Pmir report vector

The MYO1B 3’UTR (1.4 kb; chr2:192,288,687-192,290,115) was PCR amplified using the forward primer 5’-GGACTAGTAACCGTCTCCTTGAAGTTGC-3’ and the reverse primer 5′-GGAAGCTTGGCACAAGGCAAGAAGAATC-3′. The primers were designed with a SpeI restriction site on the forward primer and a HindIII site on the reverse primer to aid in directional cloning of the amplified DNA into the pMIR-REPORT^TM vector (Applied Biosystems). The orientation of the inserted fragment was confirmed by restriction enzyme digestion and sequencing.
Deletion primers and the QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies; Santa Clara, CA) was used to delete miR-363 binding site 1 (BS1) (chr2:192,288,731-192,288,738) or binding site 2 (BS2) (chr2: 192,289,618-192,289,625) from the 3’UTR of the MYO1B gene cloned into the pMIR-REPORT^TM vector (Applied Biosystems). BS1 was deleted using the forward primer 5’-CTACTTTCATGGACTTGTTCCTTTGTAATA-TGGTTTTGTTTTATTTGGGGTTCATTGTATG-3’ and the reverse primer 5’-CATACAATGAACCCCAAATAAAACAAAACCA-TATTACAAAGGAACAAGTCCATGAAAGTAG-3’. BS2 was deleted using the forward primer 5’-CCATTCAGATAGCAGTAAAACATTCTGTATGAT-AAACATCCAAGATCTTTTTTGAAAG-3’ and the reverse primer 5’-CTTTCAAAAAAGATCTTGGATGTTT-ATCATACAGAATGTTTTACTGCTATCTGAATGG-3’. Deletion mutants were confirmed by restriction enzyme digestion and DNA sequencing.

Hela cells were transfected with DNA plasmids (pLVX-DsRed, pMIR-REPORT, pRL-CMV) using Rotifect+ (Carl Roth CL21.2) and processed according to the Dual-Luciferase Reporter Assay protocol (Promega E1910). Pre-miR-502 was expressed from pLVX-DsRed-Monomer-C1 vector (Clontech) cloned downstream of the dsRED gene and 3′UTRs of Ku70, Ku80, LIG4 and XLF were fused downstream to the firefly luciferase gene in the pMIR-REPORT vector (Ambion). pRL-CMV vector was cotransfected to account for transfection efficiency and cell death (Promega).

The fragments, 3′‐UTRs of HMGB1, ATG9A, and ATG4B contained the wild‐type (wt) and the mutant (mut) binding sites of miR‐34a, were cloned into the downstream of firefly luciferase coding gene in the pMIR‐Report vector (Ambion Inc). The constructed luciferase reporters were called as pMIR‐LUC‐3′‐UTR‐WT HMGB1 and pMIR‐LUC‐3′‐UTR‐HMGB1‐MUT, pMIR‐LUC‐3′‐UTR‐WT ATG9A and pMIR‐LUC‐3′‐UTR‐ATG9A‐MUT, and pMIR‐LUC‐3′‐UTR‐WT ATG4B and pMIR‐LUC‐3′‐UTR‐ATG4B‐MUT. For luciferase assay, HCT8 cells (1 × 10⁵ cells/well) were seeded in 24‐well plates, miR‐34a mimic or mimic NC (100 nmol/L) was co‐transfected with reporter plasmids (100 ng) using Lipofectamine 3000. After 48 hours, the luciferase activities were measured using the Dual Luciferase kit (Promega). Cells transfected with 100 ng pMIR‐control vector were used as blank and Renilla luciferase was used as control.

Genomic DNA was isolated from the B7-H1 expressing melanoma cell line UKRV-Mel-14a using the QIAamp DNA Mini Kit (Qiagen) according the manufacturers’ protocol. The B7-H1 promoter was amplified by PCR with Taq DNA polymerase Kit (Invitrogen) employing the forward primer 5′-AAAGGTACCTAGAAGTTCAGCGCGGGATA-3′ and the reverse primer 5′-AAAGGATCCCAGCGAGCTAGCCAGAGATA-3′. The specific PCR product was purified and cloned into the pMiR REPORT vector (Ambion, Austin, Texas, USA) using the restriction enzymes KpnI and BamHI (Fermentas) replacing the CMV promoter as recently described [23 (link)]. For replacing the luciferase (luc) reporter gene by the red fluorescent m-cherry protein, the m-cherry sequence was amplified from the pmR-m-cherry vector (Clontech, Mountain View, CA, USA) applying the forward primer 5′-AAAGGATCCATGGTGAGCAAGGGCGAGGA-3′ and the reverse primer 5′-AATGTGGTATGGCTGATTAT-3′. The PCR product was digested with BamHI (Fermentas) and SpeI (NEB, Ipswich, MA, USA) and cloned behind the B7-H1 promoter sequence in the pMiR REPORT backbone replacing the luciferase gene. The plasmid map is shown in Additional file 1: Figure S1.