Pgl3 control vector

The targets of circ_0006916 were predicted via CircInteractome (http://circinteractome.nia.nih.gov/),26 (link) and the targets of miR-599 were predicted by starBase (http://starbase.sysu.edu.cn/).27 (link) The dual-luciferase reporter and RIP analyses were carried out to explore the target correlation of miR-599 and circ_0006916 or SRSF2. In brief, the wild-type (circ_0006916 WT or SRSF2-3ʹUTR WT) or mutant luciferase reporter vectors (circ_0006916 MUT or SRSF2-3ʹUTR MUT) were generated via inserting the corresponding sequence containing wild-type or mutant miR-599 complementary sites in luciferase gene downstream in PGL3-Control vectors (Promega, Madison, WI, USA) via endonuclease site Xba I. These constructed vectors, control vectors and miR-599 mimic or miRNA NC were transfected into Huh7 and SNU-387 cells for 24 h. The luciferase activity was measured via a dual-luciferase analysis kit (Promega) and GloMax 20/20 Luminometer (Promega).
RIP analysis was conducted with a Magna RIP kit (Sigma, St. Louis, MO, USA). 1 × 10⁷ Huh7 and SNU-387 cells were lysed and interacted with magnetic beads conjugated via anti-Ago2 for 4 h. Next, The RNA enriched on the beads was extracted and used for the detection of circ_0006916, miR-599 and SRSF2 levels. IgG was employed as a negative control, and input functioned as a positive control.

The control mimic, miR-7 mimic, control inhibitor, and miR-7 inhibitor were purchased from GenePharma (Shanghai, China) and transfected into MCF-7 and MDA-MB-231 cells using the Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocols (final concentration: 100 nM). At 48 hrs after transfection, cells were collected for measuring miR-7 expression or used for further experiments. For luciferase reporter assay, 3ʹ-UTR fragment of MRP1 and BCL2 was cloned into the pGL3-control vectors (Promega). The mutant construct was developed by using the Phusion Site-Directed Mutagenesis Kit (ThermoFisher Scientific). HEK293 cells were seeded in 24-well plates and co-transfected firefly luciferase reporter vector, control Renilla pRL-TK vector (Promega) with control mimic, miR-7 mimic, control inhibitor, or miR-7 inhibitor using the Lipofectamine 2000. At 48 hrs after transfection, cells were lysed and the activity of firefly and Renilla luciferase was assessed using the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Each treatment was performed with 4 replicates, and firefly luciferase activity was normalized to Renilla luciferase activity for each well.

The 3′untranslated region (UTR) sequences of MMP-2 containing wide-type or mutant-type miR-34a binding sites were amplified by PCR into pGL3-control vectors (Promega Corporation). The constructed reporter vector (100 ng of MMP-2 plasmid and 100 ng of miR-34a mimics) was cotransfected into U251 cells (1×10⁶ cell/ml) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Luciferase activity was detected using dual-luciferase reporter assay system (Promega Corporation) after 48 h. miR-34a was predicted to target the 3′-UTR of MMP-9 using TargetScan software version 7.1 (http://www.targetscan.org). Luciferase activity was normalized against Renilla luciferase.

Partial fragments of PPAR-γ 3′UTR containing predicted miR-128 binding sites were amplified by PCR and subcloned into pGL3-control vectors (Promega, Madison, WI, USA) to generate wild-type (WT) PPAR-γ reporter. Moreover, mutant type (MUT) PPAR-γ reporter with mutant miR-128 binding sites was produced using Quickchage Multi Site-Directed Mutagenesis kit (Stratagene, Lajolla, CA, USA). Next, constructed WT-PPAR-γ or MUT-PPAR-γ reporter was co-transfected into MCN and N2a cells together with control Renilla luciferase pRL-TK vectors (Promega) and miR-Control or miR-128. At 48 h after transfection, relative luciferase activity was determined via dualluciferase reporter assay (Promega).