Abstract
Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA. Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers. A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments. Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively. HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor. Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups. Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression. Results Restriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully. The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+ mmu-miR-30b, pmirGLO+ mmu-miR-30b inhibitor, pmirGLO-mFoxO3+ mmu-miR-30b, pmirGLO-mFoxO3+ mmu-miR-30b+ mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+ mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively. Compared with pmirGLO+ mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+ mmu-miR-30b group (P 0.05). In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+ mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05). Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05). Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b. There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA. Key words: mmu-miR-30b; Mouse FoxO3; Target gene
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