MicroRNA-29b Contributes to Collagens Imbalance in Human Osteoarthritic and Dedifferentiated Articular Chondrocytes

Figure 3

Study of miR-29b interaction with human collagens mRNAs using luciferase reporter assays. (a) Schematic construct of pGL3-collagens reporter. SV40: SV40 promoter, PA: polyadenylation signal, and WT: wild-type. The sequences predicted to be targeted by the seed sequence of miR-29b in the 3′-UTRs of human COL1A2 or COL2A1 mRNAs are highlighted. 3′-UTR of human COL10A1 is used as a control lacking any predicted target sequence and represented arbitrarily in the same sequence location as those targeted in hCOL1A2 or hCOL2A1. (b) Luciferase activities in HeLa cells cotransfected either with pcDNA3.1-pre-miR-29b or a negative control (pcDNA3.1-empty), and the internal control plasmid encoding Renilla luciferase (RLuc), and either pGL3-Luc::hCOL1A2 nor pGL3-Luc::hCOL2A1 or pGL3-Luc::hCOL10A1 sens or antisens (AS) constructs. Cotransfection with either pcDNA3.1-pre-miR-199a or a negative control (pcDNA3.1-empty) was used as a nonspecific control of IL-1β effect. Data are shown as relative luciferase activity, taking the activity measured in HeLa cells transfected with plasmid pcDNA3.1-empty as the reference 100% value for each construct. (c) miR-29b level determined by the method as described in Figure 2. (d) Luciferase::hCOL2A1 mRNA level determined by quantitative PCR and normalized to RPS29 mRNA chosen as a housekeeping gene. Data are expressed as relative levels, taking the mRNA level in HeLa cells transfected with plasmid pcDNA3.1-empty as the reference value 1. In all experiments, bars show the mean values ± SEM of at least 3 independent experiments, each being run in triplicate. versus control.

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