miR-148a-3p Promoter Regulation and Target Validation

The putative promoter region of miR-148a-3p (-1 kb) harboring the predicted myc-binding sequences were synthesized by PCR (PrimerSTAR, TaKaRa) and cloned into the pGL3 vector between HindIII and NheI. pENTER-c-myc vector (pC-MYC) or pENTER vector (pNull)-treated T24 or UM-UC-3 bladder cancer cells were co-transfected with the cloned pGL3 vector and renilla luciferase vector in the absence or presence of the c-myc inhibitor (c-myc-Inh, 10058-F4, Selleck, Houston, TX, USA). A total of 48 h after transfection, the relative luciferase activity was calculated using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
The 3′-UTRs of DNMT1, ERBB3 and AKT2 containing putative miR-148a-3p target regions were synthesized (Sangon, Shanghai, China) and cloned between the SacI and SalI sites downstream of the luciferase reporter gene in pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). In addition, the mutant miR-148a-3p putative target region was generated using the same approach. All insertions were verified by sequencing (Sangon). The dual-Luciferase report assay was performed as previously described.^{14 (link)}