lncRNA H19 promotes matrix mineralization through up-regulating IGF1 by sponging miR-185-5p in osteoblasts
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Matrix mineralization is a key stage in bone formation involving in many bone-specific genes and signaling pathways. Emerging evidence indicate that long non-coding RNA (lncRNA) and microRNAs (miRNAs) play crucial roles in regulating the mineralization process of osteoblasts. This study aims to characterize the function and mechanism of lncRNA H19/miR-185-5p/IGF1 axis in modulating matrix mineralization of osteoblasts.
H19 and IGF1 were highly expressed while miR-185-5p was lowly expressed in mineralized cells. Knocking down H19 inhibited matrix mineralization of osteoblasts, yet miR-185-5p had opposite effects. Moreover, H19 directly targeted miR-185-5p, whereas miR-185-5p repressed IGF1 expression. Meanwhile, miR-185-5p inhibition compensated the suppression of the matrix mineralization in osteoblasts by H19 knockdown.
The findings of this study showed that lncRNA H19 was upregulated in mineralized osteoblasts and promoted matrix mineralization through miR-185-5p/IGF1 axis in osteoblasts for the first time. This study may provide a new perspective for the diagnosis and treatment of diseases related to bone metabolism.
KeywordslncRNA H19 miR-185-5p IGF1 Osteoblasts Mineralization
Collagen type I alpha 2 chain
Human epithelial growth factor receptor 2
HOX transcript antisense RNA
Insulin-like growth factor 1
Long non-coding RNA
Metastasis-associated lung adenocarcinoma transcript 1
Bone metabolism is a continual cycle of bone growth and resorption that is carefully orchestrated by the dynamic relationship between osteoclasts, osteoblasts and an array of hormonal and regulatory influences [1, 2]. The steps of osteogenic proliferation, differentiation, and bone homeostasis are controlled by various markers and signaling pathways . Bone remodeling is delicately regulated by both the number and activity of osteoblasts, which are bone-forming cells, and osteoclasts, which are bone-degrading cells . The process of osteoblastogenesis can be divided into steps comprising proliferation, extracellular matrix development and maturation, mineralization, and apoptosis, which are controlled by a well-defined genetic program [5, 6].
The pathological state of various bone metabolism-related diseases such as osteoporosis is closely related to mineralization of osteoblasts . It is well known that matrix mineralization plays a critical role in bone formation . At the end of osteogenic differentiation, mineralized nodules formed in osteoblasts . Differential expression of bone-related genes and signaling molecules may be involved in the regulation of osteoblasts mineralization .
Long noncoding RNAs (lncRNAs) are RNAs longer than 200 nucleotides in length participating in a variety of cellular processes as potential biological regulators, such as gene expression, translation regulation, and signal transduction . Recently, they were also reported to participate in the regulation of osteogenic activity . H19 was the first lncRNA ever discovered, and has many diverse biological functions, participating in the regulation of cell proliferation, differentiation and metabolism . Increasing evidences indicated that H19 was involved in bone regeneration process. It could promote osteogenic differentiation by activating Wnt signaling  and it could also mediate mechanical tension-induced osteogenesis via focal adhesion kinase (FAK) . However, it has not been fully elucidated the function and mechanism in mineralization of osteoblasts. Therefore, it is of great significance to study the regulatory role of lncRNA H19 and molecular mechanisms regulating the mineralization process of osteoblasts for better understanding bone formation and bone diseases.
Recently, competitive endogenous RNA (ceRNA) has been reported as a new underlying mechanism of lncRNA, which lncRNAs act as microRNAs (miRNAs) sponges and regulate the expression of downstream target mRNA of those miRNAs . MiRNAs are approximately 22 nucleotides in length and act as gene expression regulators involved in the regulation of multiple life processes . Several miRNAs have been identified to regulate cancer growth and stemness, osteogenetic process [17, 18]. miR-185-5p was reported to be a bone specific circulating miRNAs and participated in regulation of cell growth, proliferation and apoptosis  and miR-185-5p over-expression inhibited amelogenesis and osteogenesis . While, it remains to be verified the molecular mechanisms of miR-185-5p in regulating the process of osteogenic mineralization. Insulin-like growth factor 1 (IGF1), an important factor in growth and development, played essential roles in bone matrix mineralization [21, 22]. IGF1 up-regulated Collagen Type I Alpha 2 Chain (COL1A2) protein expression and alkaline phosphatase (ALP) activity in the primary osteoblasts and IGF1 promoted osteogenic differentiation and mineralization in vitro .
In this study, we showed that lncRNA H19 was upregulated in mineralized osteoblasts and promoted matrix mineralization of osteoblasts through miR-185-5p/IGF1 axis suggesting a regulatory role for H19 and miR-185-5p in the pathophysiological process leading to bone formation in skeletal disorders for the first time. This study may provide a new perspective for the diagnosis and treatment of diseases related to bone metabolism.
Differential expression of H19, miR-185-5p and IGF1 in mineralized osteoblasts
H19 knockdown inhibits matrix mineralization of osteoblasts
H19 acts as a ceRNA by sponging miR-185-5p and regulated IGF1 expression indirectly
MiR-185-5p inhibits matrix mineralization of osteoblasts
H19 promotes matrix mineralization through miR-185-5p/IGF1 axis in osteoblasts
LncRNAs play an important role in metabolism of bone, and participate in development and conversion of bone metabolism especially in the regulation of osteogenic differentiation . LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) could promote osteogenic differentiation by targeting miR-204 . HOX transcript antisense RNA (HOTAIR) inhibited mineralization in osteoblastic osteosarcoma cells by epigenetically repressing ALPL . Moreover, H19 promoted osteogenic differentiation via Wnt/β-catenin pathway by sponging miR-141 and miR-22 . However, there is no more report of lncRNA H19 in regulation of osteoblasts activity and matrix mineralization. Here, to our knowledge, we reported the regulatory role and molecular mechanisms that H19 played in osteoblasts activity and matrix mineralization.
Studies showed that lncRNA-miRNA-mRNA network plays an important role in the regulation of cell function, including bone metabolism process. lncRNA MEG3 (maternally expressed gene 3) could enhance the expression matrix mineralization-related proteins (ALP, OCN and Collagen I) via the miR-27a-3p/IGF1 signaling pathway . And lncRNA PCAT1 (prostate cancer-associated ncRNA transcript 1)/miR-145-5p/Toll-like receptor 4 (TLR4) signal axis also functions in osteogenic differentiation process . Furthermore, H19 was reported to mediate osteogenesis by sponging miR-138, which is in agreement with the results of our study . In this study, we found that H19 suppression showed inactivation of mineralization related genes such as ALP, OCN and Collagen I, indicating it play a critical role in regulation of matrix mineralization in osteoblasts. Moreover, lncRNA H19 modulated miR-185-5p levels through its function as a miRNA sponge to trap miR-185-5p, therefore regulated the expression of IGF1. And a study have reported that miR-185-5p was associated with osteogenic differentiation through Dlx2 repression , which is consistent with our findings. According to the recovery experiment, as the downstream mediator of H19, miRNA-185-5p participates in regulating the expression of mineralization related genes. IGF1, a direct target of miRNA-185-5p, is evolutionarily conserved hormonal signaling molecule, influencing a wide array of physiological functions including bone metabolism . It has been demonstrated that IGF1 promoted osteogenic differentiation during osteoblasts maturation and stimulated osteoblasts proliferation . In the current study, we found that miR-185-5p negatively regulated IGF1 expression, and subsequently facilitated ALP activity and mineralization in MC3T3-E1 cells. Taken together, H19-miR-185-5p-IGF1 regulatory network may exist in mineralization process of osteoblasts and H19 works through ceRNA mechanism. Extracellular matrix mineralization is an important stage of osteoblasts in bone formation. These findings also give insight into the association of H19/miR-185-5p/IGF1 signal axis and osteogenesis, even bone diseases.
Because the development of MC3T3-E1 cells, including ALP activity and matrix mineralization, is similar to bone formation in vivo, thus, this cell line is often used to study bone metabolism related diseases in vitro . Although we have preliminarily explored the molecular mechanism of H19 regulating the matrix mineralization of osteoblasts in vitro, further in vivo experiments are needed to verify the feasibility of H19 as a potential therapeutic target for skeletal diseases in future studies.
MC3T3-E1 cells were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and grown in α-minimum essential medium (α-MEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, USA), 1% penicillin (100 U/ml, GIBCO, USA), streptomycin (100 μg/ml streptomycin; GIBCO, USA), in a humidified atmosphere of 5% CO2 and 95% air at 37 °C.
Mineralization induced by β-glycerophosphate and ascorbic acid
MC3T3-E1 cells were divide into 2 groups: control group (α-MEM) and mineralization group (α-MEM with 10− 8 M dexamethasone, 50 mg/L L-ascorbic acid and 10 mM β-glycerophosphate). During the culture process, the medium was replaced every three days.
For downregulation of H19, the small interfering RNA (siRNA) against H19 (si-H19) and negative control (si-NC) were designed by Genepharma (Shanghai, China). The miRNA mimics/inhibitor for miR-185-5p (mimics: 5′-UGGAGAGAAAGGCAGUUCCUGA-3′, 5′-AGGAACUGCCUUUCUCUCCAUU-3′; inhibitor: 5′-UGGAGAGAAAGGCAGUUCCUGA-3′) and negative control oligonucleotide (miR-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′; inhibitor-NC: 5′-CAGUACUUUUGUGUAGUACAA-3′) were obtained from RiboBio (Guangzhou, China). At approximately 60% cell confluence, transfection assay was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
RNA extraction and qPCR
qPCR assay were performed as described before . Briefly, total RNA was extracted using Trizol® (Invitrogen, USA) after cell treatment. ImProm-II Reverse Transcription System (Promega, USA) was used to generate First-strand cDNA. Real-time qPCR of the reverse transcription products were determined using Permix Ex Taq (Takara, Japan) and gene-specific primers were used for qPCR in an ABI 7500HT real time PCR system (Applied Biosystems, USA). The relative expression levels of RNAs were normalized with GAPDH or U6 and calculated using the comparative 2-ΔΔCT method. All experiments were performed at least three times. The primers used for qPCR were list as follow:
Osteocalcin (OCN)-Forward: 5′-AAGCAGGAGGGCAATAAGGT-3′,
Collagen I-Forward: 5′-CCCAGCCGCAAAGAGTCTAC-3′,
Collagen I-Reverse: 5′-AGCATACCTCGGGTTTCCAC-3′.
We used the methodology previously described by Wang et al. , and the membranes were incubated with primary antibodies: GAPDH (1:2000, CST, USA), OCN (1:1000, abcam, USA), ALP (1:2000, abcam, USA), and Collagen I (1:1000, abcam, USA) overnight at 4 °C. Then membranes were incubated with secondary antibody (1:5000, proteintech, USA) for 1 h at room temperature followed by detection using chemiluminescence substance (Thermo Scientific, USA). Quantity One software (Bio-Rad Laboratories, Inc., USA) was used to quantified the proteins.
Bone-specific ALP (BALP) and collagen I protein ELISA analysis
BALP and Collagen I levels in cell medium were detected by ELISA assay using BALP ELISA kit (EM0867, FineTest, China) and Collagen I ELISA kit (EM0939, FineTest, China) according to the manufacturer’s instructions. In brief, the samples were added into the wells precoated ELISA plates followed by incubation with antibody solution at 37 °C. After three times washion, colour rendering, and termination reaction, the absorbance value of each well was detected. Last, the contents were calculated from standard curve fitted by absorbance value of standard wells.
In order to assess ALP activity, the cells were prepared and washed with PBS. Then, the fixed cells were staining by ALP measurement kit (GeFan biotechnology, China). Cells were observed and photographed by a light microscope (Leica DMIRB, Germany). The staining results were confirmed by three repeated tests.
Alizarin red staining
The mineralized nodule formation was detected by Alizarin Red staining as previously described . After staining, the areas or number of positively stained mineral nodules in 3 randomly selected visual fields were measured and analyzed with Image Pro-Plus 6.0 software (Media Cybernetics, USA). Staining for each group was replicated three times.
Luciferase reporter assay
StarBase was used to predict the interaction probability and the binding sites of H19, IGF1 and miR-185-5p. The 3′-UTR of H19 (forward primer, 5′-GACTTCTTTAAGTCCGTCTCGTTCT-3′, reverse primer, 5′-ATGACTGTAACTGTATTTATTGATG-3′) and IGF1 (forward primer, 5′-TCTATGTAAACTCTGAAAAGTAACT-3′, reverse primer, 5′-ATAAAGAAACCCTGGAGCCATAGGG-3′) were amplified by PCR. Then the amplified DNA sequences were subcloned into the pGL3-control vector (Promega, USA) immediately downstream of the stop codon of the luciferase gene. After cells were seeded into 24-well plates and grown overnight to 80-90% confluence, plasmids were transiently co-transfected with miR-185-5p mimics or miR-NC into cells using lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s manual. 48 h after transfection, the Dual-Luciferase Reporter Assay Kit (Promega, USA) was performed to determine luciferase activity.
Data were all expressed as the mean ± standard deviation (SD) and analyzed with Prism 6.0 as described before . Statistical evaluation was performed using Student’s t test between two groups or one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparison. P < 0.05 was considered significantly different.
YW conceived and YJ designed the experiments. QL guaranteed the integrity of the whole research and made the clinical studies. CZL definited intellectual content, YJ did literature research, CZL made data analysis and YW contributed statistical analysis. All authors were responsible for the editing of the manuscript. All authors read and approved the final manuscript.
This Work was supported by Innovation guidance project of clinical medicine technology, Hunan provincial science and technology department (No.2018SK50715).
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The authors declare that they have no competing interests.
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