The lncRNA-AK046375 Upregulates Metallothionein-2 by Sequestering miR-491-5p to Relieve the Brain Oxidative Stress Burden after Traumatic Brain Injury

Figure 2

AK046375 upregulates MT2 expression by acting as a “sponge” for miR-491-5p. (a) Predicted binding sites of miR-491-5p and miR-505-3p with MT2-mRNA-3-UTR and dual-luciferase reporter gene assay results verifying their binding activity (/group, , vs. the mimics-NC group by -test). (b) Expression of MT2 mRNA and MT2 protein levels in response to miR-491-5p-mimics or miR-491-5p-inhibitors in primary astrocytes (/group, , vs. the mimics-NC group, ^& vs. the inhibitors-NC group by one-way ANOVA). (d–f) Cell viability, membrane damage, and oxidative burden in astrocytes of each group (/group, , vs. the mimics-NC+H₂O₂ group, ^& vs. the inhibitors-NC+H₂O₂ group by one-way ANOVA,  μm, 200x). (g) Ago2 protein level detected by western blot analysis. Quantitative PCR analysis of AK046375 and miR-491-5p levels compared to the input (/group, , vs. the IgG group by -test). (h) Potential binding sites of miR-491-5p with AK046375 (up), and luciferase activity of the wild-type and mutant AK046375 groups with miR-491-5p in 293T cells (down) (/group, , vs. the mimics-NC group by -test). (i, j) Quantitative PCR analysis of miR-491-5p levels in primary neurons and astrocytes after transfection with either overexpression or knockdown AK046375 adenovirus or their corresponding vector viruses (/group, , vs. the overexpression vector group, ^& vs. the AK046375 overexpression group by one-way ANOVA). (k, l) Quantitative PCR analysis of MT2 mRNA levels and western blotting results of MT2 in astrocytes from each group after transfection with AK046375 overexpression or knockdown adenovirus, miRN-491-5p-mimics, and miR-491-5p-inhibitor and their quantification (/group, , vs. the AK046375 overexpression group, ^& vs. the AK046375 knockdown group by one-way ANOVA).

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