Silencing of lncRNA AK045490 Promotes Osteoblast Differentiation and Bone Formation via β-Catenin/TCF1/Runx2 Signaling Axis
<p>Elevated <span class="html-italic">AK045490</span> expression in bone is accompanied by deteriorated bone microstructure and decreased bone formation in aging mice and in ovariectomized (OVX) mice. (<b>a</b>) The RNA level of long noncoding RNAs (lncRNAs) <span class="html-italic">AK045490</span> in bone isolated from the age-related osteoporotic mice. (<b>b</b>) Representative images showing the 3D architecture (Left, top) and Micro Computed Tomography (Micro CT) measurements in the distal femurs (Middle). Representative images of new bone formation assessed by double calcein labeling (Left, bottom) and quantitative analysis of mineral apposition rate (MAR) at the distal femur (Right). (<b>c</b>) The RNA level of lncRNA <span class="html-italic">AK045490</span> in bone isolated from the postmenopausal osteoporotic mice. Sham: Sham operation group. OVX: ovariectomy operation group. (<b>d</b>) Representative images showing the 3D architecture (Left, top) and Micro CT measurements in the distal femurs (Middle). Representative images of new bone formation assessed by double calcein labeling (Left, bottom) and quantitative analysis of mineral apposition rate (MAR) at the distal femur (Right). All data were expressed as mean ± SD. Student’s <span class="html-italic">t</span>-test was performed for comparison between two groups. <span class="html-italic">p</span> value less than 0.05 were considered significant in all cases (* <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01). Scale bar: 500 μm in b, d (top), 20 μm in b, d (bottom). <span class="html-italic">n</span> = 6 mice in each group.</p> "> Figure 2
<p>Silencing of <span class="html-italic">AK045490</span> promoted osteoblast differentiation. (<b>a</b>) AK045490 RNA levels of MC3T3-E1 cells treated with <span class="html-italic">AK045490</span> siRNA, negative control RNA (si-NC) or without treatment (Control), as detected by RT-PCR. (<b>b</b>) <span class="html-italic">Alp, Ocn, and Col-</span><span class="html-italic">I</span> expression levels of MC3T3-E1 cells treated with <span class="html-italic">AK045490</span> siRNA, as detected by RT-PCR. (<b>c</b>) ALP staining (up) and Alizarin Red staining (bottom) in MC3T3-E1 cells treated with <span class="html-italic">AK045490</span> siRNA. All data were expressed as mean ± SD. Student’s <span class="html-italic">t</span>-test was performed for comparison between two groups. <span class="html-italic">p</span> values less than 0.05 were considered significant in all cases (** <span class="html-italic">p</span> < 0.01). <span class="html-italic">n</span> = 3 in each group.</p> "> Figure 3
<p>Knockdown of <span class="html-italic">AK045490</span> promoted β-catenin nuclear translocation and up-regulated the expression of TCF1, LEF1, and Runx2. (<b>a</b>) IHF staining showed the location of β-catenin in the cells transfected with <span class="html-italic">AK045490</span>-siRNA (si-<span class="html-italic">AK045490</span>) or scrambled-control-siRNA (si-NC), respectively. β-catenin was stained as red and nuclei were stained by DAPI showing blue. Bar (1, 2, 3): 20 µm. Bar (4): 50 µm. (<b>b</b>) Representative western blots of the nuclear translocation of β-catenin. The nuclear (nucleus) and cytosolic (cytosol) fractions of proteins isolated from <span class="html-italic">AK045490</span> knockdown cells and control cells were probed for β-catenin. Lamin B1 and GAPDH were used as internal controls for nuclear and cytosol fractions, respectively. Full unedited gels available in the <a href="#app1-ijms-20-06229" class="html-app">Supplementary file</a>. (<b>c</b>) Quantification of nuclear and cytosol levels of β-catenin with Lamin B1 and GAPDH as internal control, respectively. (<b>d</b>) mRNA expression of TCF1, LEF1, and Runx2 as detected by real time PCR. (<b>e</b>) TCF1 activity in MC3T3-E1 cells transfected with <span class="html-italic">AK045490</span> siRNA, as detected by TOPflash luciferase reporter assay. (<b>f</b>) Pattern diagram showed the network of lncRNA–microRNA–RNA interaction. All data were expressed as mean ± SD. Student’s <span class="html-italic">t</span>-test was performed for comparison between two groups. <span class="html-italic">p</span> values less than 0.05 were considered significant in all cases (* <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01). <span class="html-italic">n</span> = 3 in each group.</p> "> Figure 4
<p>Promoting effect of AK045490 siRNA on calvarial bone formation in OVX mice. (<b>a</b>) Representative images showing calvarial mineral apposition rate of C57BL/6 mice after OVX treatment and siRNA transfection. White segments showed the width between the two lines. Scale bar = 20 µm. (<b>b</b>) Calvarial mineral apposition rates of C57BL/6 mice after OVX treatment and siRNA transfection. All data were expressed as mean ± SD. Student’s <span class="html-italic">t</span>-test was performed for comparison between two groups. <span class="html-italic">p</span> value less than 0.05 were considered significant in all cases (** <span class="html-italic">p</span> < 0.01). <span class="html-italic">n</span> = 5 in each group.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Elevated AK045490 Expression in Bone Was Accompanied by Deteriorated Bone Microstructure and Decreased Bone Formation in Osteoporotic Mice
2.2. AK045490 Inhibited Osteoblast Differentiation
2.3. Knockdown of AK045490 Promoted β-Catenin Nuclear Translocation and Up-Regulates the Expression of TCF1, LEF1 and Runx2
2.4. Promoting Effect of AK045490 siRNA on Calvaria Bone Formation in OVX Mice
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Mice Model
4.3. RNA Isolation and Real-Time PCR (RT-PCR) Analysis
4.4. MicroCT Analysis
4.5. Bone Histology and Histomorphometry
4.6. siRNA Transfection In Vitro
4.7. Western Blot
4.8. ALP Staining
4.9. Alizarin Red Staining
4.10. Immunohistofluorescence (IHF) Staining
4.11. siRNA Transfection In Vivo
4.12. Analysis of lncRNA–microRNA–mRNA Interaction
4.13. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
ALP | alkaline phosphatase |
BFR | bone formation rate |
BL | baseline |
BMD | bone mineral density |
BMSC | bone-derived mesenchymal stem cell |
ceRNA | competing endogenous RNA |
Col Iα1 | collagen type I |
ECL | electro-chemi-luminescence |
IHF | immunohistofluorescence |
LEF1 | lymphoid enhancer-binding factor 1 |
lncRNA | long noncoding RNA |
Linc-ROR | large intergenic noncoding RNA regulator of reprogramming |
MAR | mineralizing apposition rate |
Micro CT | micro computed tomography |
MSC | mesenchymal stem cell |
Sham | sham-operated |
Ocn | osteocalcin |
OVX | ovariectomized |
PFA | paraformaldehyde |
Runx2 | runt-related transcription factor 2 |
TCF1 | transcription factor T cell factor 1 |
WB | western blot |
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Target gene | Primer Direction | Sequence (5′–3′) |
---|---|---|
AK045490 | Forward: | GCATTGTATCTCGCTCCACA |
Reverse: | TGTTGCCTACCTGCTTACTGC | |
Alp | Forward: | GTTGCCAAGCTGGGAAGAACAC |
Reverse: | CCCACCCCGCTATTCCAAAC | |
Ocn | Forward: | GAAGGCAACAGTCGATTCACC |
Reverse: | GACTGTCTTGCCCCAAGTTCC | |
Col Ⅰα1 | Forward: | GAAGGCAACAGTCGATTCACC |
Reverse: | GACTGTCTTGCCCCAAGTTCC | |
Tcf1 | Forward: | CAGAATCCACAGATACAGCA |
Reverse: | CAGCCTTTGAAATCTTCATC | |
Let1 | Forward: | GATCCCCTTCAAGGACGAAG |
Reverse: | GGCTTGTCTGACCACCTCAT | |
Runx2 | Forward: | CGCCCCTCCCTGAACTCT |
Reverse: | TGCCTGCCTGGGATCTGTA | |
Gapdh | Forward: | TGCACCACCAACTGCTTAG |
Reverse: | GGATGCAGGGATGATGTTC |
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Li, D.; Tian, Y.; Yin, C.; Huai, Y.; Zhao, Y.; Su, P.; Wang, X.; Pei, J.; Zhang, K.; Yang, C.; et al. Silencing of lncRNA AK045490 Promotes Osteoblast Differentiation and Bone Formation via β-Catenin/TCF1/Runx2 Signaling Axis. Int. J. Mol. Sci. 2019, 20, 6229. https://doi.org/10.3390/ijms20246229
Li D, Tian Y, Yin C, Huai Y, Zhao Y, Su P, Wang X, Pei J, Zhang K, Yang C, et al. Silencing of lncRNA AK045490 Promotes Osteoblast Differentiation and Bone Formation via β-Catenin/TCF1/Runx2 Signaling Axis. International Journal of Molecular Sciences. 2019; 20(24):6229. https://doi.org/10.3390/ijms20246229
Chicago/Turabian StyleLi, Dijie, Ye Tian, Chong Yin, Ying Huai, Yipu Zhao, Peihong Su, Xue Wang, Jiawei Pei, Kewen Zhang, Chaofei Yang, and et al. 2019. "Silencing of lncRNA AK045490 Promotes Osteoblast Differentiation and Bone Formation via β-Catenin/TCF1/Runx2 Signaling Axis" International Journal of Molecular Sciences 20, no. 24: 6229. https://doi.org/10.3390/ijms20246229