lncRNA GAS5 regulates angiogenesis by targeting miR‑10a‑3p/VEGFA in osteoporosis
- Authors:
- Published online on: August 9, 2021 https://doi.org/10.3892/mmr.2021.12350
- Article Number: 711
Abstract
Introduction
Osteoporosis is a metabolic bone disease characterized by reduced bone tissue volume per unit volume and degeneration of the bone tissue microstructure, resulting in decreased bone strength, increased brittleness and the risk of fracture (1). The morbidity of osteoporosis increases with age in both males and postmenopausal females (2), mainly due to advanced age and estrogen deficiency (3), affecting >50% of females >50 years of age (4). Other pathological factors are associated with immune responses, including chronic inflammation (5) or immune regulation (6), as well as an imbalance in osteoclast or osteoblast differentiation (7). Various efforts have been taken to improve the diagnosis and treatment of osteoporosis; however, high recurrence rates, high cost of treatment, poor patient compliance, poisonous side effects of treatment drugs and malabsorption render the clinical efficacy suboptimal (8). Therefore, there are a number of challenges associated with the prevention and treatment of osteoporosis. Accordingly, exploring the molecular mechanisms underlying osteoporosis is necessary and meaningful to identify a new target and treatment direction.
Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs without protein-coding function (9). They are typically 200–100,000 nucleotides in length and produced by RNA polymerase II (10). However, lncRNAs are involved in multiple cellular processes and serve various roles in epigenetic, transcriptional and post-transcriptional regulation of gene expression (10–15). Certain studies have reported that lncRNAs play important regulatory roles in the occurrence and development of osteoporosis. For example, Zhang et al (16) demonstrated that lncRNA MSC-antisense (AS)1 promotes the osteogenic differentiation of bone marrow-derived stromal cells (BMSCs) and alleviates the progression of osteoporosis by sponging miR-140-5p to upregulate bone morphogenic protein (BMP)2. Conversely, Wang et al (17) reported that lncRNA maternally expressed 3 (MEG3) inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells from postmenopausal osteoporosis (PMOP) by targeting the expression of microRNA (miRNA/miR)-133a-3p. Ma et al (18) showed that lncRNA neighboring enhancer of FOXA2 is downregulated in PMOP and associated with the course of treatment and recurrence, which may be involved in the inhibition of IL-6 secretion. lncRNA DANCR is upregulated in blood mononuclear cells and promotes bone resorption by releasing TNF-α and IL-6, resulting in osteoporosis (19). Downregulation of lncRNA ANCR promotes osteoblast differentiation by targeting enhancer of zeste homolog 2 and regulating runt-related transcription factor 2 (RUNX2) expression (20,21). These observations suggest that lncRNAs are closely associated with the development of osteoporosis. lncRNA growth arrest-specific 5 (GAS5) isolated at the lymphoma-associated chromosomal locus (1q25) and exerts an important regulatory role in tumorigenesis (22). Additionally, GAS5 negatively regulates cell survival, participates in the development of bone diseases and is upregulated in patients with osteoarthritis (23). However, Feng et al (24) reported that GAS5 is downregulated in patients with osteoporosis, and GAS5 overexpression promotes the osteogenic differentiation of human mesenchymal stem cells by regulating miR-498 to upregulate RUNX2 expression, which alleviates the development of osteoporosis. Thus, the mechanisms of GAS5 in osteoporosis require further exploration.
miRNAs are functional non-coding RNAs that can recognize specific target genes by incomplete base pairing, inhibiting the translation of these target genes (25). As potential therapeutic targets or biomarkers, certain miRNAs have gained increasing attention in osteoporosis, including miR-144-3p (26) and miR-132-3p (27). However, only one study has reported the role of miR-10a-3p in osteoporosis. Kaempferol promotes BMSC osteogenic differentiation and improves osteoporosis by downregulating miR-10a-3p (28), a topic that requires further exploration. Vascular endothelial growth factor A (VEGFA), originally identified as an endothelial-specific mitogen and permeability factor, serves a critical role in promoting angiogenesis, which is essential and dependent on bone formation (29,30). Additionally, VEGFA is expressed during osteoblast differentiation, and the exogenous addition of VEGFA can stimulate osteoblast-like cell differentiation, which can attenuate osteoporosis (31). Angiogenesis plays a positive regulatory role in attenuating osteoporosis by enhancing bone formation (29). However, the association between miR-10a-3p and VEGFA remains unclear, and the effects of miR-10a-3p on VEGFA regulation in angiogenesis warrant further exploration.
The present study investigated the effect of knockdown or overexpression GAS5 or miR-10a-3p on angiogenesis of osteoblasts. The present study aimed to verify whether GAS5 overexpression regulates angiogenesis via miR-10a-3p/VEGFA, which may provide novel targets and pathways for the clinical treatment of osteoporosis.
Materials and methods
Clinical sample collection
Blood samples were obtained (March 2019 to June 2019; Brain Hospital of Hunan Province, Changsha, China) from median cubital vein of patients with osteoporosis (n=10; 6 females and 4 males; age, 56–73 years) and healthy subjects (n=10; 5 females and 5 males; age, 57–72 years). The inclusion criteria were as follows: Patients who were diagnosed with osteoporosis via X-ray examination and without other diseases; and healthy subjects who exhibited normal bone density via X-ray examination and without other diseases. The basic information of the patients and healthy controls is presented in Table I. Patients with rheumatoid arthritis and other metabolic diseases were excluded. The healthy subjects had no bone diseases and could walk freely. The blood samples were centrifuged (1,000 × g, 5 min at room temperature), serum was collected and was frozen and stored at −80°C. The study was approved by the Brain Hospital of Hunan Province (approval no. 2019058). Informed consent was obtained from all the participants before sample collection.
Table I.Basic information of patients with osteoporosis and healthy individuals in the present study. |
Detection of bone mineral density (BMD)
The BMD of the patients with osteoporosis and healthy subjects was determined via peripheral dual-energy X-ray absorptiometry (PIXImus II; Lunar; GE Healthcare). They were arranged in the prone position, and an image was acquired in 5 min. The BMD (g/cm2) of the entire body was determined except the head.
Cell culture
Human osteoblasts (hFOB1.19) and human umbilical vein endothelial cells (HUVECs) were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin and 0.5 µg/ml fungizone at 37°C in a humidified 5% CO2 incubator.
Cell transfection
pcDNA-GAS5 or pcDNA-VEGFA (pcDNA; control), short hairpin RNA (sh)GAS5 (shNC; control) were synthesized by GeneCopoeia, Inc. miR-10a-3p mimics (5′-CAAAUUCGGAUCUACAGGGUAUU-3′), miR-10a-3p inhibitor (5′-CACAAAUUCGGAUCUACAGGGUA-3′), NC (mimics NC, 5′-UUCUCCGAACGUGUCACGUTT-3′); inhibitor NC, (5′-CAGUACUUUUGUGUAGUACAA-3′) were purchased from Sangon Biotech Co., Ltd. Briefly, 2Then, 1×104 hFOB1.19 cells were transfected with pcDNA-GAS5 or pcDNA-VEGFA (0.5 µg), miR-10a-3p mimics/inhibitor (50 nM) or mimics NC/inhibitor NC (50 nM) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h, during which time the medium was not replaced. The expression of the relative genes in transfected cells was subsequently determined via reverse transcription-quantitative (RT-q)PCR.
RT-qPCR
Total RNA in the cells was extracted using TRIzol® reagent (Takara Bio, Inc.). The RNA concentration and purity were detected via spectrophotometry. Subsequently, 1 µg total RNA was reverse transcribed into cDNA using a PrimeScript™ RT Reagent kit with gDNA Eraser (Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was performed according to the manufacturer's instructions of an SYBR Premix Ex Taq II kit (Takara Bio, Inc.) using an ABI Prism 7500 HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction procedures were as follows: Pre-denaturation at 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension for 60 sec at 72°C. The relative expression levels were calculated using the 2−ΔΔCq method (32) and were normalized to GAPDH or U6. The primer sequences were as follows: GAS5 forward, 5′-CTTGCCTGGACCAGCTTAAT-3′ and reverse, 5′-CAAGCCGACTCTCCATACCT-3′; miR-10a-3p forward, 5′-GCGCGCAAATTCGTATCTAGG-3′ and reverse, 5′-GTCGTATCCAGTGCAGGGTCC-3′; VEGFA forward, 5′-CCCGGGCCTCGGTTCCAG-3′ and reverse, 5′-GTCGTGGGTGCAGCCTGGG-3′; GAPDH forward, 5′-GAGTCAACGGATTTGGTCGTT-3′ and reverse, 5′-TTGATTTTGGAGGGATCTCG-3′; and U6 forward, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′.
ELISA
The cells were treated with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology). VEGFA was examined using a Human VEGF ELISA kit (Abcam; cat. no. ab222510) according to the manufacturer's protocols.
Western blot analysis
Total proteins in cells were extracted using RIPA lysis buffer containing protease and phosphatase inhibitors (Selleck Chemicals). Protein concentrations were determined using a BCA kit. The protein samples (25 µg/lane) were separated via 12% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked in 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. SW3015) for 1 h at room temperature and incubated with anti-VEGFA (Abcam; 1:1,000; cat. no. ab1316) and anti-GAPDH (Abcam; 1:1,000; cat. no. ab8245) antibodies at 4°C overnight. On the following day, the membranes were incubated with HRP-conjugated secondary antibodies diluted at 1:3,000 (Abcam; cat. no. ab97040) at room temperature for 2 h. The protein bands were visualized using an Immobilon Western Chemilum HRP Substrate (EMD Millipore, WBKLS0100) and analyzed using ImageJ software 1.52a (National Institutes of Health).
Matrigel angiogenesis
Conditioned medium (CM) was collected from hFOB1.19 cells transfected with GAS5 or miR-10a-3p. Matrigel was slowly melted at 4°C overnight, and 10 µl was added to each well of the angiogenic microslide. After Matrigel solidification, 2×105 cells/ml of a HUVEC cell suspension was prepared. Next, 50 µl cell suspension (~10,000 cells) was added to the Matrigel and then blocked with a coverslip. After incubating with 100 µl/well of the indicated CM for 8 h at 37°C, tubular structures were captured in five fields of view using a light inverted microscope (cat. no. IX73; Olympus Corporation) at ×100 magnification and analyzed using ImageJ software 1.52a (National Institutes of Health).
Dual-luciferase reporter assay
The interaction between GAS5 and miR-10a-3p, and VEGFA and miR-10a-3p was predicted by StarBase V2.0 (starbase.sysu.edu.cn). To determine the relationship between GAS5 and miR-10a-3p, as well as between miR-10a-3p and VEGFA, wild-type (WT) or mutant (MUT) sequences of the 3′-untranslated region of GAS5 or VEGFA were inserted into pmirGLO luciferase vectors to generate GAS5-WT, GAS5-MUT, VEGFA-WT and VEGFA-MUT vectors (Promega Corporation). Osteoblasts were co-transfected with the above plasmids and miR-10a-3p or mimics NC using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The transfected cells were washed with PBS, and the luciferase activity was measured after 48 h using a dual-luciferase assay system (Promega Corporation). The firefly luciferase activity was normalized to Renilla luciferase activity.
Statistical analysis
For statistical analyses, one-way ANOVA (multiple comparisons) and Student's t-test (two comparisons) were performed using SPSS software (v12.0; SPSS, Inc.). Tukey's post hoc test was used for multiple comparisons. Correlation was assessed by Pearson's coefficient. The results were presented as the mean ± SD of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.
Results
lncRNA GAS5 is downregulated and miR-10a-3p is upregulated in patients with osteoporosis
The bone mineral density of patients with osteoporosis and healthy subjects was detected, revealing that patients exhibited significantly reduced bone mineral density compared with healthy subjects (Fig. 1A). Representative X-ray images of patients with osteoporosis and healthy subjects are shown in Fig. S1. Serum was collected from the blood samples of patients with osteoporosis and healthy subjects to detect the levels of GAS5 and miR-10a-3p via RT-qPCR. The expression of GAS5 was significantly downregulated in patients compared with the healthy subjects (Fig. 1B). Conversely, the levels of miR-10a-3p were significantly increased in patients (Fig. 1C). Additionally, there was a negative linear correlation between the expression of GAS5 and miR-10a-3p (Fig. 1D). These results indicated that GAS5 was downregulated and miR-10a-3p was upregulated in osteoporosis.
Overexpression of lncRNA GAS5 effectively promotes angiogenesis
GAS5 was overexpressed in osteoblasts to investigate the effects of GAS5. RT-qPCR analysis that GAS5 expression is significantly increased in osteoblasts after GAS5 transfection compared with in the untransfected and empty vector control groups (Fig. 2A), indicating that GAS5 was successfully transfected in osteoblasts. The levels of VEGFA were detected via ELISA and western blotting. The levels of VEGFA were significantly elevated in osteoblasts after GAS5 transfection (Fig. 2B and C), indicating that GAS5 induced the upregulation of VEGFA. Additionally, a Matrigel angiogenesis assay revealed increased angiogenesis for HUVECs treated with CM from the GAS5 group compared with the control and empty vector groups (Fig. 2D), implying that GAS5 induced angiogenesis via VEGFA. Thus, GAS5 overexpression promoted angiogenesis by increasing the levels of VEGFA.
lncRNA GAS5 acts as a sponge of miR-10a-3p
A negative correlation was observed between GAS5 and miR-10a-3p, but the upstream or downstream relationship remains unknown. Therefore, the binding site between miR-10a-3p and GAS5 was predicted using StarBase V2.0 (Fig. 3A). A dual-luciferase reporter assay showed that the luciferase activity in the GAS5-WT group was significantly decreased following co-transfection with miR-10a-3p compared with mimics NC, indicating that GAS5 interacted with miR-10a-3p. When GAS5 was mutated, luciferase activity was not notably affected by transfection with miR-10a-3p compared with mimics NC (Fig. 3B). These results indicated an association between GAS5 and miR-10a-3p.
The expression levels of GAS5 and miR-10a-3p were detected in osteoblasts following transfection with GAS5 or shGAS5. The expression of GAS5 was upregulated in osteoblasts after transfection with GAS5 (Fig. 3C), while the expression of miR-10a-3p was downregulated (Fig. 3D). Opposing effects were observed after transfection with shGAS5 (Fig. 3C and D). These results indicated that GAS5 targeted miR-10a-3p.
VEGFA is a target gene of miR-10a-3p
The above findings indicated that GAS5 induced downregulation of miR-10a-3p. However, whether a targeting relationship exists between miR-10a-3p and VEGFA remains unknown. Therefore, a binding site between miR-10a-3p and VEGFA was predicted by StarBase V2.0 (Fig. 4A). A dual-luciferase reporter assay showed that miR-10a-3p bound to VEGFA in a targeted manner (Fig. 4B). After transfection with miR-10a-3p mimics, miR-10a-3p was upregulated and VEGFA mRNA was downregulated. When the miR-10a-3p inhibitor was transfected, opposing effects were observed (Fig. 4C and D). Similar findings were observed at the protein level (Fig. 4E). These results suggested that miR-10a-3p inhibited the expression of its target VEGFA.
lncRNA GAS5 promotes angiogenesis via the miR-10a-3p/VEGFA axis
Next, the mechanism underlying the promotion of angiogenesis by GAS5 was explored. After transfection with GAS5 or VEGFA overexpression vectors (Fig. S2), the expression of GAS5 was significantly increased, while miR-10a-3p was significantly downregulated (Fig. 5A and B), and the mRNA and protein levels of VEGFA were increased (Fig. 5C-E). After transfection with miR-10a-3p mimics, miR-10a-3p was significantly increased (Fig. 5B), and the mRNA and protein levels of VEGFA were decreased (Fig. 5C-E). When GAS5 and miR-10a-3p mimics were transfected simultaneously, their effects were offset; however, the addition of VEGFA attenuated the inhibition of VEGFA expression by miR-10a-3p mimics (Fig. 5C-E). These results demonstrated that GAS5 regulated the expression of VEGFA by inhibiting miR-10a-3p.
Moreover, GAS5 overexpression promoted angiogenesis, whereas miR-10a-3p mimics exhibited an inhibitory effect. The presence of miR-10a-3p suppressed angiogenesis after GAS5 overexpression. However, the overexpression of VEGFA reversed the inhibition by miR-10a-3p mimics of angiogenesis (Fig. 5F), indicating that GAS5 promoted angiogenesis by increasing the expression of VEGFA.
In summary, GAS5 was downregulated in patients with osteoporosis, which induced upregulation of its target miR-10a-3p. Subsequently, miR-10a-3p inhibited the expression of its target VEGFA to suppress angiogenesis. Thus, the present study indicated that GAS5 promoted angiogenesis via the miR-10a-3p/VEGFA axis in osteoporosis.
Discussion
Osteoporosis is a severe bone disease, resulting in decreased bone strength, and increased fragility and risk of fracture, resulting in substantial harm to postmenopausal women and older men (33). Anti-resorptive agents (such as bisphosphonates and selective estrogen receptor modulators) and anabolic drugs that stimulate bone formation (including parathyroid hormone analogues and sclerostin inhibitors) are current treatment strategies for osteoporosis (34). Previously, no study has compared the anti-fracture efficacy of different bisphosphonates, but recent evidence suggests that zoledronate treatment is more effective than risedronate or alendronate (35). Alendronate and calcium have been reported to exhibit higher efficacy when combined with oral Chinese herbal medicines in the treatment of senile osteoporosis (36). Despite their efficacy, long-term adherence remains a challenge due to the severe side effects and loss of potency (34).
A number of studies have investigated osteoporosis at the genetic level, and it has been reported that lncRNAs serve important roles in influencing osteoporosis. Examples of lncRNAs that promote osteogenic differentiation and alleviate the progression of osteoporosis include lncRNA MSC-AS1 (16) and GAS5 (23,24). Conversely, lncRNA MEG3 (17), lncRNA DANCR (19) and lncRNA-ANCR (20,21) inhibit osteogenic differentiation, resulting in osteoporosis. lncRNA GAS5 is reported to alleviate the development of osteoporosis via the miR-498/RUNX2 (24) and miR-135a-5p/FOXO1 pathways (37). However, treatments for osteoporosis using lncRNA GAS5-targeted drugs have not been reported. In the present study, it was demonstrated that GAS5 overexpression downregulated its target miR-10a-3p, which subsequently induced the upregulation of VEGFA and promoted angiogenesis, indicating potential for the treatment of osteoporosis. The study described a novel mechanism and novel targets with relevance for the treatment of osteoporosis.
miRNAs decrease the expression of their targets via specific binding, thus serving regulatory roles in cells. miR-133a promotes bone loss by altering the serum levels of osteoclastogenesis-related factors, decreasing lumbar spine BMD and altering bone histomorphology (38). miR-208a-3p, miR-155-5p and miR-637 were significantly upregulated in postmenopausal and premenopausal patients with osteoporosis, suggesting their association with disease pathogenesis (39). miR-19b-3p promotes the proliferation and osteogenic differentiation of BMSCs, revealing a role for miR-19b-3p in postmenopausal osteoporosis (40). However, no study has reported on the pro-angiogenic role of miR-10a-3p in osteoporosis. In the present study, the downregulation of miR-10a-3p by GAS5 overexpression increased the levels of VEGFA. These data are the first to indicate potential roles for GAS5 and miR-10a-3p in osteoporosis.
Several signaling pathways have been reported to be involved in the occurrence and development of osteoporosis, such as the JNK pathway (41), Wnt/β-catenin pathway (42), Janus kinase 2/STAT3 signaling pathway (43) and BMP6/Smad1/5/9 pathway (44), through which various proteins are targeted to regulate cell proliferation and osteogenic differentiation. VEGFA can stimulate the differentiation of osteoblast-like cells (27) and influence BMD in osteoporosis (45). In the present study, VEGFA was upregulated via the inhibition of miR-10a-3p by GAS5 overexpression; subsequently, VEGFA promoted osteoblastic angiogenesis. The present study further elucidated the role of VEGFA in osteoporosis and described a novel miR-10a-3p/VEGFA signaling pathway, providing new leads for understanding the mechanisms underlying the occurrence and development of osteoporosis.
The etiology of osteoporosis is complex, involving endocrine, immune, lifestyle, environmental and nutritional factors (46). Numerous studies have focused on the genetic level and have described several molecular signaling pathways. Therefore, investigating these molecular mechanisms may provide improved understanding of osteoporosis pathogenesis and potential therapeutic interventions. It was found that GAS5 overexpression upregulated the level of VEGFA by inhibiting miR-10a-3p, thus promoting angiogenesis. The present study is the first to describe the targeted relationship between GAS5, miR-10a-3p and VEGFA, increasing knowledge concerning this signaling pathway and providing a possible therapeutic target for osteoporosis.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
WW conceived and designed the study. WW and YFL performed experiments and acquired data. WW and YL analyzed the data. QL assisted in data acquisition and interpretation and revised the manuscript. WW and YL prepared the manuscript. WW and YL confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the Brain Hospital of Hunan Province (approval no. 2019058). Informed consent was obtained from all participants prior to sample collection.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
lncRNA |
long non-coding RNA |
GAS5 |
growth arrest-specific 5 |
VEGFA |
vascular endothelial growth factor A |
PMOP |
postmenopausal osteoporosis |
miRNA/miR |
microRNA |
RT-qPCR |
reverse transcription-quantitative PCR |
CM |
conditioned medium |
References
Ahlborg HG, Rosengren BE, Järvinen TL, Rogmark C, Nilsson JA, Sernbo I and Karlsson MK: Prevalence of osteoporosis and incidence of hip fracture in women-secular trends over 30 years. BMC Musculoskelet Disord. 11:482010. View Article : Google Scholar : PubMed/NCBI | |
Baccaro LF, Conde DM, Costa-Paiva L and Pinto-Neto AM: The epidemiology and management of postmenopausal osteoporosis: A viewpoint from Brazil. Clin Interv Aging. 10:583–591. 2015. View Article : Google Scholar : PubMed/NCBI | |
Syed FA and Ng AC: The pathophysiology of the aging skeleton. Curr Osteoporos Rep. 8:235–240. 2010. View Article : Google Scholar : PubMed/NCBI | |
Xue F, Wagman RB, Yue S, Smith S, Arora T, Curtis JR, Ehrenstein V, Sørensen HT, Tell G, Kieler H, et al: Incidence rate of potential osteonecrosis of the jaw among women with postmenopausal osteoporosis treated with prolia or bisphosphonates: Abstract number 348. Arthritis Rheum. 67:492–495. 2015. | |
Montalcini T, Romeo S, Ferro Y, Migliaccio V, Gazzaruso C and Pujia A: Osteoporosis in chronic inflammatory disease: The role of malnutrition. Endocrine. 43:59–64. 2013. View Article : Google Scholar : PubMed/NCBI | |
Zhao R: Immune regulation of osteoclast function in postmenopausal osteoporosis: A critical interdisciplinary perspective. Int J Med Sci. 9:825–832. 2012. View Article : Google Scholar : PubMed/NCBI | |
Zaidi M: Skeletal remodeling in health and disease. Nat Med. 13:791–801. 2007. View Article : Google Scholar : PubMed/NCBI | |
Beaupre LA, Majumdar SR, Dieleman S, Au A and Morrish DW: Diagnosis and treatment of osteoporosis before and after admission to long-term care institutions. Osteoporos Int. 23:573–580. 2012. View Article : Google Scholar : PubMed/NCBI | |
Xu Y, An JJ, Tabys D, Xie YD, Zhao TY, Ren HW and Liu N: Effect of lactoferrin on the expression profiles of long non-coding RNA during osteogenic differentiation of bone marrow mesenchymal stem cells. Int J Mol Sci. 20:48342019. View Article : Google Scholar : PubMed/NCBI | |
Ponting CP, Oliver PL and Reik W: Evolution and functions of long noncoding RNAs. Cell. 136:629–641. 2009. View Article : Google Scholar : PubMed/NCBI | |
Yoon JH, Abdelmohsen K and Gorospe M: Posttranscriptional gene regulation by long noncoding RNA. J Mol Biol. 425:3723–3730. 2013. View Article : Google Scholar : PubMed/NCBI | |
Jarroux J, Morillon A and Pinskaya M: History, discovery, and classification of lncRNAs. Adv Exp Med Biol. 1008:1–46. 2017. View Article : Google Scholar : PubMed/NCBI | |
Cao J: The functional role of long non-coding RNAs and epigenetics. Biol Proced Online. 16:112014. View Article : Google Scholar : PubMed/NCBI | |
Wapinski O and Chang HY: Long noncoding RNAs and human disease. Trends Cell Biol. 21:354–361. 2011. View Article : Google Scholar : PubMed/NCBI | |
Jin D, Wu X, Yu H, Jiang L, Zhou P, Yao X, Meng J, Wang L, Zhang M and Zhang Y: Systematic analysis of lncRNAs, mRNAs, circRNAs and miRNAs in patients with postmenopausal osteoporosis. Am J Transl Res. 10:1498–1510. 2018.PubMed/NCBI | |
Zhang N, Hu X, He S, Ding W, Wang F, Zhao Y and Huang Z: lncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140-5p to upregulate BMP2. Biochem Biophys Res Commun. 519:790–796. 2019. View Article : Google Scholar : PubMed/NCBI | |
Wang Q, Li Y and Zhang Y, Ma L, Lin L, Meng J, Jiang L, Wang L, Zhou P and Zhang Y: lncRNA MEG3 inhibited osteogenic differentiation of bone marrow mesenchymal stem cells from postmenopausal osteoporosis by targeting miR-133a-3p. Biomed Pharmacother. 89:1178–1186. 2017. View Article : Google Scholar : PubMed/NCBI | |
Ma X, Guo Z, Gao W, Wang J, Liu Y, Gao F, Sun S, Zhou X, Yang Z and Zheng W: lncRNA-NEF is downregulated in postmenopausal osteoporosis and is related to course of treatment and recurrence. J Int Med Res. 47:3299–3306. 2019. View Article : Google Scholar : PubMed/NCBI | |
Tong X, Gu PC, Xu SZ and Lin XJ: Long non-coding RNA-DANCR in human circulating monocytes: A potential biomarker associated with postmenopausal osteoporosis. Biosci Biotechnol Biochem. 79:732–737. 2015. View Article : Google Scholar : PubMed/NCBI | |
Zhu L and Xu PC: Downregulated lncRNA-ANCR promotes osteoblast differentiation by targeting EZH2 and regulating Runx2 expression. Biochem Biophys Res Commun. 432:612–617. 2013. View Article : Google Scholar : PubMed/NCBI | |
Cai N, Li C and Wang F: Silencing of lncRNA-ANCR promotes the osteogenesis of osteoblast cells in postmenopausal osteoporosis via targeting EZH2 and RUNX2. Yonsei Med J. 60:751–759. 2019. View Article : Google Scholar : PubMed/NCBI | |
Nakamura Y, Takahashi N, Kakegawa E, Yoshida K, Ito Y, Kayano H, Niitsu N, Jinnai I and Bessho M: The GAS5 (growth arrest-specific transcript 5) gene fuses to BCL6 as a result of t(1;3)(q25;q27) in a patient with B-cell lymphoma. Cancer Genet Cytogenet. 182:144–149. 2008. View Article : Google Scholar : PubMed/NCBI | |
Song J, Ahn C, Chun CH and Jin EJ: A long non-coding RNA, GAS5, plays a critical role in the regulation of miR-21 during osteoarthritis. J Orthop Res. 32:1628–1635. 2014. View Article : Google Scholar : PubMed/NCBI | |
Feng J, Wang JX and Li CH: lncRNA GAS5 overexpression alleviates the development of osteoporosis through promoting osteogenic differentiation of MSCs via targeting microRNA-498 to regulate RUNX2. Eur Rev Med Pharmacol Sci. 23:7757–7765. 2019.PubMed/NCBI | |
Saliminejad K, Khorram Khorshid HR, Soleymani Fard S and Ghaffari SH: An overview of microRNAs: Biology, functions, therapeutics, and analysis methods. J Cell Physiol. 234:5451–5465. 2019. View Article : Google Scholar : PubMed/NCBI | |
Wang C, He H, Wang L, Jiang Y and Xu Y: Reduced miR-144-3p expression in serum and bone mediates osteoporosis pathogenesis by targeting RANK. Biochem Cell Biol. 96:627–635. 2018. View Article : Google Scholar : PubMed/NCBI | |
Hu Z, Zhang L, Wang H, Wang Y, Tan Y, Dang L, Wang K, Sun Z, Li G, Cao X, et al: Targeted silencing of miRNA-132-3p expression rescues disuse osteopenia by promoting mesenchymal stem cell osteogenic differentiation and osteogenesis in mice. Stem Cell Res Ther. 11:582020. View Article : Google Scholar : PubMed/NCBI | |
Liu H, Yi X, Tu S, Cheng C and Luo J: Kaempferol promotes BMSC osteogenic differentiation and improves osteoporosis by downregulating miR-10a-3p and upregulating CXCL12. Mol Cell Endocrinol. 520:1110742021. View Article : Google Scholar : PubMed/NCBI | |
Fu R, Lv WC, Xu Y, Gong MY, Chen XJ, Jiang N, Xu Y, Yao QQ, Di L, Lu T, et al: Endothelial ZEB1 promotes angiogenesis-dependent bone formation and reverses osteoporosis. Nat Commun. 11:4602020. View Article : Google Scholar : PubMed/NCBI | |
Yang M, Li CJ, Sun X, Guo Q, Xiao Y, Su T, Tu ML, Peng H, Lu Q, Liu Q, et al: miR-497~195 cluster regulates angiogenesis during coupling with osteogenesis by maintaining endothelial Notch and HIF-1α activity. Nat Commun. 8:160032017. View Article : Google Scholar : PubMed/NCBI | |
Deckers MM, Karperien M, van der Bent C, Yamashita T, Papapoulos SE and Löwik CW: Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology. 141:1667–1674. 2000. View Article : Google Scholar : PubMed/NCBI | |
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI | |
Ensrud KE and Crandall CJ: Osteoporosis. Ann Intern Med. 167:ITC17–ITC32. 2017. View Article : Google Scholar : PubMed/NCBI | |
Li H, Xiao Z, Quarles LD and Li W: Osteoporosis: Mechanism, molecular target, and current status on drug development. Curr Med Chem. 28:1489–1507. 2021. View Article : Google Scholar : PubMed/NCBI | |
Compston J: Practical guidance for the use of bisphosphonates in osteoporosis. Bone. 25:1153302020. View Article : Google Scholar : PubMed/NCBI | |
Wang H, Mo S, Yang L, Wang P, Sun K, Xiong Y, Liu H, Liu X, Wu Z, Ou L, et al: Effectiveness associated with different therapies for senile osteoporosis: A network meta-analysis. J Tradit Chin Med. 40:17–27. 2020. View Article : Google Scholar : PubMed/NCBI | |
Wang X, Zhao D, Zhu Y, Dong Y and Liu Y: Long non-coding RNA GAS5 promotes osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the miR-135a-5p/FOXO1 pathway. Mol Cell Endocrinol. 496:1105342019. View Article : Google Scholar : PubMed/NCBI | |
Li Z, Zhang W and Huang Y: miRNA-133a is involved in the regulation of postmenopausal osteoporosis through promoting osteoclast differentiation. Acta Biochim Biophys Sin (Shanghai). 50:273–280. 2018. View Article : Google Scholar : PubMed/NCBI | |
Ismail SM, El Boghdady NA, Hamoud HS and Shabayek MI: Evaluation of circulating miRNA-208a-3p, miRNA-155-5p and miRNA-637 as potential non-invasive biomarkers and the possible mechanistic insights into pre- and postmenopausal osteoporotic females. Arch Biochem Biophys. 684:1083312020. View Article : Google Scholar : PubMed/NCBI | |
Xiaoling G, Shuaibin L and Kailu L: MicroRNA-19b-3p promotes cell proliferation and osteogenic differentiation of BMSCs by interacting with lncRNA H19. BMC Med Genet. 21:112020. View Article : Google Scholar : PubMed/NCBI | |
Meng YC, Lin T, Jiang H, Zhang Z, Shu L, Yin J, Ma X, Wang C, Gao R and Zhou XH: miR-122 exerts inhibitory effects on osteoblast proliferation/differentiation in osteoporosis by activating the PCP4-mediated JNK pathway. Mol Ther Nucleic Acids. 20:345–358. 2020. View Article : Google Scholar : PubMed/NCBI | |
Liu TJ and Guo JL: Overexpression of microRNA-141 inhibits osteoporosis in the jawbones of ovariectomized rats by regulating the Wnt/β-catenin pathway. Arch Oral Biol. 113:1047132020. View Article : Google Scholar : PubMed/NCBI | |
Fu Y, Xu Y, Chen S, Ouyang Y and Sun G: miR-151a-3p promotes postmenopausal osteoporosis by targeting SOCS5 and activating JAK2/STAT3 signaling. Rejuvenation Res. 23:313–323. 2020. View Article : Google Scholar : PubMed/NCBI | |
Wang T, Zhang C, Wu C, Liu J, Yu H, Zhou X, Zhang J, Wang X, He S, Xu X, et al: miR-765 inhibits the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting BMP6 via regulating the BMP6/Smad1/5/9 signaling pathway. Stem Cell Res Ther. 11:622020. View Article : Google Scholar : PubMed/NCBI | |
Lee HS and Park T: Nuclear receptor and VEGF pathways for gene-blood lead interactions, on bone mineral density, in Korean smokers. PLoS One. 13:e01933232018. View Article : Google Scholar : PubMed/NCBI | |
Lane NE: Epidemiology, etiology, and diagnosis of osteoporosis. Am J Obstet Gynecol. 194 (Suppl 2):S3–S11. 2006. View Article : Google Scholar : PubMed/NCBI |