Identification of Serum Exosomal MicroRNA Expression Profiling in Menopausal Females with Osteoporosis by High-throughput Sequencing

Abstract

Estrogen deficiency, which mainly occurs in postmenopausal women, is a primary reason for osteoporosis in clinical diagnosis. However, the molecular regulation of osteoporosis in menopausal females is still not adequately explained in the literature, with the diagnosis and treatment for osteoporosis being limited. Herein, exosomal microRNAs (miRNAs) were used to evaluate their diagnosis and prediction effects in menopausal females with osteoporosis. In this study, 6 menopausal females without osteoporosis and 12 menopausal females with osteoporosis were enrolled. The serum exosomes were isolated, and the miRNA expression was detected by miRNA high-throughput sequencing. Exosomal miRNA effects were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The miRNA-targeted genes were evaluated by Targetscan 7.2 and the protein-protein interactions (PPI) by STRING. Hub genes were analyzed by the CytoHubba app of Cytoscape. The results showed that 191 aberrant miRNAs were found in the group of menopausal females with osteoporosis, including 72 upregulated miRNAs and 121 downregulated miRNAs. Aberrant miRNAs were involved in many signaling pathways, such as the Wnt, MAPK, and Hippo pathways. Based on PPI network analysis, FBXL3, FBXL13, COPS2, UBE2D3, DCUN1D1, DCUN1D4, CUL3, FBXO22, ASB6, and COMMD2 were the 10 most notable genes in the PPI network. In conclusion, aberrant serum exosomal miRNAs were associated with an altered risk of osteoporosis in menopausal females and may act as potential biomarkers for the prediction of risk of osteoporosis in menopausal females.

Keywords

exosome / diagnosis / biomarker / osteoporosis

References

[1] RachnerTD, KhoslaS, HofbauerLC. Osteoporosis: Now and the future. Lancet, 2011, 377(9773): 1276-1287

[2] CuiZ, MengX, FengH, et al.. Estimation and projection about the standardized prevalence of osteoporosis in mainland China. Arch Osteoporos, 2019, 15(1): 2

[3] LewieckiEM, BilezikianJP, BonewaldL, et al.. Osteoporosis update: Proceedings of the 2013 Santa Fe bone symposium. J Clin Densitom, 2014, 17(3): 330-343

[4] CooperC, AtkinsonEJ, JacobsenSJ, et al.. Population-based study of survival after osteoporotic fractures. Am J Epidemiol, 1993, 137(9): 1001-1005

[5] TrevisanC, GallinariG, KlumppR, et al.. Year to year comparison of 2000–2015 in hip fracture management: Same survival rate despite older and more fragile patients. Aging Clin Exp Res, 2019, 31(8): 1097-1103

[6] BlackDM, RosenCJ. Clinical practice. Postmenopausal osteoporosis. N Engl J Med, 2016, 374(3): 254-262

[7] KellerS, SandersonMP, StoeckA, et al.. Exosomes: From biogenesis and secretion to biological function. Immunol Lett, 2006, 107(2): 102-108

[8] ShanSK, LinX, LiF, et al.. Exosomes and bone disease. Curr Pharm Des, 2019, 25(42): 4536-4549

[9] MurphyC, WithrowJ, HunterM, et al.. Emerging role of extracellular vesicles in musculoskeletal diseases. Mol Aspects Med, 2018, 60: 123-128

[10] XieY, GaoY, ZhangL, et al.. Involvement of serum-derived exosomes of elderly patients with bone loss in failure of bone remodeling via alteration of exosomal bone-related proteins. Aging Cell, 2018, 17(3): e12758

[11] ZhangY, CaiF, LiuJ, et al.. Transfer RNA-derived fragments as potential exosome trna-derived fragment biomarkers for osteoporosis. Int J Rheum Dis, 2018, 21(9): 1659-1669

[12] van der PolE, BoingAN, HarrisonP, et al.. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev, 2012, 64(3): 676-705

[13] WuZ, WangL, LiJ, et al.. Extracellular vesicle-mediated communication within host-parasite interactions. Front Immunol, 2018, 9: 3066

[14] LiuJ, DangL, WuX, et al.. MicroRNA-mediated regulation of bone remodeling: A brief review. JBMR Plus, 2019, 3(9): e10213

[15] LiQQ, WeiQ, ZhaiXC, et al.. MiRNA-7b-5p attenuates the progression of osteoporosis by inhibiting adipose differentiation of hMSCs via regulating IRS2. Eur Rev Med Pharmacol Sci, 2019, 23(21): 9207-9214

[16] BottaniM, BanfiG, LombardiG. Perspectives on mirnas as epigenetic markers in osteoporosis and bone fracture risk: A step forward in personalized diagnosis. Front Genet, 2019, 10: 1044

[17] KanisJA. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis: Synopsis of a who report. Who study group. Osteoporos Int, 1994, 4(6): 368-381

[18] AgarwalV, BellGW, NamJW, et al.. Predicting effective microRNA target sites in mammalian mRNAs. Elife, 2015, 4: e05005

[19] McClungMR, LewieckiEM, CohenSB, et al.. Denosumab in postmenopausal women with low bone mineral density. N Engl J Med, 2006, 354(8): 821-831

[20] ChenX, LiangH, ZhangJ, et al.. Secreted microRNAs: A new form of intercellular communication. Trends Cell Biol, 2012, 22(3): 125-132

[21] ZhengC, BaiC, SunQ, et al.. Long noncoding RNA XIST regulates osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting mir-9-5p. Mech Dev, 2020, 162: 103612

[22] DengJ, WuJ, ZhuY. Inhibition of microRNA-9 improves fracture healing by modulating the bone morphogenetic protein-7 pathway. Pharmacology, 2019, 104(5–6): 352-358

[23] SunT, LeungF, LuWW. Mir-9-5p, mir-675-5p and mir-138-5p damages the strontium and lrp5-mediated skeletal cell proliferation, differentiation, and adhesion. Int J Mol Sci, 2016, 17(2): 236

[24] EskildsenT, TaipaleenmakiH, StenvangJ, et al.. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. Proc Natl Acad Sci USA, 2011, 108(15): 6139-6144

[25] MartinPJ, HarenN, GhaliO, et al.. Adipogenic RNAs are transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles (evs). BMC Cell Biol, 2015, 16: 10

[26] YanTB, LiC, JiaoGJ, et al.. Timp-1 suppressed by mir-138 participates in endoplasmic reticulum stress-induced osteoblast apoptosis in osteoporosis. Free Radic Res, 2018, 52(2): 223-231

[27] ZhengH, RamnaraignD, AndersonBA, et al.. MicroRNA-138 inhibits osteogenic differentiation and mineralization of human dedifferentiated chondrocytes by regulating RhoC and the actin cytoskeleton. JBMR Plus, 2019, 3(2): e10071

[28] WuJ, ZhaoJ, SunL, et al.. Long non-coding RNA H19 mediates mechanical tension-induced osteogenesis of bone marrow mesenchymal stem cells via FAK by sponging miR-138. Bone, 2018, 108: 62-70

[29] AnJH, OhnJH, SongJA, et al.. Changes of microRNA profile and microrna-mRNA regulatory network in bones of ovariectomized mice. J Bone Miner Res, 2014, 29(3): 644-656

[30] YuY, YaoP, WangZ, et al.. Down-regulation of FTX promotes the differentiation of osteoclasts in osteoporosis through the notch1 signaling pathway by targeting miR-137. BMC Musculoskelet Disord, 2020, 21(1): 456

[31] CaiWL, ZengW, ZhuBY, et al.. MiR-137 affects bone mineral density in osteoporosis rats through regulating RUNX2. Eur Rev Med Pharmacol Sci, 2020, 24(3): 1023-1029

[32] LiuX, XuX. MicroRNA-137 dysregulation predisposes to osteoporotic fracture by impeding ALP activity and expression via suppression of leucine-rich repeat-containing G-protein-coupled receptor 4 expression. Int J Mol Med, 2018, 42(2): 1026-1033

[33] ChenY, YangYR, FanXL, et al.. MiR-206 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targetting glutaminase. Biosci Rep, 2019, 39(3): BSR20181108

[34] ZhangY, LeiW, YanW, et al.. MicroRNA-206 is involved in survival of hypoxia preconditioned mesenchymal stem cells through targeting PIM-1 kinase. Stem Cell Res Ther, 2016, 7(1): 61

[35] Amjadi-Moheb F, Akhavan-Niaki H. Wnt signaling pathway in osteoporosis: Epigenetic regulation, interaction with other signaling pathways, and therapeutic promises. J Cell Physiol, 2019. doi: https://doi.org/10.1002/jcp.28207. Online ahead of print

[36] ZhengS, WangYB, YangYL, et al.. LncRNA MALAT1 inhibits osteogenic differentiation of mesenchymal stem cells in osteoporosis rats through MAPK signaling pathway. Eur Rev Med Pharmacol Sci, 2019, 23(11): 4609-4617

[37] Li X, Ning L, Zhao X, et al. MicroRNA-543 promotes ovariectomy-induced osteoporosis through inhibition of AKT/p38 MAPL signaling pathway by targeting YAF2. J Cell Biochem, 2018. doi: https://doi.org/10.1002/jcb.28143. Online ahead of print

[38] YangBC, KuangMJ, KangJY, et al.. Human umbilical cord mesenchymal stem cell-derived exosomes act via the miR-1263/Mob1/Hippo signaling pathway to prevent apoptosis in disuse osteoporosis. Biochem Biophys Res Commun, 2020, 524(4): 883-889

[39] YuT, LiuK, WuY, et al.. MicroRNA-9 inhibits the proliferation of oral squamous cell carcinoma cells by suppressing expression of CXCR4 via the Wnt/β-catenin signaling pathway. Oncogene, 2014, 33(42): 5017-5027

[40] YiJ, GaoZF. MicroRNA-9-5p promotes angiogenesis but inhibits apoptosis and inflammation of high glucose-induced injury in human umbilical vascular endothelial cells by targeting CXCR4. Int J Biol Macromol, 2019, 130: 1-9

[41] WangY, KongD. Knockdown of lncRNA MEG3 inhibits viability, migration, and invasion and promotes apoptosis by sponging miR-127 in osteosarcoma cell. J Cell Biochem, 2018, 119(1): 669-679

[42] LiQ, GeYL, LiM, et al.. MiR-127 contributes to ventilator-induced lung injury. Mol Med Rep, 2017, 16(4): 4119-4126

[43] HigashiT, HayashiH, IshimotoT, et al.. MiR-9-3p plays a tumour-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells. Br J Cancer, 2015, 113(2): 252-258

[44] FungE, RichterC, YangHB, et al.. FBXl13 directs the proteolysis of CEP192 to regulate centrosome homeostasis and cell migration. EMBO Rep, 2018, 19(3): e44799

[45] ZhangW, NiP, MouC, et al.. Cops2 promotes pluripotency maintenance by stabilizing nanog protein and repressing transcription. Sci Rep, 2016, 6: 26 804

[46] LiP, DingN, ZhangW, et al.. COPS2 antagonizes OCT4 to accelerate the G2/M transition of mouse embryonic stem cells. Stem Cell Reports, 2018, 11(2): 317-324

[47] CaiH, LiuA. Spop promotes skeletal development and homeostasis by positively regulating IHH signaling. Proc Natl Acad Sci USA, 2016, 113(51): 14 751-14 756

[48] HungKF, LiaoPC, ChenCK, et al.. ASB6 promotes the stemness properties and sustains metastatic potential of oral squamous cell carcinoma cells by attenuating ER stress. Int J Biol Sci, 2019, 15(5): 1080-1090