Dual light luminescent reporter gene assay

The miR-203a mimics/inhibitor and corresponding NC were purchased from GenePharma (Shanghai, China). The potential binding site between Smad3 and miR-203a was searched using TargetScan (Figure 4A) (http://www.targetscan.org). The Smad3 3ʹ-UTR with wild-type (wt) and mutant (mut) containing the putative binding site of miR-203a were constructed (Figure 4B) and subsequently cloned into a pMIR-REPORT luciferase reporter vector (Ambion, U.S.A.). The mutated plasmid, pMIR-Smad3-mut-3ʹ-UTR was generated by a QuikChange Kit (Qiagen). The chondrocytes were co-transfected with 400 ng of the reporter construct (pMIR-Smad3-3ʹ-UTR or pMIR-Smad3-3ʹ-UTR) and 50 ng miR-203a mimic/inhibitor or corresponding NC using Lipofectamine 2000 reagent (Invitrogen). Forty-eight hours after transfection, the relative firefly luciferase activity normalized with Renilla luciferase was measured using the Dual-Light luminescent reporter gene assay (Applied Biosystems).

To generate luciferase reporter vectors, a pair of oligonucleotide with wide type 3′-UTR region of HNF4G (containing binding site with miR-34a) were designed and synthesized (F: 5′-cATTATATTTTTATATACTGAACTGCCATAGATGCTGGAAAAAAGGAAg-3′; R: 5′-tcgacTTCCTTTTTT  CCAGCATCTA  ACCGTCATCA  GTATATAAAA  ATATAATgagct-3′) (RiboBio, Guangzhou, China). The oligonucleotide pair was annealed by heating at 90°C for 3 min and then at 37°C for 15 min. Then the duplex was inserted between the SacI and SalI sites of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). This expression constructed was designated as Luc-HNF4G. The same method was applied to generate Luc-HNF4G-mut with mutant 3′-UTR region of HNF4G by using the following oligonucleotide pair: F: 5′-cATTATATTTTTATATACTGATGACGGTTAGATGCTGGAAAAAAGGAAg-3′; R: 5′-tcgacTTCCTTTTTTCCAGCATCTAACCGTCACAGTATATAAAAATATAATgagct-3′. The insertion was verified by sequencing. miR-34a mimics or the control was cotransfected with Luc-HNF4G or Luc-HNF4G-mut into HEK 293T cells. Cells were lysed 48 h after transfection. Luciferase activity of the lysates was detected by using the Dual-Light luminescent reporter gene assay (Applied Biosystems).

The potential binding site between PTEN and miR-106b was identified using TargetScan (http://www.targetscan.org). The miR-106b mimics/inhibitor and corresponding negative control (NC) were synthesized by RiboBio (Guangzhou, China). The wild-type (wt) PTEN-3′-UTR and mutant (mut) PTEN-3′-UTR containing the putative binding site of miR-106b were established (Figure 5A) and cloned in the firefly luciferase expressing vector pMIR-REPORT (Ambion, U.S.A.). Site-directed mutagenesis of the PTEN 3′-UTR at the putative miR-106b binding site was performed by a QuikChange Kit (Qiagen). For the luciferase assay, Ishikawa cells at a density of 2 × 10⁵ per well were seeded into 24-well plates and co-transfected with 0.8 μg of pMIR-PTEN-3′-UTR or pMIR-PTEN-mut-3′-UTR, 50 nM miR-106b mimic/inhibitor or corresponding mimic NC using Lipofectamine 2000 reagent (Invitrogen). The relative firefly luciferase activity normalized with Renilla luciferase was measured 48 h after transfection by using the Dual-Light luminescent reporter gene assay (Applied Biosystems).