Psicheck 2 dual luciferase reporter vector

The psiCheck-2 dual-luciferase reporter vector (Promega) harboring the 3′-untranslated region (UTR) of MAVS was inserted into the XhoI and PmeI restriction sites at the 3′-end of Renilla, and was used to check the effect of miR-22 on Renilla luciferase activity. The 3′-UTR and the coding region of MAVS were amplified from U251 cell genomic DNA with specific primers and cloned into psiCheck-2 and pCDNA4, respectively (Life Technologies). The psiCheck-2 mutant MAVS 3′-UTR construct was generated by inducing point mutations with overlap-extension PCR method. Table 2 lists all primer sequences. All constructs were verified with sequencing.

Eight miR-205 candidate target genes were selected using TargetScan and miRWalk 2.0 databases combined with mRNA-seq data, which were BTBD3, CADM1, HS3ST1, NAA25, SLC35B3, SRSF10, and RAP2B. The 3′-UTR of target genes containing wild-type (WT) or mutant (Mut) in the seed region were cloned (the primers are presented in Supplementary Table S2). All amplified sequences were ligated to the psi-CHECK2 dual-luciferase reporter vector (Promega, Madison, AL, United States) using T4 DNA ligase (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Sequencing was performed for validating all the constructs.
All the luciferase reporter plasmid vectors were transfected into HeLa cells by Lipofectamine 2000 along with miR-205 mimics or NC. The Renilla and Firefly luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega, Madison, AL, United States). All the experiments were performed in triplicates at least. The Renilla luciferase activity was employed for normalizing the signal value.

Full-length 3′-UTRs of the miR-138-regulated targets ABCA13, EZH2 and Sox4 were amplified from human genomic DNA and individually cloned into psiCHECK-2 dual luciferase reporter vector (Promega). After transfection for 48 h, the ratio of Renilla to firefly luciferase was measured with the dual luciferase assay (Promega). All transfection and reporter assays were performed in triplicate.

FZD3, CaMKIIδ gene involving Wnt signal pathway and FRS2 gene in FGF signal pathway were considered as the potential targets of chi-miR-30b-5p by TargetScan software. The FZD3–3’UTR, CaMKIIδ-3’UTR and FRS2–3’UTR sequences including chi-miR-30b-5p binding site were amplified by PCR, then the amplified fragments and psi-CHECK-2 dual-luciferase reporter vector (Promega, Madison, WI, USA) were digested by Xho I/Bsa I and Not I enzymes (NEB, New England) and subsequently ligated by T4 DNA ligase (NEB, New England). The constructed vectors were further verified by sequencing. The PCR primer information was listed in Additional file 8.

The psi-CHECK™-2 dual-luciferase reporter vector (Promega, Madison, WI, USA) harboring the wild-type and mutant YWHAZ 3′-UTR, which were inserted into the Xho I and Not I restriction sites 3′ to the end of the Renilla gene, were used to check the effect of gga-miR-451 on Renilla activity. The full length of the wild-type YWHAZ 3′-UTR or fragments covering the putative gga-miR-451-binding site were amplified by RT-PCR following cDNA extraction from the lung tissues of chicken. The psi-CHECK™-2 mutant YWHAZ 3′-UTR construct was generated by inducing a point mutation using the overlap extension PCR method. The recombinant wild-type and mutant plasmids were named Luc-WT-YWHAZ and Luc-Mut-YWHAZ, respectively. The primers are listed in Table 1. All constructs were verified by sequencing.

The pcDNA3 EGFP and pcDNA3 turboGFP were constructed by substituting the neomycin gene of pcDNA3 (Invitrogen, Carlsbad, CA, USA) with the EGFP or turboGFP genes, respectively. The pri-miR-155 (FL-BIC), obtained from A. van den Berg and described in [18 (link)], and pri-miR-497 (isolated from BJ-hTert) were cloned in the pcDNA3 EGFP. The pri-miR-155 and pri-miR-497 containing the sORFs fused in frame with EGFP were cloned in the pcDNA3 turboGFP. The miPEPs expression constructs were obtained by cloning the sORFs placed downstream from a Kozak consensus sequence in the pIRES2 turboGFP (constructed by replacing the EGFP gene of pIRES2 EGFP vector—from Clontech—with the turboGFP gene) (for miPEP155) or pcDNA3 EGFP (for miPEP497 and miPEP200a). The miR sensor plasmids were constructed by cloning three repeat reverse complementary sequences corresponding to miR-155-5p or miR-497-5p or miR-200a-3p or miR-200b-3p (synthesized by Eurofins Genomics, Ebersberg, Germany) into the end of Renilla luciferase of the psiCHECK-2 dual luciferase reporter vector (Promega, Madison, WI, USA). The hsa-miR-155-5p, hsa-miR-497-5p, hsa-miR-200a-3p, and hsa-miR-200b-3p pre-miR miRNA precursors, pre-miR negative control #2, hsa-miR-155-5p, hsa-miR-497-5p miRVana miRNA inhibitors, and anti-miR negative control #1 were purchased from Ambion (Thermo Fisher Scientific).