Pmirglo luciferase reporter

The wild types of SMAD7-3′UTR, including the binding bite of miR-581 and the site-directed mutant type, were synthesized and inserted into the pmiR-GLO™ luciferase reporter plasmid (Promega, Madison, WI, USA). The WT, or MUT vector, was co-transfected with miR-581 mimics or negative controls in 24-well plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h, the cells were harvested and assayed by a Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) according to the manuals.

The HUVEC cells underwent 24 h of treatment with 50 μg/ml of ox-LDL that was harvested for luciferase assay and RIP assay. In order to produce the SNHG1-WT, vectors of pmirGLO luciferase reporter (Promega, Madison, WI) were utilized to insert the sequence of SNHG1 flanking the complementary sequence encompassing miR-556-5p element. At the same time, SNHG1-Mut was acquired by the mutation of the sequences complementary to the seed region of miR-556-5p. Besides, the 3′UTR sequences of GNAI2 and PCBP1 as well as their mutated sequences without miR-556-5p binding sites were acquired and inserted to pmirGLO vectors for luciferase assays. After the cotransfection was completed, luciferase reporter system (Promega) was used to analyze the luciferase activity of indicated HUVEC cells. With regard to RIP assay, HUVEC cells were lysed in RIP lysis buffer containing proteinase inhibitors and RNase inhibitors. Magna RNA immunoprecipitation kit (Millipore, Bedford, MA) was applied to capture RNAs that could be precipitated by anti-Ago2 in different HUVEC cells. Then qRT-PCR was applied to estimate the enrichment of precipitated RNAs.

Luciferase reporter assay was to proceed as previously described [8 (link)]. The wild-type or mutated HMMR-AS1 fragment and HMGA2 3’-UTR fragment were constructed into the PmirGLO luciferase reporter (Promega, USA). The reporter plasmid and Renilla luciferase (hRlucneo) control plasmid were co-transfected into cells with either miR-627-3p mimic or miR-NC in a 12-well plate (1 × 10^5 cells/well) using Lipofectamine 3000 reagent (Invitrogen, USA). Forty-eight-hour post transfection, the relative luciferase activities were measured by Dual-Luciferase Reporter Assay Kit (Promega, USA) on a luminescence microplate reader.