The human pri-miR-31 sequence containing miR-31 pre-miRNA and its flanking sequences on both sides was amplified from genomic DNA using the primers listed in Supplementary Table 1 , and was subsequently cloned into pcDNA3.0 vector and lentivirus shuttle vector plenti6 (Invitrogen). For knockdown of miR-31 expression, tough decoy (TuD) RNA against miR-31 (5′-CGggatccGACGGCGCTAGGATCATC AACAGCTATGCCAGATCTCATCTTGCCTCAAGTATTCTGGTCACAGAATACAACAGCTATGCCAGATCTCATCTTGCCTCAAGATGATCCTAGCGCCGTCTTTTTTctcgagCGG-3′) were designed according to reference [25 (link)]. The TuD sequence was cloned into lentiviral shuttle vector plenti6-U6 vector.
For miR-31 sensor vector, the 3′-UTR of ISL1 gene including potential miR-31 binding sites was amplified from cDNAs prepared from Hep-12 cells, and the corresponding mutant 3′-UTRs were obtained by overlap-extension PCR. These amplification products were subsequently cloned into the downstream of firefly luciferase gene of the pGL3-control plasmid (Promega, Madison, WI).
Lentiviral constructs were transfected with the ViraPower Packaging Mix (Invitrogen) into 293FT cells to generate lentivirus. Cells infected with virus are selected by 5 μg/ml blasticidin (Invitrogen). The pool of antibiotic-resistance cells were used for subsequent assay.
For miR-31 sensor vector, the 3′-UTR of ISL1 gene including potential miR-31 binding sites was amplified from cDNAs prepared from Hep-12 cells, and the corresponding mutant 3′-UTRs were obtained by overlap-extension PCR. These amplification products were subsequently cloned into the downstream of firefly luciferase gene of the pGL3-control plasmid (Promega, Madison, WI).
Lentiviral constructs were transfected with the ViraPower Packaging Mix (Invitrogen) into 293FT cells to generate lentivirus. Cells infected with virus are selected by 5 μg/ml blasticidin (Invitrogen). The pool of antibiotic-resistance cells were used for subsequent assay.
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