Pmirglo dual luciferase microrna target expression vector

The 3′ UTR of MECP2 with the hsa-miR-1275 binding region was cloned and inserted into the pmirGLO Dual-Luciferase MicroRNA Target Expression Vector (Promega, USA). The mutant construct with deletion of the binding region of hsa-miR-1275 was an NTC. Primers used for PCR are shown in Table 5. 24 h after transfection with 2 μg of these constructions, the U251 cells were then cotransfected with hsa-miR-1275 mimics, inhibitor, and mimic NTCs. Cells were analyzed for luciferase activity after 48 h by the Luciferase Reporter Assay Kit (Promega, USA), and each construct was compared to that of the pmirGLO vector no-insert control. For each transfection, luciferase activity was averaged from three replicates.Table 5

hsa-miR-1275 Gene Ontology

Gene GO	Number of Genes	Percentage	p Value (%)	Fold Enrichment
GO: 0019226 ∼ nerve impulse conduction	43	8.29	0	3.90
GO: 0007268 ∼ synaptic transmission	35	6.74	0	3.73
GO: 0007267 ∼ intercellular signal	52	10.02	0	2.75
GO: 0016337 ∼ intercellular adhesion	28	5.39	0	3.22
GO: 0021700 ∼ maturation	16	3.08	0	5.03
GO: 0030182 ∼ neuronal differentiation	35	6.74	0	2.54

Luciferase reporter assay was used to validate the targeting relationships between miR-193a-5p and CASC9 or 3ʹ-UTR of ERBB2. The wild type (WT) CASC9 sequence or the WT 3ʹ-UTR fragment from ERBB2 mRNA including the predicted binding site of miR-193a-5p were amplified and inserted into the pmirGLO dual-luciferase microRNA target expression vector (Promega, Madison, WI, USA) to construct the report vector pmirGLO-CASC9-WT or pmirGLO-ERBB2-WT. The presumed binding sites of miR-193a-5p in CASC9 or ERBB2 3ʹ-UTR were mutated by GeneArt Site-Directed Mutagenesis PLUS System (cat. No. A14604; Thermo Fisher Science, Inc.). Mutant (MUT) CASC9 sequence or ERBB2 3′-UTR sequence were inserted into the pmirGLO vector to construct report vector pmirGLO-CASC9-MUT or pmirGLO-ERBB2-MUT. The corresponding reporter vectors and miR-193a-5p or NC mimics were co-transfected into SW480 and HCT116 cells and incubated for 48 hrs. Luciferase activity was then measured via Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).