment of ONFH

The pathological process of ONFH refers to the ischemic changes in the components of the femoral head, including bone, endothelium, fat, and hematopoietic cells,resulting in necrosis and apoptosis of the cells [30-32]. The disease presents the characteristics of progression and destruction,which if not treated promptly,would eventually lead to the collapse of the femoral head; approximately 70%patients would require hip replacement [33, 34].Previous studies demonstrated that a group of miRNAs played a fundamental role in the regulation of various genes in ONFH [35].Thus, it was estimated that miRNAs could regulate >5300 human genes,accounting for approximately 30% of the human genome[36].

miRNA and bone marrow mononuclear cells

Bone marrow contains various subgroups of progenitor cells. Reyes et al. [65] reported that human bone marrow mononuclear cells (BM-MNCs) could differentiate into not only mesenchymal cells but also endothelial cells. Hisatome et al. [66] found that autologous BM-MNC expressed CD31 and the vascular endothelial cell marker that could effectively induce and promote neovascularization after BM-MNC implantation.Several studies[67]showed that BM-MNCs could secrete a variety of growth factors and stimulate the growth of vascular smooth muscle cells,fibroblasts,and endothelial cells. Yan et al. [68] suggested that the transplantation of MSCs could promote bone formation; however, BM-MNC implantation directly participated in the regeneration of blood vessels of the femoral head. Furthermore, BMMNC transplantation could increase the osteogenic activity of rabbit femoral head,and the repair signs of local necrosis could be observed in the histological sections. When BM-MNC was increased,a large number of osteoblasts could be observed, which might be attributed to the formation of osteoblasts stimulated by BM-MNC implantation in the femoral head.On the other hand,accelerated vascularization of the femoral head also contributed to bone regeneration. miRNA could modulate the functions of BM-MNCs and play a crucial role in local vascular pathology and physiological stress conditions[69-71].Chan et al.[72]demonstrated that miR-34a,let-7,and miR-210 could activate the BM-MNCs and inhibit the apoptosis and separation of these cells.Among them, miR-210 could inhibit the mitochondrial function and promote angiogenesis and cell migration [73]; whereas, let-7 family members,including let-7a/b/c/d/f, could activate the expression of vascular endothelial growth factor(VEGF) and release IL6. The release of proangiogenic cytokines was considered as a crucial mechanism underlying BM-MNC-mediated neovascularization repair.Moreover,the increase in the number of let-7 family cytokines could assist the BM-MNCs in repairing the necrotic areas of the femoral head[74,75].

miRNA and osteoblasts

Cx43 is the major connecting protei n in bone cells and also a molecular target of miR-206.miR-206 and Cx43 have been confirmed as the key factors in osteoblast differentiation,maturation,and bone cell metabolism[37].Inose et al.[37]speculated that miR-206 was a specific muscular miRNA with a decreasing disposition during the osteogenic differentiation of C2C12 cells.Cx43 could repair the function and gene expression of osteoblasts in culture[38].Liu et al.[42]demonstrated that when the expression of miR-206 was increased, the expression of Cx4 protein was reduced in animal models of ONFH. These results suggested that Cx43/miR-206 was related to the pathogenesis of steroid-induced ONFH. In osteoblast cultures,alkaline phosphatase,osteopontin,and osteocalcin inhibited the osteoblastic differentiation and labeling[39,42].Runx2 plays a crucial role in the early stage of osteoblast formation and differentiation.When the expression of Runx2 was up-regulated,the activity of ALP was also increased in order to promote the expression of osteoclast by mRNA[40,41].The result of osteoblast differentiation was the key factor for bone formation and the gain of bone mass.The evaluation of the expression of osteoblast differentiation markers revealed that the increase in Runx2 could effectively activate the number of osteoblasts[42].Wnt/β-catenin signaling pathway was involved in the regulation of osteoblast proliferation,survival,differentiation,and apoptosis[43],and the activation of this signaling pathway could promote the formation of osteoblasts and inhibit the differentiation of osteoclasts[44,45].In addition,early studies have shown that Cx43 was a functional target of Wnt signaling pathway,and the activation of the pathway could,in turn,enhance the expression of Cx43/mRNA proteins[46].

miRNA and bone marrow MSCs

Bone marrow MSCs could directionally differentiate into osteoblasts,adipocytes,chondrocytes,and muscle cells in a local microenvironment,secrete a variety of osteogenic activity factors, and then promote neovascularization and new bone formation in the necrotic area,thereby enhancing the repair process of ONFH[47].The number of MSCs in bone marrow of patients with steroid-induced ONFH was decreased significantly.This decrease showed that while there were no MSCs in the necrotic area,the number of MSCs was decreased significantly in the non-necrotic area[48].A previous study demonstrated that pre-mir-2861 mutation could lead to primary osteonecrosis of the femoral head,as it could enhance the degradation of osteogenic transcription factors by inhibiting HDAC5(Histone deacetylase5),thereby promoting the differentiation of Runx2 to osteoblasts[49].Recent studies found that miR-21,miR-23a,miR-24,and miR-25 showed an upward trend in the serum and bone tissues in patients with ONFH,suggesting that they could promote the formation of new bone in the necrotic area[50].miR-140 is present in the articular cartilage tissues of MSCs; therefore, it has been considered as a cartilage-specific miRNA involved in the early skeletal development, with JAG1 and Tmem119 as the functional targets[51-53].JAG1 is a ligand of Notch receptor that induced the differentiation of MSCs into osteoblasts by activating Runx2 and ALP. Tmem119 could induce the transcriptional activity of SMAD1/5 and Runx2 and further promote the osteogenic differentiation[54,55].Chen et al.[56]found that the generation of miR-126 and miR-296 in MSCs could be promoted by the expression of EMVs （ extracellular membrane vesicles）,in which,miR-126 repaired the blood vessels in the ischemic areas of the femoral head mediated by the Akt/Erk-related signaling pathways.Endothelial progenitor cells(EPCs)are spindle-like stem cells that can differentiate into mature vascular endothelial cells.The cells could be isolated from bone marrow and peripheral blood.First,EPCs differentiated into vascular endothelial cells and promoted neovascularization;second,the EPCs promoted the mobilization and growth of MSCs through angiopoietin 1-Tie2 signaling pathway, further promoting the repair of the femoral head[57].Asahara et al.[58]described another subtype of EPCs with distinct morphological features and diffusion patterns;for instance,endothelial colony forming cells(ECFCs)with a high proliferative potential and self-renewal ability that can form new blood vessels in vivo.As a potential source of cells,EPCs can effectively promote neovascularization in the ischemic areas of the femoral head.miRNAs were considered as key regulatory factors of neovascularization with positive or negative regulatory effects on the differentiation function of endothelial cells,thereby promoting vascularization and development in adult embryos [59].Recently,miRNA and stem cells have been under intensive research focus.Meng et al. [60] found that miR-126 up-regulated the damaged EPCs in patients with ONFH,and the target site of the damaged EPCs was Spred-1.miR-150 was a monocyte-enriched miRNA that played a critical role in cell proliferation, migration,differentiation, and embryonic development [61,62].Some studies [63] found that miR-150 constituted the miRNA expression profiling spectrum for expressing EPCs,human umbilical vein endothelial cells, and coronary artery endothelial cells.Therefore,the functions of miR-150 in different aspects of the cells were explored.Zhang et al.[64]demonstrated that monocytes with miR-150 secretion could promote the migration of human microvascular endothelial cells and vascularization.

Prospects

In recent years,miRNA has gradually become the focus of research in bone science. With an increasing number miRNA studies, miRNA has been speculated to have a promising prospect in orthopedic research owing to the specific structure of the femoral head tissues.The detection and analysis of miRNA opened a new research direction for the studies on pathogenesis,diagnostic methods,and treatment approaches of ONFH, thereby postulating the molecular biology and genetic mechanism underlying ONFH. As different pathological factors could lead to differential expressions of different miRNAs, the detection of miRNA could be used to identify the pathogenesis of different ONFH,rendering a targeted treatment and improving the cure rate of patients with ONFH.In addition,the present study proposed that the strategies for prevention and treatment of ONFH could be divided into 2 directions in the future:(1)silencing the highly expressed disease-related genes through miRNA or similar drugs; (2)silencing the highly expressed disease-related miRNA through anti-miRNA molecules.Therefore,miRNA could not only guide doctors in the clinical treatment but also aid in designing an efficient miRNA-targeting drug[76].

Presently, the studies on the prevention and treatment of ONFH are still at the preliminary stage.A majority of the target genes and regulatory pathways related to ONFH have not yet been elucidated.The application of miRNA technique in the treatment of ONFH is still at the experimental stage,and the precise role of miRNA in the occurrence, development,prognosis, and treatment of ONFH needs further studies.However,owing to the rapid development of miRNA detection technology and the biological characteristics based on the regulation of gene and chromosome level, novel approaches for the prevention and treatment of ONFH would be available in the future.

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