FIGURE 4.

Silencing of H19 promoted bone morphogenetic protein 2 (BMP2)-induced hypertrophic differentiation of mesenchymal stem cells (MSCs) in vitro. (A) C3H10T1/2 cells infected with AdGFP, AdBMP2, AdBMP2, and AdsimH19 were subjected to micromass culture. Bright field, GFP (AdGFP, AdBMP2) and RFP (AdsimH19) fluorescence fields were recorded at day 3 after infection; the fluorescence indicated high efficiency in single or combination infection, scale bar = 1,000 μm. (B) Effective knockdown of mouse H19 expression. The expression level of H19 and BMP2 messenger RNA (mRNA) at day 3 in different groups were detected by reverse transcription and quantitative real-time PCR (RT-qPCR); AdsimH19 silences the expression of H19 without influencing the expression of BMP2. All samples were normalized with the reference gene Gapdh. Each assay condition was done in triplicate. (C) Silencing H19 promoted BMP2-induced collagen 10α1 and MMP13 mRNA expression. Subconfluent MSCs were infected with AdBMP9 or AdGFP and/or AdsimH19 and subjected to micromass culture. At the indicated time points, total RNA was isolated and subjected to qPCR analysis using primers for mouse collagen 10α1 and MMP13; each assay condition was done in triplicate. (D) Silencing H19 promoted BMP2-induced collagen 10α1 and MMP13 protein expression. Western blot for the expression of collagen 10α1 and MMP13 were conducted at day 7 after transduction of indicated recombinant adenoviruses (a). Relative protein expression was analyzed by Image Lab software using β-actin as control (b). “*” and “**” respectively mean p < 0.05 and p < 0.01 compared with AdGFP group; “#” and “##” respectively mean p < 0.05 and p < 0.01 compared with AdBMP2 group. Each assay was done in triplicate and/or carried out in three independent experiments at least; representative results are shown.