Identifying miR-93 Targets in TLR4 Regulation

miRNA target prediction tools, including miRanda (https://miranda.org.uk/) and TargetScan Release 7.0 (http://targetscan.org/) were used to search for the putative targets of miR-93. The dual-luciferase reporter assay was performed as described previously (32 (link)). Briefly, partial sequences of TLR4 3'-UTR containing miR-93 binding sites was amplified by PCR and cloned into the dual-luciferase reporter vector (pmirGLO; Promega Corporation) (wild-type pmirGLO-TLR4-3'-UTR, wt). Mutation of the predicted binding site (mutation pmirGLO-TLR4-mut-3'-UTR) was generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.) according to the manufacturer's instructions. RAW264.7 cells were co-transfected with miR-93 mimics, miR-93 inhibitor and the luciferase reporter plasmids using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, luciferase activities were detected using the dual luciferase reporter kit (Beyotime Institute of Biotechnology) with Renilla luciferase activity as an internal control.

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Gao H., Xiao D., Gao L, & Li X. (2020). MicroRNA-93 contributes to the suppression of lung inflammatory responses in LPS-induced acute lung injury in mice via the TLR4/MyD88/NF-κB signaling pathway. International Journal of Molecular Medicine, 46(2), 561-570.

Publication 2020

pmirGLO QuikChange Site-Directed Mutagenesis kit Lipofectamine 2000 dual luciferase reporter kit

Assay Binding site Lipofectamine 2000 Luciferase Mirna Mutation Plasmids Promega Raw264 7 cells Renilla luciferase Site directed mutagenesis Transfection Vector

Corresponding Organization :

Other organizations : Sichuan University, West China Second University Hospital of Sichuan University

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Variable analysis

independent variables

MiR-93 mimics
MiR-93 inhibitor

dependent variables

Luciferase activities

control variables

Renilla luciferase activity

controls

Positive control: wild-type pmirGLO-TLR4-3'-UTR (wt)
Negative control: mutation pmirGLO-TLR4-mut-3'-UTR

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