Int J Stem Cells.  2021 Aug;14(3):286-297. 10.15283/ijsc20188.

The Role of miR-34c-5p in Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

• Liu B
• Gan W
• Jin Z
• Wang M
• Cui G
• Zhang H
• Wang H

• KMID: 2519371
• DOI: http://doi.org/10.15283/ijsc20188

Abstract

Background and Objectives
Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a critical role in the success of lumbar spinal fusion with autogenous bone graft. This study aims to explore the role and specific mechanism of miR-34c-5p in osteogenic differentiation of BMSCs.
Methods and Results
Rabbit model of lumbar fusion was established by surgery. The osteogenic differentiation dataset of mesenchymal stem cells was obtained from the Gene Expression Omnibus (GEO) database, and differentially expressed miRNAs were analyzed using R language (limma package). The expressions of miR-34c-5p, miR-199a-5p, miR-324-5p, miR-361-5p, RUNX2, OCN and Bcl-2 were determined by qRT-PCR and Western blot. ELISA, Alizarin red staining and CCK-8 were used to detect the ALP content, calcium deposition and proliferation of BMSCs. The targeted binding sites between miR-34c-5p and Bcl-2 were predicted by the Target database and verified using dual-luciferase reporter assay. MiR-34c-5p expression was higher in rabbit lumbar fusion model and differentiated BMSCs than normal rabbit or BMSCs. The content of ALP and the deposition of calcium increased with the osteogenic differentiation of BMSCs. Upregulation of miR-34c-5p reduced cell proliferation and promoted ALP content, calcium deposition, RUNX2 and OCN expression compared with the control group. The effects of miR-34c-5p inhibitor were the opposite. In addition, miR-34c-5p negatively correlated with Bcl-2. Upregulation of Bcl-2 reversed the effects of miR-34c-5p on ALP content, calcium deposition, and the expressions of RUNX2 and OCN.
Conclusions
miR-34c-5p could promote osteogenic differentiation and suppress proliferation of BMSCs by inhibiting Bcl-2.

Keyword

miR-34c-5p; Bcl-2; BMSCs; Osteogenic differentiation; Proliferation

Figure

• 

  Fig. 1 During osteogenic differentiation of BMSCs, miR-34c-5p expression, ALP content and calcium deposition were promoted. (A) Abnormal expressions of miRNAs during osteogenic differentiation of mesenchymal stem cells obtained from the GEO database (GSE115197 and GSE19232). (B) The expressions of miR-34c-5p, miR-199a-5p, miR-324-5p and miR-361-5p in rabbit lumbar fusion model rabbit or normal rabbit were determined by qRT-PCR. (C) qRT-PCR was used to detect the expression of miR-34c-5p in BMSCs with different differentiation times (0, 3, 7 and 14 day). (D) ELISA was used to measure the content of ALP in BMSCs at different differentiation times (0, 3, 7 and 14 day). (E) Alizarin red staining of BMSCs at different differentiation times (0, 3, 7 and 14 day). Scales: 200 μm; magnifications: ×200. ^vs. Control, *vs. 0 days. ^^^ or ***p<0.001. BMSCs, bone marrow mesenchymal stem cells; ALP, alkaline phosphatase; GEO, gene expression omnibus; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ELISA, enzyme linked immune sorbent assay. The experiment was independently repeated three times.

• 

  Fig. 2 miR-34c-5p decreased cell proliferation and increased ALP content and calcium deposits of BMSCs. (A) The expression of miR-34c-5p in BMSCs transfected with miR-34c-5p mimic or mimic control was detected by qRT-PCR. (B) The expression of miR-34c-5p in BMSCs transfected with miR-34c-5p inhibitor or inhibitor control was detected by qRT-PCR. (C) The cell proliferation of BMSCs transfected with miR-34c-5p mimic or mimic control was identified by CCK-8 at different times (1, 3, 5 and 7 day). (D) The cell proliferation of BMSCs transfected with miR-34c-5p inhibitor or inhibitor control was identified by CCK-8 at different times (1, 3, 5 and 7 day). (E) The ALP content of BMSCs transfected with miR-34c-5p mimic or mimic control was detected by ELISA. (F) The ALP content of BMSCs transfected with miR-34c-5p inhibitor or inhibitor control was detected by ELISA. (G) Alizarin red staining of BMSCs transfected with miR-34c-5p mimic or mimic control at 14 days. Scales: 200 μm; magnifications: ×200. (H) Alizarin red staining of BMSCs transfected with miR-34c-5p inhibitor or inhibitor control at 14 days. Scales: 200 μm; magnifications: ×200. *vs. MC; ^vs. IC. * or ^p<0.05; ** or ^^p<0.01; *** or ^^^p<0.001. BMSCs, bone marrow mesenchymal stem cells; qRT-PCR, quantitative reverse transcription polymerase chain reaction; CCK-8, cell counting 8; ALP, alkaline phosphatase; ELISA, enzyme linked immune sorbent assay; I, miR-34c-5p inhibitor; IC, inhibitor control; M, miR-34c-5p mimic; MC, mimic control. The experiment was independently repeated three times.

• 

  Fig. 3 MiR-34c-5p promoted the expression of RUNX2 and OCN of BMSCs. (A, B) The expression of RUNX2 and OCN of BMSCs transfected with miR-34c-5p mimic or mimic control was detected by Western blot. (C) The expressions of RUNX2 and OCN of BMSCs transfected with miR-34c-5p mimic or mimic control were detected by qRT-PCR. (D, E) The expressions of RUNX2 and OCN of BMSCs transfected with miR-34c-5p inhibitor or inhibitor control were detected by Western blot. (F) The expressions of RUNX2 and OCN of BMSCs transfected with miR-34c-5p inhibitor or inhibitor control were detected by qRT-PCR. *vs. MC; ^vs. IC. ^^p<0.01; *** or ^^^p<0.001. BMSCs, bone marrow mesenchymal stem cells; RUNX2, Runt-related transcription factor 2; OCN, osteocalcin; qRT-PCR, quantitative reverse transcription polymerase chain reaction; I, miR-34c-5p inhibitor; IC, inhibitor control; M, miR-34c-5p mimic; MC, mimic control. The experiment was independently repeated three times.

• 

  Fig. 4 MiR-34c-5p decreased cell activity and increased ALP content of BMSCs by inhibiting Bcl-2 expression. (A) The binding site of miR-34c-5p and Bcl-2 was predicted by TargetScan. (B, C) Dual-luciferase reporter gene assay was used to verify the targeted binding relationship between miR-34c-5p and Bcl-2. (D) The cell proliferation of BMSCs transfected with or untransfected miR-34c-5p mimic and/or Bcl-2 was detected by CCK-8 at 1, 3, 5 and 7 day. (E) The cell proliferation of BMSCs treated or untreated with miR-34c-5p inhibitor and/or siBcl-2 was detected by CCK-8 at 1, 3, 5 and 7 day. (F) The ALP content of BMSCs treated or untreated with miR-34c-5p mimic and/or Bcl-2 was detected by ELISA. (G) The ALP content of BMSCs treated or untreated with miR-34c-5p inhibitor and/or siBcl-2 was detected by ELISA. *vs. MC, #vs. M, †vs. BCL2, ^vs. IC, §vs. I, ‡vs. siBCL2. † or ^ or ‡ or §p<0.05; ## or ^^ or §§p<0.01; *** or ^^^ or ††† or ###p<0.001. BMSCs, bone marrow mesenchymal stem cells; CCK-8, cell counting 8; ALP, alkaline phosphatase; ELISA, enzyme linked immune sorbent assay; Bcl-2, B cell lymphoma/lewkmia-2; I, miR-34c-5p inhibitor; IC, inhibitor control; M, miR-34c-5p mimic; MC, mimic control. The experiment was independently repeated three times.

• 

  Fig. 5 MiR-34c-5p promoted calcium deposits and the expression of RUNX2 and OCN of BMSCs by inhibiting Bcl-2 expression. (A) Alizarin red staining of BMSCs treated or untreated with miR-34c-5p mimic and/or Bcl-2 at 14 days. Scales: 200 μm; magnifications: ×200. (B) Alizarin red staining of BMSCs treated or untreated with miR-34c-5p inhibitor and/or siBcl-2 at 14 days. Scales: 200 μm; magnifications: ×200. (C, D) The expression of Bcl-2, RUNX2 and OCN of BMSCs treated or untreated with miR-34c-5p mimic and/or Bcl-2 were detected by Western blot. (E) The expressions of Bcl-2, RUNX2 and OCN of BMSCs treated or untreated with miR-34c-5p mimic and/or Bcl-2 were detected by qRT-PCR. (F) The expressions of Bcl-2, RUNX2 and OCN of BMSCs treated or untreated with miR-34c-5p inhibitor and/or siBcl-2 were detected by qRT-PCR. (G, H) The expressions of Bcl-2, RUNX2 and OCN of BMSCs treated or untreated with miR-34c-5p inhibitor and/or siBcl-2 were detected by Western blot. *vs. MC, #vs. M, †vs. BCL2, ^vs. IC, §vs. I, ‡vs. siBCL2. † or * or ‡p<0.05; ^^ or ††p<0.01; *** or ^^^ or ‡‡‡ or ### or §§§p<0.001. BMSCs, bone marrow mesenchymal stem cells; CCK-8, cell counting 8; ALP, alkaline phosphatase; ELISA, enzyme linked immune sorbent assay; Bcl-2, B cell lymphoma/lewkmia-2; I, miR-34c-5p inhibitor; IC, inhibitor control; M, miR-34c-5p mimic; MC, mimic control. The experiment was independently repeated three times.

Reference

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