Pgl3 control

The entire 3′ UTR of target gene Sus Scrofa cyclin E (XM_003127005) were amplified from porcine mRNA by RT-PCR, and then cloned into downstream of the luciferase open reading frame (ORF) in the vector pGL3-control (Invitrogen). The primers (CCNE F: 5′-GCTCTAGA CTGCAGCAGAGGCCTGCAT-3′ and CCNE R: 5′-GCTCTAGACCCTCAACGAACCCATACATAC-3′, and CCNE MF: 5′- GGACGACATCGTCTCTCCGTTTTTTAATAAAGATGACACTGTC-3′ and CCNE MR: 5′-ACGGAGAGACGATGTCGTCCTTACAAAACAATAGTTC CNE-3′) were used to create the plasmids pGL3-control-CCNE-3′ UTR (wild-type) and pGL3-control-CCNE M-3′ UTR (mutant), respectively. The single restriction enzyme cutting site is XbaI. The vector pRL-TK (Promega) was also used as the co-transfection vector for dual luciferase analysis experiments.

Prediction of miRNA target relied on the databases TargetScan (WWW.targetscan.org/) and miRanda (WWW.microrna.org/). The full length 3’UTR of HDAC6 was amplified by PCR from genomic DNA using specific primers (Supporting information: Tables C and D in S1 File) and cloned into pGEM-T cloning vector (Promega, Madison, WI, USA) then sub-cloned into pGL3 Control vector (Promega, Madison, WI, USA). The constructed vector was confirmed by DNA sequencing. Co-transfection of HDAC6 3’UTR/pGL3 Control and miR-22/pIRES-2-EGFP expression vectors was performed using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). After 48 h, luciferase activity was measured using a dual luciferase reporter assay system according to the manufacturer’s protocol (Promega, Madison, WI, USA).

A full length of the human HSD17B6 3′-untranslated region (449 bp) with the miR-31-5p targeting sequence was cloned downstream of the firefly luciferase gene in pGL3-control (Invitrogen) to construct pGL3-luc-HSD17B6. Then, the luciferase activity was determined as previously described [63 (link)].