Detection of the targeting relationship between TRAF6 and miR-146-5p by dual luciferase reporter system

doi:10.3969/j.issn.2095-4344.1392

Abstract

Abstract:

BACKGROUND: Preliminary study has found that miR-146-5p is up-regulated in osteobIasts induced by sodium fIuoride.
OBJECTIVE: To verify the targeting relationship between miR-146-5p and its potential target gene TNF receptor associated factor 6 (TRAF6) using a dual luciferase reporter gene through constructing a luciferase reporter plasmid for the 3' non-coding region (3'UTR) of TRAF6 gene.
METHODS: Bioinformatics methods were used to predict the binding sites of miR-146-5p and TRAF6 genes. The 3'UTR fragment of TRAF6 gene was amplified by PCR and cloned into pYr-MirTarget vector to construct a wild type recombinant dual-luciferase reporter plasmid. There were six groups: (1) 6a-5p-nucleoside analogs + TRAF6-W 3'UTR co-transfection group; (2) non-nucleoside analogs mimics + TRAF6-W 3'UTR co-transfection group (control group); (3) miR-146a-5p inhibitor + TRAF6-W group; (4) TRAF6-W 3'UTR co-transfection group; (5) empty pYr-MirTarget transfection group; (6) normal cell group. The co-transfection of recombinant luciferase reporter plasmid and miR-146-5p or nucleoside analogs (mimics) with Saos-2 was made, respectively. At the same time, nucleoside analogs mimics (control group), miR-146-5p inhibitor (negative control group) and empty pYr-MirTarget-W 3'UTR and TRAF6-W 3'UTR were added simultaneously to detect luciferase activity in six groups of cells. Mimics, miR-146-5p inhibitor and nucleoside analogs mimics were transfected to the human osteoblast Saos-2 respectively. The protein expression level was detected by western blot assay after lysing cells to extract protein.
RESULTS AND CONCLUSION: (1) Luciferase activity in Saos-2 cells co-transfected with miR-146-5p mimics was 10.103 0±0.558 5; the control group’s (NC mimics-TRAF6 3'UTR) luciferase activity was 13.140 0±0.720 4; the inhibitor group's luciferase activity was 13.707 1±0.434 8; and luciferase activity in Saos-2 cells of the wild-type TRAF6 3'UTR plasmid was 13.202 1±0.456 5. The luciferase activity of the cells transfected with the empty plasmid alone was 14.706 2±0.441 6. The original luciferase activity in Saos-2 cells was 1.126 4±0.126 2. The number of miR-146-5p mimics co-transfected significantly decreased (F=715.789, P < 0.000 1). The other three groups were compared with the control group, showing no significant difference (P > 0.05). (2) Western blot assay showed that compared with the control group, the expression level of TRAF6 protein was significantly down-regulated after transfection of miR-146-5p mimics. (3) To conclude, there is a targeted relationship between TRAF6 and miR-146-5p.

Key words: microRNA, tumor necrosis family, dual luciferase, TRAF6 gene, luciferase

CLC Number:

Zhang Yalou1, Deng Qiang2, Zhou Yangjunjie2, Zhao Yang1, Guo Qiong1, Jiang Xiangju3, Yue Mingming1, Chen Long1, Ma Wenjing4. Detection of the targeting relationship between TRAF6 and miR-146-5p by dual luciferase reporter system [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(27): 4397-4401.

Figures/Tables 2

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