Pmirglo e1330

The mouse FAIM proximal promoter genomic region (NC_000075.7:98866745-98868446 as described in the National Center for Biotechnology Information database, −1681 to +20, −520 to +20 and the CRE element mutant promoter-reporter constructs −1681 to +20) were inserted upstream of a luciferase reporter gene with a flanking sequence into the BglII and apaI-digested pmirGLO (E1330, Promega) vector to generate pmirGLO-FAIM (WT, mutant, truncated), respectively. The coding region of the mouse Creb1 gene (Mus musculus, transcript variant C, NM_001037726.1) was cloned into the pcDNA3.1(+) vector (pcDNA3.1(+)-CREB). The plasmids were constructed and sequenced by Genewiz (Suzhou, CHINA). AML12 were transfected with FAIM promoter-luciferase reporter plasmids and pcDNA3.1(+)-CREB vector using Lipofectamine 3000 according to the manufacturer’s instructions. In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 μM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17-6, Selleck Chemicals, USA). Cells then lysed in 100 μl lysis buffer and detected using the Dual-Luciferase Reporter Assay System (E1910, Promega). Renilla luciferase activity was used as a control.

The bioinformatics website TargetScan 7.2 (www.targetscan.org) was used to identify COPB2 as the target gene of miR-335-3p. For the luciferase reporter assay, the cDNAs of wild-type COPB2-3−UTR (5−-TTTCCTTTCTCAATAATGAAAAT-3−) and COPB2-3−UTR-mut (5−-TTTCCTTTCTCAATATACTTTTT-3−) were synthesized by TsingKe Biotech Co., Ltd. (Beijing, China) and cloned into the luciferase reporter gene vector pmirGLO (E1330) (Promega, Madison, WI, USA) to construct the COPB2-3−-UTR plasmid and the COPB2-3−-UTR-mut plasmid. Firefly luciferase was used as the reporter for miRNA regulation of the 3−UTR. Briefly, the NCI-H1975 cells were maintained in 96-well plates and co-transfected with a luciferase reporter plasmid (0.05 μg/well) and mimic (5 pmol). Cells were divided into four groups: cells transfected with control mimic and COPB2-3−UTR plasmid; cells transfected with miR-335-3p mimic COPB2-3−UTR plasmid; cells transfected with miR-335-3p mimic and COPB2-3−UTR-mut plasmid; and cells transfected with control mimic and COPB2-3−UTR-mut plasmid. After incubation at 37°C for 72 h, the firefly luciferase activity was measured by using the luciferase reporter assay system (N1610) (Promega, Madison, WI, USA) with a GloMax Discover System (GM3000) (Promega, Madison, WI, USA). The relative firefly reporter activity was calculated through normalization to the Renilla luciferase control.