Dual lucy assay kit

The SMAD4 3’UTR wild-type (WT) or mutant (MUT) with miRNA-182-5p binding sequences was amplified and cloned into the pHS-AVC-ZQ480 and pHS-AVC-ZQ481 luciferase reporter vector to synthesize SMAD4-WT or -MUT luciferase reporter vector by Beijing Syngentech Co., LTD. TransIntro EL transfection reagent was used to co-transfect the HFs with the respective WT or MUT vectors (FT201, TransGen). Following 48 hr of transfection, the Dual-Lucy Assay Kit (D0010, Solarbio) was used to measure luciferase activity using a microplate reader (Synergy2, BioTek) according to the manufacturer’s recommendations.

The potential target of circ_0007142 was searched by starBase v3.0 (http://starbase.sysu.edu.cn/). Also, the putative candidate target of miR-122-5p was predicted by starBase v3.0. The wild type (containing the complementary binding sites) and mutant sequences of circ_0007142 and 3ʹ-untranslated regions (3ʹUTR) of CDC25A were inserted into pmirGLO vector (Promega, Madison, WI, USA) to construct luciferase reporter. The luciferase reporter and miR-122-5p or miR-NC was co-transfected into HT-29 and HCT-116 cells. The luciferase activity was detected using Dual-Lucy Assay Kit (Solarbio, Beijing, China). This test was performed according to the previous description.21 (link)

The fragments of circFIG 4 or E2F3 3′UTR possessing the presumptive or mutant target sites of miR‐493‐5p were severally introduced into pmirGLO vector (Promega), forming wild‐type circFIG 4 or E2F3 (circFIG 4 WT or E2F3 3′UTR WT) and mutant‐type circFIG 4 or E2F3 (circFIG 4 MUT or E2F3 3′UTR MUT) luciferase reporter vectors. Subsequently, the constructed reporter vectors and miR‐NC or miR‐493‐5p were transfected into EC cells. Forty‐eight hours later, the luciferase intensity was tested via a Dual‐Lucy Assay Kit (Solarbio).

The interaction between miR-325-3p and MEG3 or TRPV4 was predicted using LncBase Predicted (version 2; carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-predicted) or Targets can Human 7.1 (www.targetscan.org) online databases, respectively. The wild-type (WT) and mutant (MUT) sequences of MEG3 or the 3′-untranslated regions (3′-UTRs) of TRPV4 were inserted into the pmirGLO vector (Hunan YouBio Medical Device Co., Ltd.) to construct WT-MEG3, MUT-MEG3, WT-TRPV4-3′UTR and MUT-TRPV4-3′UTR luciferase reports, respectively. H9c2 cells (4×10⁵ cells/well) were co-transfected with WT-MEG3 (2 µg), MUT-MEG3 (2 µg), WT-TRPV4-3′UTR (2 µg) or MUT-TRPV4-3′UTR (2 µg) and miR-325-3p (25 nM at final concentration) or miR-NC (25 nM at final concentration) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were replaced with fresh medium 6 h after transfection before the hypoxia group received hypoxia treatment 24 h after transfection for subsequent experiments. Luciferase activities were detected 48 h after transfection using the Dual-Lucy Assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Renilla luciferase activities were normalized as control.

The fragment of circ-ATP5H or TNFAIP3 3ʹUTR containing miR-138-5p binding sites was cloned to pmirGLO vector (LMAI Bio) to generate WT-circ-ATP5H or TNFAIP3 3ʹUTR-WT. To form the mutant reporters (MUT-circ-ATP5H or TNFAIP3 3ʹUTR-MUT), the binding sequence was mutated. Then, the constructed plasmids were transfected into HBV-infected cells together with miR-NC or miR-138-5p. Final, the luciferase intensity was examined via Dual-Lucy Assay Kit (Solarbio).

The binding sequence between miR-145-5p and circANKRD11 or BRD4 was severally predicted by circinteractome (https://circinteractome.nia.nih.gov/api/v2/mirnasearch?circular_rna_query=hsa_circ_0040929&mirna_query=&submit=miRNA+Target+Search) and MicroT_CDS online databases (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/results&keywords=hsa-miR-145-5p%20%20%20ENSG00000141867&genes=ENSG00000141867%20&mirnas=hsa-miR-145-5p%20&descr=&threshold=0.1&page=1). Built luciferase reporter plasmids were mixed with miR-145-5p or NC, and transfected into HPMECs with TurboFect reagent (Thermo Fisher). At the second day after transfection, cells were collected and firefly luciferase activity was quantified by Dual-Lucy Assay Kit (Solarbio) with Renilla luciferase activity as a control.

Wild-type (WT) or mutant-type (MUT) sequences of hsa_circ_0012634 or HIPK2 3’UTR were cloned into the pmirGLO vector to construct WT/MUT-hsa_circ_0012634 or WT/MUT-HIPK2 3’UTR vectors. The indicated vectors were transfected into PDAC cells with miR-147b mimics or NC mimics. After 48 h, Dual-Lucy Assay Kit (Solarbio) was used to examine luciferase activity.