miR-9-5p, miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation, Differentiation, and Adhesion
Abstract
:1. Introduction
2. Results
2.1. Strontium Altered the Expression Levels of miRNAs in Vitro and in Vivo
2.2. miR-9-5p, miR-675-5p, and miR-138-5p Reduced Skeletal Cell Count and Cell Proliferation
2.3. miR-9-5p, miR-675-5p, and miR-138-5p Impaired Skeletal Cell Differentiation and Cell Adhesion
2.4. miR-9-5p and miR-675-5p Induced MC3T3-E1 Cell Apoptosis More Specifically than miR-138-5p
2.5. miR-9-5p, miR-675-5p, and miR-138-5p Targeted GSK3β, ATP8A2, and EIF4EBP1
2.6. LRP5 Played Positive Roles in Skeletal Development
3. Discussion
4. Experimental Section
4.1. Cell Cultures and Reagents
4.2. Plasmid Construction
4.3. Transfection
4.4. Clonogenic Assay (Colony Formation Assay)
4.5. Cell Viability Assay
4.6. Cell Cytotoxicity Assay
4.7. TUNEL Cell Apoptosis Assay
4.8. Animal
4.9. RNA Extraction
4.10. Poly (A) Polymerase Tailing
4.11. Reverse Transcription
4.12. Quantitative Real-Time PCR (qPCR)
Primer Name | Sequences (5′–3′) |
---|---|
RACE Reverse | GCGAGCACAGAATTAATACGAC |
miR-9-5p Forward | GCGTCTTTGGTTATCTAGCTGTA |
miR-675-5p Forward | TGGTGCGGAAAGGGCCCACAGT |
miR-138-5p Forward | AGCTGGTGTTGTGAATCAGGCCG |
miR-370 Forward | GCCTGCTGGGGTGGAACCTGGT |
miR-23a Forward | ATCACATTGCCAGGGATTTCC |
miR-103 Forward | ACACTCCAGCTGGGAGCAGCATTGTAC |
GAPDH Forward | ATTGTCAGCAATGCATCCTG |
GAPDH Reverse | ATGGACTGTGGTCATGAGCC |
ALP Forward | AACCCAGACACAAGCATTCC |
ALP Reverse | GCCTTTGAGGTTTTTGGTCA |
COL1A1 Forward | GCCAAGAAGACATCCCTGAA |
COL1A1 Reverse | GCCATTGTGGCAGATACAGA |
RUNX2 Forward | AAGTGCGGTGCAAACTTTCT |
RUNX2 Reverse | ACGCCATAGTCCCTCCTTTT |
GSK3β Forward | TTGGACAAAGGTCTTCCGGC |
GSK3β Reverse | AAGAGTGCAGGTGTGTCTCG |
ATP8A2 Forward | ACGAGGGACGTGCTCATGAAGC |
ATP8A2 Reverse | CCTCAAGTGTCACCAGCAGGCT |
EIF4EBP1 Forward | CTAGCCCTACCAGCGATGAG |
EIF4EBP1 Reverse | CCTGGTATGAGGCCTGAATG |
LRP5 Forward | TGCCACTGGTGAGATTGAC |
LRP5 Reverse | ACTGCTGCTTGATGAGGAC |
Cyclin-D1 Forward | GCGTACCCTGACACCAATCT |
Cyclin-D1 Reverse | CTCCTCTTCGCACTTCTGCT |
CyclinA Forward | CAGAGGCCGAAGACGAGAC |
CyclinA Reverse | TCAGCTGGCTTCTTCTGAGC |
CyclinE Forward | GTTATAAGGGAGACGGGGAG |
CyclinE Reverse | TGCTCTGCTTCTTACCGCTC |
PCNA Forward | TTTGAGGCACGCCTGATCC |
PCNA Reverse | GGAGACGTGAGACGAGTCCAT |
Integrin α2 Forward | AAGTGCCCTGTGGACCTACCCA |
Integrin α2 Reverse | TGGTGAGGGTCAATCCCAGGCT |
Integrin α5 Forward | ACCACCTGCAGAAACGAGAGGC |
Integrin α5 Reverse | TGGCCCAAACTCACAGCGCA |
Integrin Αv Forward | TCCCACCGCAGGCTGACTTCAT |
Integrin Αv Reverse | TCGGGTTTCCAAGGTCGCACAC |
Integrin β1 Forward | TTCAGACTTCCGCATTGGCT |
Integrin β1 Reverse | AATGGGCTGGTGCAGTTTTG |
Fibronectin Forward | TGCAGTGGCTGAAGTCGCAAGG |
Fibronectin Reverse | GGGCTCCCCGTTTGAATTGCCA |
FAK Forward | AGCACCTGGCCACCTAAGCAAC |
FAK Reverse | CATTGGACCGGTCAAGGTTGGCA |
Vinculin Forward | TCAAGCTGTTGGCAGTAGCCGC |
Vinculin Reverse | TCTCTGCTGTGGCTCCAAGCCT |
Osteocalcin Forward | AGCAGGAGGGCAATAAGGTAGT |
Osteocalcin Reverse | TCGTCACAAGCAGGGTTAAGC |
Osteonectin Forward | ATGTCCTGGTCACCTTGTACGA |
Osteonectin Reverse | TCCAGGCGCTTCTCATTCTCAT |
Osteopontin Forward | TGATTCTGGCAGCTCAGAGGA |
Osteopontin Reverse | CATTCTGTGGCGCAAGGAGATT |
Type I collagen Forward | CTCCTGACGCATGGCCAAGAA |
Type I collagen Reverse | TCAAGCATACCTCGGGTTTCCA |
Type III collagen Forward | CCCTGGCTCAAATGGCTCACCA |
Type III collagen Reverse | CCTTTCCACCAGGACTGCCGTT |
Cadherin 11 Forward | GGCCCAAACAGGTATCATCA |
Cadherin 11 Reverse | TTGGTTGTCCCTGAGAGTCC |
MAPK1 Forward | ATCCGGGCACCAACCATTGAGC |
MAPK1 Reverse | GTGGTCATTGCTGAGGTGCTGTGT |
NFkB Forward | AACCCATCGCCTTGGCATCCAC |
NFkB Reverse | AGTCGAAAAGGGCGTTGGCGT |
FasL Forward | GAGAATTGCTGAAGACATGACAATCC |
FasL Reverse | ATGGCTGGAACTGAGGTAGTTTTCAC |
p53 Forward | TAACAGTTCCTGCATGGGCGGC |
p53 Reverse | CGGAGGCCCATCCTCACCATCATCA |
HEY1 Forward | GCATACGGCAGGAGGGAAA |
HEY1 Reverse | CTGGGAAGCGTAGTTGTTGAGAT |
Sost Forward | GGAATGATGCCACAGAGGTCAT |
Sost Reverse | CCCGGTTCATGGTCTGGTT |
Smad7 Forward | GGACGCTGTTGGTACACAAG |
Smad7 Reverse | GCTGCATAAACTCGTGGTCATTG |
SMAD6 Forward | GCTCTAGGAATGCAGACGCTG |
SMAD6 Reverse | CAACAGGCAGTCAGCACAGTC |
4.13. Luciferase Reporter Assay
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sun, T.; Leung, F.; Lu, W.W. 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, 236. https://doi.org/10.3390/ijms17020236
Sun T, Leung F, Lu WW. miR-9-5p, miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation, Differentiation, and Adhesion. International Journal of Molecular Sciences. 2016; 17(2):236. https://doi.org/10.3390/ijms17020236
Chicago/Turabian StyleSun, Tianhao, Frankie Leung, and William W. Lu. 2016. "miR-9-5p, miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation, Differentiation, and Adhesion" International Journal of Molecular Sciences 17, no. 2: 236. https://doi.org/10.3390/ijms17020236