The starBase v2.0 (http://starbase.sysu.edu.cn/ ) was used in the bioinformatics analysis to predict the binding sites between GATA3-AS1 and miR-30b-5p, and between miR-30b-5p and Tex10 (15 (link)). The sequences of GATA3-AS1 wild-type (wt, chr10:8,092,655-8,092,876) and Tex10 wt (chr9:103,064,259-103,064,483), as well as their corresponding mutant sequences were synthesized and inserted into the pGL3 vectors by Shanghai Sangon Biotech Co., Ltd., and named as pGL3-GATA3-AS1 wt, pGL3-GATA3-AS1 mut, pGL3-Tex10 wt, and pGL3-Tex10 mut plasmids, respectively. Cells (4×104) were co-transfected with indicated pGL3 plasmids and miR-30b-5p mimic or mimic NC, along with pRL-TK plasmids (Promega Corporation), using Lipofectamine 2000 according to the manufacturer's instructions. The luciferase activity was monitored 48 h post transfection using Dual-Glo Luciferase assay (Promega Corporation) according to the manufacturer's instructions. The relative firefly luciferase activity was normalized to Renilla luciferase activity.
Protocol detail
Bioinformatic Analysis of lncRNA-miRNA-mRNA Interactions
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Assay
Binding sites
Cells
Firefly luciferase
Gata3
Lipofectamine 2000
Luciferase
Pgl3
Plasmids
Promega
Renilla luciferase
Tex10
Transfection
Vectors
Corresponding Organization :
Other organizations : First Affiliated Hospital of Xi'an Jiaotong University
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Variable analysis
independent variables
- Transfection with miR-30b-5p mimic or mimic NC
- Luciferase activity
- Co-transfection with pRL-TK plasmids (Promega Corporation)
- Co-transfection with indicated pGL3 plasmids (pGL3-GATA3-AS1 wt, pGL3-GATA3-AS1 mut, pGL3-Tex10 wt, and pGL3-Tex10 mut)
- Positive control: pRL-TK plasmids (Promega Corporation)
- Negative control: mimic NC
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