Pgl3 vector

NEAT1-mutant and BCL2L11-mutant plasmids were designed and purchased from GeneScript (Nanjing, China). The 3′-UTR of lncRNA NEAT1 and BCL2L11 cRNA fragments containing the potential binding sequences of miR-29a sites were amplified from PCR and inverted into pGL3 vector (Ambion, Inc., Austin, TX, USA).
HEK-293 cells were cultured in 24-well plates and co-transfected with vectors and miR-29a mimics or miR-nc. Afterward, cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific., Inc, Waltham, MA, USA). Finally, the luciferase activities were detected using a dual-luciferase reporter assay system (Promega, Madison, WI) following the manufacturer’s instructions.

Prediction of potential miR-152 target genes was performed using the four public bioinformatics algorithms: miRanda, TargetScan, miRBase, and PicTar. One of the identified possible targets, namely, E2F3, was chosen for experimental verification of its ability to bind miR-152 via luciferase assays. The human E2F3-3′-UTR, containing the putative miR-152 binding site, was amplified and subcloned into the pGL3 vector (Ambion, Austin, TX, USA) and named as WT-E2F3-3′-UTR. A mutated E2F3-3′-UTR with an altered binding sequence was created using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Dallas, TX, USA), referred to as MT-E2F3-3′-UTR. For the luciferase reporter assays, U2OS cells were cotransfected with an oligo (miR-152 or miR-NC) and one of the two reporter plasmids (WT-E2F3-3′-UTR or MT-E2F3-3′-UTR), using Lipofectamine 2000 according to the manufacturer’s protocol. Forty-eight hours after cotransfection, cells were lysed, and the luciferase activity was determined using the Dual-Luciferase Reporter Assay Kit (Promega) per the manufacturer’s protocol. Renilla luciferase activity was normalized to that of firefly luciferase.

The wild type of LPL 3′-untranslated regions fragment (LPL WT 3′UTR) containing the potential target site of miR-138 at position 1481–1503 was mutated. Then, LPL WT 3′UTR and its mutant (LPL MUT 3′UTR) were cloned into pGL3 vector (Invitrogen), respectively. The LPL MUT 3′UTR was constructed by replacing the UGAUU with the complementary sequence ACUAA in the LPL sequence. 293T cells were transfected with LPL WT 3′UTR+miR-NC mimics, LPL WT 3′UTR+miR-138 mimics, LPL MUT 3′UTR+miR-NC mimics, LPL MUT 3′UTR+miR-138 mimics, LPL WT 3′UTR+anti-miR-NC, LPL WT 3′UTR+anti-miR-138, LPL MUT 3′UTR+anti-miR-NC, or LPL MUT 3′UTR+anti-miR-138. After 48-h incubation, cells were collected to measure luciferase activity using the dual-luciferase reporter assay system (Promega, Madison, WI, USA).

DianaTools (Diana Lab, University of Thessaly, Thessaly, Greece) with microT-CDS algorithm (33 (link)) determined that there was a potential target binding site for miR-23a-3p in the 3'untranslated region (3'UTR) of HMGB1. For determination, the fragment of the HMGB1 3'UTR containing the putative sequence (positions 649-669) was mutated by replacing the AAU…GC…UGUGAU of the complementary sequence. Then, the wild type and mutant of HMGB1 3'UTR (HMGB1 WT/MUT) were cloned into a pGL3 vector (Invitrogen; Thermo Fisher Scientific, Inc.). RAW264.7 cells were co-transfected with HMGB1 WT/MUT (2 µg) and miR-23a-3p/NC mimic (30 nM), or co-transfected with HMGB1 WT/MUT (2 µg) and in-miR-23a-3p/NC (30 nM) using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). pRL-TK plasmids were used as an internal control and co-transfected with those at a dose of 50 ng. Every transfection group was carried out in triplicate. After 48 h, transfected cells were collected to measure Firefly and Renilla luciferase activity using a dual-luciferase reporter assay system (Promega Corporation), and relative luciferase activity was the ratio of Firefly and Renilla.

Luciferase assays were implemented by adopting the Dual-Luciferase Reporter Assay System (Promega Corporation) in accordance with the manufacturer's instructions. The wild type (WT) or mutant (MT) SOCS1 3′-UTR sequences including the miR-155 targeting site were inserted into the pGL3 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to construct pGL3-luc-SOCS1-WT and pGL3-luc-SOCS1-MT. AML12 cells were transfected with 50 ng pGL3-luc-SOCS1-WT/MT, 5 pmol miR-155 mimic or NC mimics (control,) and 5 ng Renilla luciferase using adopting Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and seeded in a 96-well plate. The luciferase activities were detected 48 h post-transfection on a GloMax system (Promega Corporation).