Pmir report basic vector

The 3’-UTR of BTG2 containing the predicted binding site of miR-6875-3p was amplified from normal fetal genomic DNA using PCR specific primers. The PCR product was restricted and inserted between the restriction sites SpeI and HindIII into pMIR-REPORT-basic vector (Applied Biosystems, USA). The consensus miR-6875-3p binding site was mutated via PCR using a QuikChange II XL site-directed mutagenesis kit (Stratagene, USA). All clones were verified by DNA sequencing.Then, miR-6875-3p mimic or negative control was co-transfected with luciferase reporter vectors into HCC cells as described above, followed by the detection of luciferase activity via a luminescence reporter gene assay system (PerkinElmer, Norwalk, CT, USA) according to the manufacturer’s instructions.

The 3′-UTR of EZH2, which contained the predicted binding site of miR-506, was amplified from normal fetal genomic DNA via PCR using specific primers (EZH2– 3′-UTR forward 5′-catggcatactagtcatctgctacctcc-3′, reverse 5′-cctagcgcataagcttaaaacactttgc-3′). The PCR product was restricted and inserted between the restriction sites SpeI and HindIII into pMIR-REPORT-basic vector (Applied Biosystems, USA). The consensus miR-506 binding site was mutated via PCR using a QuikChange II XL site-directed mutagenesis kit (Stratagene, USA). All clones were verified by DNA sequencing. For the luciferase reporter assay, cells were seeded on 24-well plates. Then, 20 nM miRNA mimic or negative control was co-transfected with 100 ng of luciferase reporter vectors into SW620 cells using Lipofectamine 2000. At 48 hours after transfection, the cells were washed with Dulbecco's PBS (DPBS) and resuspended in lysis buffer, followed by the detection of luciferase activity using a luminescence reporter gene assay system (PerkinElmer, Norwalk, CT, USA) according to the manufacturer's instructions.

We identified the miR-644a target site in the HSF1–3’UTR with TargetScan 6.2 (http://www.targetscan.org/vert_71/). We then designed primers to generate the mutant and wild type HSF1 based on the HSF1 mRNA sequence in NCBI GenBank. We PCR amplified the miR-644a target sequence in HSF1 from the total RNA from the HCC cells. The mutant version of the HSF1 3’UTR was generated by overlapping PCR with mutagenic primers. We cloned both wild-type and mutant HSF1–3’UTR sequences into the pMIR-REPORT-basic vector (Applied Biosystems, USA) and confirmed the products by DNA sequencing. For the luciferase reporter assay, we seeded 3 × 10³ cells in 24-well plates and co-transfected 100 ng of the luciferase reporter vector with 20 nM miR-644a mimic or miRNA negative control into SMMC-7721 cells. The cells were lysed after 48 h, and the dual luciferase activities were determined using the luminescence reporter gene assay system (PerkinElmer, Norwalk, CT, USA) according to the manufacturer’s instructions.