Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells by regulating the lncRNA H19/miR-29b-3p/SOX9 axis

Abstract

Platelet lysate (PL) has been shown to induce chondrogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs). However, the underlying mechanism is still not clear. The aim of this study was to investigate whether long noncoding RNA H19 is involved in this process. hUCMSCs were isolated, identified and cultured in 5% PL-supplemented chondrogenic differentiation medium. Chondrogenic differentiation was assessed by Alcian blue staining. The expressions of H19, miR-29b-3p, SRY-related high-mobility-group box 9 (SOX9), collagen II and aggrecan were determined by quantitative real-time PCR and western blot. The interaction between miR-29b-3p and H19 or SOX9 was analyzed by luciferase reporter assay. During PL-induced chondrogenic differentiation of hUCMSCs, expressions of H19 and SOX9 were increased, whereas miR-29b-3p expression was decreased. H19 overexpression promoted, whereas H19 silencing attenuated the PL-induced chondrogenic differentiation of hUCMSCs. SOX9 was identified as a direct target of miR-29b-3p, and H19 was observed to act as a sponge of miR-29b-3p to up-regulate SOX9 expression. The chondrogenic differentiation-promoting effect of H19 overexpression was negated when miR-29b-3p expression was up-regulated by Lenti-miR-29b-3p infection. In conclusion, PL induced chondrogenic differentiation of hUCMSCs by regulating the H19/miR-29b-3p/SOX9 axis.

Keywords: H19; SOX9; chondrogenic differentiation; mesenchymal stem cells; miR-29b-3p; platelet lysate.

Fig. 1

H19 expression was increased during PL‐induced chondrogenic differentiation of hUCMSCs. (A) hUCMSCs at passage 2 were characterized by flow cytometry. Forward scatter (FSC) and side scatter (SSC) distribution of gated cells (left). The cells were positive for CD105 and CD44, but negative for CD45 and CD34 (right). (B) qRT‐PCR analysis of H19 expression in hUCMSCs cultured in 5% PL‐supplemented chondrogenic differentiation medium for 0, 7, 14 and 21 days. *P < 0.05, **P < 0.01, vs. 0 day (ANOVA). The quantitative statistics were presented as the mean ± standard deviation (n = 3).

Fig. 2

Effects of H19 overexpression and silencing on PL‐induced chondrogenic differentiation of hUCMSCs. (A) qRT‐PCR analysis of H19 expression in hUCMSCs at 48 h after infection with Lenti‐NC, Lenti‐H19, Lenti‐shRNA and Lenti‐sh‐H19. (B, C) The infected hUCMSCs as described in (A) were cultured in 5% PL‐supplemented chondrogenic differentiation medium for 21 days. (B) Chondrogenic differentiation was assessed by Alcian blue staining (scale bar: 100 μm). (C) The protein levels of collagen II and aggrecan in hUCMSCs were examined by western blot. *P < 0.05, **P < 0.01, vs. Lenti‐NC; ^# P < 0.05, ^## P < 0.01, vs. Lenti‐shRNA (Student's t‐test). The quantitative statistics were presented as the mean ± standard deviation (n = 3).

Fig. 3

Interaction between miR‐29b‐3p and H19 or SOX9. qRT‐PCR analysis of (A) miR‐29b‐3p expression and (B) SOX9 mRNA level, and (C) western blot analysis of SOX9 protein level in hUCMSCs cultured in 5% PL‐supplemented chondrogenic differentiation medium for 0, 7, 14 and 21 days. *P < 0.05, **P < 0.01, vs. 0 day (Student's t‐test). (D, E) qRT‐PCR analysis of SOX9 mRNA level (D) and western blot analysis of SOX9 protein level (E) in hUCMSCs infected with Lenti‐NC, Lenti‐H19, Lenti‐shRNA and Lenti‐sh‐H19. **P < 0.01, vs. Lenti‐NC; ^## P < 0.01, vs. Lenti‐shRNA (Student's t‐test). (F) The WT (H19 WT) and mutation (H19 Mut) of binding sites between H19 and miR‐29b‐3p. Results of luciferase activity assay verified the direct binding between H19 and miR‐29b‐3p. (G) The WT (SOX9 WT) and mutation (SOX9 Mut) of binding sites between SOX9 and miR‐29b‐3p. Results of luciferase activity assay verified the direct binding between SOX9 3′‐UTR and miR‐29b‐3p. **P < 0.01, vs. mimic NC (Student's t‐test). The quantitative statistics were presented as the mean ± standard deviation (n = 3).

Fig. 4

H19 promoted PL‐induced chondrogenic differentiation of hUCMSCs by regulating the miR‐29b‐3p/SOX9 axis. (A, B) qRT‐PCR analysis of SOX9 mRNA level (A) and western blot analysis of SOX9 protein level (B) in hUCMSCs transfected with miR‐29b‐3p mimic, miR‐29b‐3p inhibitor or corresponding controls. **P < 0.01, vs. mimic NC; ^## P < 0.01, vs. inhibitor NC (Student's t‐test). (C, D) hUCMSCs were coinfected with Lenti‐NC/Lenti‐H19 and Lenti‐miR‐NC/Lenti‐miR‐29b‐3p, and then cultured in 5% PL‐supplemented chondrogenic differentiation medium for 21 days. (C) Chondrogenic differentiation was assessed by Alcian blue staining (scale bars: 100 μm). (D) The protein levels of SOX9, collagen II and aggrecan in hUCMSCs were examined by western blot. *P < 0.05, vs. Lenti‐miR‐NC + Lenti‐NC; ^# P < 0.05, vs. Lenti‐miR‐NC + Lenti‐H19 (ANOVA). The quantitative statistics were presented as the mean ± standard deviation (n = 3).