Int J Stem Cells.  2022 Aug;15(3):301-310. 10.15283/ijsc21035.

YBX1 Promotes the Inclusion of RUNX2 Alternative Exon 5 in Dental Pulp Stem Cells

• Shen J
• She W
• Zhang F
• Guo J
• Jia R

• KMID: 2532404
• DOI: http://doi.org/10.15283/ijsc21035

Abstract

Background and Objectives
RUNX2 plays an essential role during the odontoblast differentiation of dental pulp stem cells (DPSCs). RUNX2 Exon 5 is an alternative exon and essential for RUNX2 transcriptional activity. This study aimed to investigate the regulatory mechanisms of RUNX2 exon 5 alternative splicing in human DPSCs.
Methods and Results
The regulatory motifs of RUNX2 exon 5 were analyzed using the online SpliceAid program. The alternative splicing of RUNX2 exon 5 in DPSCs during mineralization-induced differentiation was analyzed by RT-PCR. To explore the effect of splicing factor YBX1 on exon 5 alternative splicing, gaining or losing function of YBX1 was performed by transfection of YBX1 overexpression plasmid or anti-YBX1 siRNA in DPSCs. Human RUNX2 exon 5 is evolutionarily conserved and alternatively spliced in DPSCs. There are three potential YBX1 binding motifs in RUNX2 exon 5. The inclusion of RUNX2 exon 5 and YBX1 expression level increased significantly during mineralization-induced differentiation in DPSCs. Overexpression of YBX1 significantly increased the inclusion of RUNX2 exon 5 in DPSCs. In contrast, silence of YBX1 significantly reduced the inclusion of exon 5 and the corresponding RUNX2 protein expression level. Knockdown of YBX1 reduced the expression of alkaline phosphatase (ALP) and osteocalcin (OC) and the mineralization ability of DPSCs, while overexpression of YBX1 increased the expression of ALP and OC and the mineralization ability of DPSCs.
Conclusions
Human RUNX2 exon 5 is conserved evolutionarily and alternatively spliced in DPSCs. Splicing factor YBX1 promotes the inclusion of RUNX2 exon 5 and improves the mineralization ability of DPSCs.

Keyword

RUNX2; Exon 5; YBX1; Dental pulp stem cells

Figure

• 

  Fig. 1 Alternative splicing of RUNX2 mRNA in dental pulp stem cells (DPSC). (A) Schematic diagram of isoforms of human RUNX2. Exon 5 and 7 are two alternative exons. (B) Analysis of alternative splicing of RUNX2 mRNA in DPSCs by RT-PCR. GAPDH served as a loading control. Diagrams on the left and right show the structure and size of RUNX2 pre-mRNA and spliced pro-ducts, and the location of primers (short lines above or below exons). The sizes of marker bands from the top to the bottom are 700 bp, 500 bp, 400 bp, 300 bp, and 200 bp, respectively. (C) Flow cytometric analysis of the purity of cultured dental pulp stem cells with stem cell biomarkers. CD90, CD29 and CD146 are mesenchymal stem cell markers, whereas CD34 and CD45 are hematopoietic stem cell markers.

• 

  Fig. 2 Conservation of RUNX2 exon 5 in human, mouse, and rat. (A) Schematic diagram of RUNX2 exon 5 splicing in human, mouse, and rat. (B) Alignment of human RUNX2, mouse Runx2, and rat Runx2 exon 5 DNA and encoded protein sequence. (C) Predicted YBX1 binding sites with scores in human RUNX2 exon 5 RNA sequence by SpliceAid online program. Positive score represents promoting exon inclusion.

• 

  Fig. 3 RUNX2 exon 5 inclusion and YBX1 expression are enhanced after mineralization induction. (A) Alizarin red staining of DPSCs treated with or without mineralization induction for 21 days. (B, C) OC and DSPP expression (B), and alternative splicing of RUNX2 exon 5 in DPSCs after three days of mineralization induc-tion (C) were analyzed by RT-PCR. GAPDH served as loading control. (D) Summary of four independent experiments for the ratios of inclusion versus exclusion of RUNX2 exon 5 in DPSCs after mineralization induction. L and S represent products with or without exon 5. (E) YBX1 protein expression increased after three days of mineralization induction in DPSCs. (F) Histogram showed quantification of three independent experiments for the relative levels of YBX1. Actin served as loading control. Statistical comparisons of means were performed with Student’s t-test.

• 

  Fig. 4 YBX1 promotes RUNX2 exon 5 inclusion in DPSCs. (A∼C) Overexpression of YBX1 promoted RUNX2 exon 5 inclusion in HEK 293 cells. (D∼F) Overexpression of YBX1 promoted RUNX2 exon 5 inclusion in DPSCs. (B, E) Overexpression of Myc-tagged YBX1 was confirmed by Western blot. (C, F) Summary of three independent experiments for the ratios of inclusion versus exclusion of RUNX2 exon 5 in HEK 293 (C) or DPSCs (F). (G) Knockdown of YBX1 reduced RUNX2 exon 5 inclusion in DPSCs. (H) Histogram showed quantification of three independent experiments for the ratios of inclusion versus exclusion of RUNX2 exon 5 in DPSCs. (I) Knockdown efficiency of YBX1 and the expression of RUNX2 were analyzed by Western blot. GAPDH served as loading control. (J) Histogram showed quantification of three independent experiments for the expression of RUNX2 protein in DPSCs.

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  Fig. 5 YBX1 promotes the mineralization ability of DPSCs. (A∼D) Overexpression of YBX1 increased ALP and OC expression (A), and mineral formation by DPSCs in differential medium (C). (B, D) Summary of three independent experiments for ALP and OC expression (B) or mineral formation (D) by DPSCs. (E∼H) Knockdown of YBX1 suppressed ALP and OC expression (E), and mineral formation by DPSCs in differential medium (G). (F, H) Summary of three independent experiments for ALP and OC expression (F) or mineral formation (H) by DPSCs. DPSCs were transfected with siRNA or plasmid for 24 hours, and then cultured in differential medium. ALP and OC expression were analyzed in Day 5. Mineral formation was analyzed by alizarin red staining in Day 21.

• 

  Fig. 6 YBX1 binds RUNX2 exon 5 RNA. The interaction between YBX1 and RUNX2 transcripts in DPSCs were analyzed by RNA-immunoprecipitation assay with anti-YBX1 antibody. (A) Immunopre-cipitated RNA was analyzed by RT-PCR with a pair of primers amplifying RUNX2 exon 5. (B) Immunoprecipitated YBX1 protein was confirmed by Western blot.

Reference

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