The target gene was predicted by TargetScan (http://www.targetscan.org/). A 450-bp fragment of the 3′-untranslated region (3′-UTR) of HIF-1α mRNA containing the target sequence (GCACTTT) of miR-20b was amplified by RT-PCR (HIF-1α, sense 5′-CTCTGAGCTCTATCTGGAAGGTATGTG-3′, antisense 5′-CCTCAAGCTTCAGTTAGTGTTAGACCC-3′). The fragment was designated HIF-1α 3′-UTR and inserted into the pMIR-REPORT™ luciferase reporter vector (SacI and HindIII restriction enzyme sites; Ambion). Another expressing vector was also constructed by the insertion of a mutated HIF-1α 3′-UTR in which the target sequence of miR-20b was mutated into GCAATTT using the QuikChangeH Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) (22 (link)). The recombinant reporter vectors with normal and or mutated HIF-1α 3′-UTR were cotransfected with miR-20b mimic, miR-20b mimic control, miR-20b inhibitor, or miR-20b inhibitor control into MG63 using TransMessenger™ Transfection Reagent (Qiagen, Germany). The luciferase assay was performed according to the manufacturer’s instructions. The relative luciferase activities were normalized to that of the control cells.