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Signature of microRNA expression during osteogenic differentiation of bone marrow MSCs reveals a putative role of miR-335-5p in osteoarthritis.

Tornero-Esteban P, Rodríguez-Rodríguez L, Abásolo L, Tomé M, López-Romero P, Herranz E, González MA, Marco F, Moro E, Fernández-Gutiérrez B, Lamas JR - BMC Musculoskelet Disord (2015)

Bottom Line: Two miRNAs, hsa-miR-210 and hsa-miR-335-5p out of 21 used for validation showed a significant downregulated expression during induced osteogenesis.To our knowledge, this study represents the most comprehensive assessment to date of miRNA expression profiling in BM-MSCs from OA patients and their role during osteogenic differentiation.We describe the existence of a correlation between miR-335-5p expression and OA indicating the putative role of this miRNA in OA features.

View Article: PubMed Central - PubMed

Affiliation: Rheumatology Service, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC). UGC de Reumatología, Hospital Clínico San Carlos, 4a Planta, Ala Norte. C/ Profesor Martín Lagos s/n, 28040, Madrid, Spain. mptornero@gmail.com.

ABSTRACT

Background: The aim of this study was to evaluate, the existence of a signature of differentially expressed microRNAs (miRNAs) during osteogenic differentiation of bone marrow MSCs from OA and healthy donors and to describe their possible implication in joint regeneration through modulation of molecular mechanisms involved in homeostatic control in OA pathophysiology.

Methods: Following phenotypic assessment of BM-MSCs obtained from OA diagnosed patients (n = 10) and non-OA (n = 10), total small RNA was isolated after osteogenic induction for 1, 10 and 21 days, miRNA profiles were generated using a commercial expression array of 754 well-characterized miRNAs. MiRNAs, with consistent differential expression were selected for further validation by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.

Results: A total of 246 miRNAs were differentially expressed (fold change ≥ ± 2, P ≤0.05) between OA and non-OA BM-MSC samples; these miRNAs showed variable interactions depending on the cell and differentiation status. Two miRNAs, hsa-miR-210 and hsa-miR-335-5p out of 21 used for validation showed a significant downregulated expression during induced osteogenesis. In particular hsa-miR-335-5p, a critical regulator in bone homeostasis, was further studied. hsa-miR-335-5p downregulation in OA-MSCs, as well as their host coding gene, MEST, were also assessed.

Conclusions: To our knowledge, this study represents the most comprehensive assessment to date of miRNA expression profiling in BM-MSCs from OA patients and their role during osteogenic differentiation. We describe the existence of a correlation between miR-335-5p expression and OA indicating the putative role of this miRNA in OA features. These findings, may contribute to our understanding of the molecular mechanisms involved in MSCs mediated homeostatic control in OA pathophysiology that could be applicable in future therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus

Two percent agarose gel of DKK1 and SFRP amplification products from OA-MSCs and Control-MSCs. After synthesizing the first strand cDNA, it was amplified using PCR. The PCR product was analyzed by agarose gel electrophoresis. PCR reactions (25ul) were loaded as follow: Lanes [1–6] ACTB [8–13] DKK1. [15–20] ACTB + SFRP1. Lanes 7 and 14 correspond to the molecular markers (GeneRuler™ Low Range DNA Ladder, Fermentas). This result shows that single specific PCR product was obtained with expected molecular amplicon sizes. No differences were found in band intensity between OA-MSCs and Control-MSCs
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Fig4: Two percent agarose gel of DKK1 and SFRP amplification products from OA-MSCs and Control-MSCs. After synthesizing the first strand cDNA, it was amplified using PCR. The PCR product was analyzed by agarose gel electrophoresis. PCR reactions (25ul) were loaded as follow: Lanes [1–6] ACTB [8–13] DKK1. [15–20] ACTB + SFRP1. Lanes 7 and 14 correspond to the molecular markers (GeneRuler™ Low Range DNA Ladder, Fermentas). This result shows that single specific PCR product was obtained with expected molecular amplicon sizes. No differences were found in band intensity between OA-MSCs and Control-MSCs

Mentions: Increased bone formation is one of the main OA features. In addition osteoinduction through Wnt signaling can be promoted by inhibition of Wnt antagonists such as DKK1 and SFRP1, two known predicted targets of miR-335-5p. We next aimed to confirm if differential expression of miR-335-5p in OA-MSCs correlated with a reduced expression of: DKK1 and SFRP1. Our data revealed that independently of miR-335-5p expression, mRNA levels of antagonists were similar both in undifferentiated OA- or Control-MSCs (Fig. 4).Fig. 4


Signature of microRNA expression during osteogenic differentiation of bone marrow MSCs reveals a putative role of miR-335-5p in osteoarthritis.

Tornero-Esteban P, Rodríguez-Rodríguez L, Abásolo L, Tomé M, López-Romero P, Herranz E, González MA, Marco F, Moro E, Fernández-Gutiérrez B, Lamas JR - BMC Musculoskelet Disord (2015)

Two percent agarose gel of DKK1 and SFRP amplification products from OA-MSCs and Control-MSCs. After synthesizing the first strand cDNA, it was amplified using PCR. The PCR product was analyzed by agarose gel electrophoresis. PCR reactions (25ul) were loaded as follow: Lanes [1–6] ACTB [8–13] DKK1. [15–20] ACTB + SFRP1. Lanes 7 and 14 correspond to the molecular markers (GeneRuler™ Low Range DNA Ladder, Fermentas). This result shows that single specific PCR product was obtained with expected molecular amplicon sizes. No differences were found in band intensity between OA-MSCs and Control-MSCs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526194&req=5

Fig4: Two percent agarose gel of DKK1 and SFRP amplification products from OA-MSCs and Control-MSCs. After synthesizing the first strand cDNA, it was amplified using PCR. The PCR product was analyzed by agarose gel electrophoresis. PCR reactions (25ul) were loaded as follow: Lanes [1–6] ACTB [8–13] DKK1. [15–20] ACTB + SFRP1. Lanes 7 and 14 correspond to the molecular markers (GeneRuler™ Low Range DNA Ladder, Fermentas). This result shows that single specific PCR product was obtained with expected molecular amplicon sizes. No differences were found in band intensity between OA-MSCs and Control-MSCs
Mentions: Increased bone formation is one of the main OA features. In addition osteoinduction through Wnt signaling can be promoted by inhibition of Wnt antagonists such as DKK1 and SFRP1, two known predicted targets of miR-335-5p. We next aimed to confirm if differential expression of miR-335-5p in OA-MSCs correlated with a reduced expression of: DKK1 and SFRP1. Our data revealed that independently of miR-335-5p expression, mRNA levels of antagonists were similar both in undifferentiated OA- or Control-MSCs (Fig. 4).Fig. 4

Bottom Line: Two miRNAs, hsa-miR-210 and hsa-miR-335-5p out of 21 used for validation showed a significant downregulated expression during induced osteogenesis.To our knowledge, this study represents the most comprehensive assessment to date of miRNA expression profiling in BM-MSCs from OA patients and their role during osteogenic differentiation.We describe the existence of a correlation between miR-335-5p expression and OA indicating the putative role of this miRNA in OA features.

View Article: PubMed Central - PubMed

Affiliation: Rheumatology Service, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC). UGC de Reumatología, Hospital Clínico San Carlos, 4a Planta, Ala Norte. C/ Profesor Martín Lagos s/n, 28040, Madrid, Spain. mptornero@gmail.com.

ABSTRACT

Background: The aim of this study was to evaluate, the existence of a signature of differentially expressed microRNAs (miRNAs) during osteogenic differentiation of bone marrow MSCs from OA and healthy donors and to describe their possible implication in joint regeneration through modulation of molecular mechanisms involved in homeostatic control in OA pathophysiology.

Methods: Following phenotypic assessment of BM-MSCs obtained from OA diagnosed patients (n = 10) and non-OA (n = 10), total small RNA was isolated after osteogenic induction for 1, 10 and 21 days, miRNA profiles were generated using a commercial expression array of 754 well-characterized miRNAs. MiRNAs, with consistent differential expression were selected for further validation by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.

Results: A total of 246 miRNAs were differentially expressed (fold change ≥ ± 2, P ≤0.05) between OA and non-OA BM-MSC samples; these miRNAs showed variable interactions depending on the cell and differentiation status. Two miRNAs, hsa-miR-210 and hsa-miR-335-5p out of 21 used for validation showed a significant downregulated expression during induced osteogenesis. In particular hsa-miR-335-5p, a critical regulator in bone homeostasis, was further studied. hsa-miR-335-5p downregulation in OA-MSCs, as well as their host coding gene, MEST, were also assessed.

Conclusions: To our knowledge, this study represents the most comprehensive assessment to date of miRNA expression profiling in BM-MSCs from OA patients and their role during osteogenic differentiation. We describe the existence of a correlation between miR-335-5p expression and OA indicating the putative role of this miRNA in OA features. These findings, may contribute to our understanding of the molecular mechanisms involved in MSCs mediated homeostatic control in OA pathophysiology that could be applicable in future therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus