Pmir report mirna expression reporter vector system

To evaluate the binding efficiency of the top deregulated miRNAs onto their predicted targets, the pMiR-REPORT miRNA Expression Reporter Vector System (AMBION, Texas) was used. The AB8/13 undifferentiated podocyte cells were incubated at 33°C at 5% CO₂ and cultured in RPMI medium, supplemented with 10% Fetal Bovine Serum (FBS) (Invitrogen, CA), 1% of 100 units/ml Penicillin/Streptomycin (Invitrogen, CA) and 1% Insulin-Transferrin-Selenium (Invitrogen, CA). For the luciferase reporter system experiments, AB8/13 cells were transfected with equal amounts of the pMIR-REPORT luciferase and β-gal vectors (100 ng each), as well as with 25 nM of miRNA inhibitors, mimics, or the AllStars Negative Control scrambled sequence LNA (Qiagen, West Sussex, UK), using lipofectamine 2000 (Invitrogen, CA). The β-gal vector was used for normalization. Every experiment was performed in quadruplicates in 12-well cell culture plates with the appropriate controls. Cells were harvested 16 hours after transfection. The Dual-Light Assay Kit (Applied Biosystems, CA) was used for the quantification of both luciferase and β-gal in an automated luminometer (Sirius, Berthold Detection Systems, Pforzheim, Germany).

JHOC-9 and OVISE cells were seeded in triplicates (or greater) in 96-well plates at densities of 1 × 10⁴ and 9 × 10³ cells, respectively. After 24 h, Lipofectamine^® 2000 Transfection Reagent (Thermo Fisher Scientific) was used to co-transfect cells with an pMIR control luciferase vector, pMIR wild-type CDH1 3′-UTR luciferase vector (CDH1 3′-UTR wt), or pMIR mutant-type CDH1 3′-UTR luciferase vector (CDH1 3′-UTR mut; addgene, Cambridge, MA, USA) and pMIR-REPORT^™ β-gal Control Plasmid (Applied Biosystems). At 24 h of post-transfection, the activities of firefly luciferase and β-galactosidase were measured using the pMIR-REPORT^™ miRNA Expression Reporter Vector System (Applied Biosystems), according to the manufacturer’s instructions. All experiments were performed in triplicates, and values are presented as means ± SDs.

To identify the rno-miR-376a-binding site at the 3′-end of GRP78 mRNA, we generated a direct-match miRNA target site and cloned the insert into the multiple cloning site in the luciferase reporter vector from the pMIR-REPORT miRNA Expression Reporter Vector System (Applied Biosystems). The sense and antisense strands of the oligonucleotides were annealed by adding 2 µg of each oligonucleotide to 46 µL of annealing solution (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4 and 2 mM magnesium acetate) and incubating at 90°C for 5 min and then at 37°C for 1 h. The annealed oligonucleotides were digested with HindIII and SpeI and ligated into the HindIII and SpeI restriction sites of pMIR-REPORT vector. The sequences of the inserts were confirmed by sequence analysis using a PRISM 3100 genetic analyzer (Applied Biosystems).
The following oligonucleotides were used in this studies: (1) miR-376a,
5′-aatgcactagtACGAGGATTTTCCTCTACGATaagcttaatgc-3′ and
5′-gcattaagcttATCGTAGAGGAAAATCCTCGTactagtgcatt-3′, and
(2) rno-miR-376a-binding site sequence at the 3′-end of GRP78 mRNA,
5′-aatgcactagtATGGTAGAAAAAAGTTCCTACaagcttaatgc-3′ and
5′-gcattaagcttGTAGGAACTTTTTTCTACCATactagtgcatt-3′

The CCDC170 3' UTR containing the rs9383935 G allele was amplified by PCR from human genomic DNA carrying the GG homozygous genotype template with the following primers: sense 5'-AGACGCGTTAAGTCAGGGGCTTTACTAGC-3' and antisense 5'-GCAAGCTTCTGCTGAGTAGTTGGGATTACA-3'. The PCR products were separated in agarose gel, extracted, purified and cloned into the pMIR-REPORT™ miRNA expression reporter vector system (Applied Biosystems, Foster City, CA, USA) with MluI and HindIII digestion and then were ligated by T4 DNA ligase to the recombinant constructs (Additional file 2: Figure S2). The plasmid with the rs9383935 G allele was used as the template for the mutation G → A. The site-directed mutagenesis for the plasmid with the A allele construct was generated using a Mut Express Fast Mutagenesis kit (Vazyme Biotech, Nanjing, China). All PCR amplifications and constructs were sequenced to confirm the accuracy of cloning.

We transfected HEK293 cells with 200 ng of each vector (pMIR-REPORT luciferase vectors, as described above, and the pMIR-REPORT βgal vector as a control for transfection normalization) and 50 nM precursor or inhibitor using Lipofectamine 2000 Transfection Reagent according to the manufacturer's protocol. To measure the luciferase activity, we harvested the cells 24 h after transfection and conducted the assay using the pMIR-REPORT miRNA Expression Reporter Vector System (Applied Biosystems).