Pmir rb report vector

The DNA sequences of JAG1, transforming growth factor beta 2 (TGFB2) and SRY‐box 6 (SOX6) 3′‐UTR, were amplified by PCR using HEK293T genomic DNA as a template. The amplified DNA sequences were inserted into pmiR‐RB‐REPORT™ vectors (RiboBio) to generate wild‐type (WT) JAG1, TGFB2 and SOX6 3′‐UTR, with a mutated (MUT) JAG1 3′‐UTR luciferase vector generated using site‐directed mutagenesis. For the reporter assay, HEK293T cells were cultured in a 96‐well plate with 1.5 × 10⁴ cells/well in 100 μl of culture medium/well for 24 hrs. Cells were then co‐transfected with 50 nM miR‐199b‐5p mimic and 100 ng of vector per well and cultured in fresh medium for an additional 48 hrs. The luciferase reporter assay was carried out using the Dual‐Glo^® Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions, and luminescence was quantified using a Veritas™ 9100‐002 luminometer (Promega).

The binding site of miR-384 and Smad5 mRNA was predicted on StarBase (http://starbase.sysu.edu.cn/). Then the wide type (WT) sequence and the corresponding mutant type (MUT) sequence based on the predicted binding site between miR-384 and 3’UTR of Smad5 were inserted into the pMIR-RB-REPORT™ vectors (RiboBio) to construct pMIR-RB-REPORT^TM-Smad5-WT and Smad5-MUT vectors. Well-designed WT and MUT vectors were co-transfected with mimic control or miR-384 mimic into HEK-293T cells. Cells were lysed 48 hours later, and the relative luciferase activity was detected using a luciferase assay kit (RG005, Beyotime Biotechnology Co., Ltd. Shanghai, China).

The region of Mettl8 3′ UTR flanking the miR-208b binding site was amplified from mouse genomic DNA using PCR. The target sequence GGGAGCT (800–806 bp) was mutated to CCCTCGA using overlap PCR. Primers were showed in Supplementary Table S1. The PCR product was cloned into the vector downstream of the Renilla Luciferase open reading frame using the NotI and XhoI restriction sites. We obtained two pmiR-RB-REPORT vectors (RiboBio) with wide-type and mutant 3′ UTR of Mettl8.

LUESCC or 3′-UTR of NRSN2 sequence containing the putative miR-6785-5p binding site as well as its mutant form were designed, synthesized and inserted into pmiR-RB-Report™ vectors (RiboBio), which were named as LUESCC (WT)-luc, NRSN2 (WT)-luc, LUESCC (MT)-luc, and NRSN2 (MT)-luc, respectively. The luciferase reporter (50 ng), negative control miRNA mimic (miR-NC) or miRNA mimic (40 nM) were co-transfected into ESCC cells seeded in 48-well plates. The firefly luciferase activity was determined 48 h after transfection with renilla luciferase activity as an internal reference using a dual luciferase reporter assay system (Promega, USA) in line with the manufacturer’s instructions.