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Acupuncture May Exert Its Therapeutic Effect through MicroRNA-339/Sirt2/NFkappaB/FOXO1 Axis |
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Our bioinformatics study suggests an association between these miRNAs and proteins, which include miR-339 and sirtuin 2 (Sirt2). |
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In this paper, we aimed to investigate whether Sirt2 was a direct target of miR-339 in neurons. |
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In human SH-SY5Y cells, the luciferase assay implied that Sirt2 was likely a target of miRNA-339. |
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Overexpression of miR-339 downregulated Sirt2 expression, while knockdown of miR-339 upregulated Sirt2 expression in human SH-SY5Y cells and rat PC12 cells. |
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In addition, overexpression of miR-399 increased the acetylation of nuclear factor-kappaB (NF-kappaB) and forkhead box protein O1 (FOXO1) in SH-SY5Y cells, which are known targets of Sirt2. |
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Our findings demonstrate that miR-339 regulates Sirt2 in human and rat neurons. |
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Since Sirt2 plays a critical role in multiple important cellular functions, our data imply that acupuncture may act through epigenetic changes and subsequent action on their targets, such as miRNA-339/Sirt2/NF-kappaB/FOXO1 axis. |
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Some physiological level changes of neurons after altering the miR-339 levels are needed to validate the suggested therapeutic role of miR-339/Sirt2/NF-kappaB/FOXO1 axis in response to acupuncture therapy in the future work. |
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Luciferase Assay Implies That Sirt2 Is Likely a Direct Target of miRNA-339 in SH-SY5Y Cells |
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To validate whether Sirt2 is a likely target of miR-339, we performed computational miRNA target analysis, which showed that miRNA-339 was able to bind to the Sirt2 mRNA 3'-UTR, suggesting this gene might be a potential target for miRNA-339 (Figure 1(a)). |
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Moreover, to examine whether miR-339 could repress Sirt2 expression through direct 3'-UTR interaction, we cloned Sirt2 3'-UTR luciferase reporter plasmid and performed reporter analysis in SH-SY5Y cells. |
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Our present data demonstrated that cotransfection of miR-339 mimic with Sirt2 3'-UTR reporter resulted in dose-dependent inhibition of luciferase activity in SH-SY5Y cells (Figure 1(b), P < 0.05 versus control). |
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However, miR-339 failed to repress the activity of Sirt2-3'-UTR reporter with a mutated miR-339 seed sequence (Figure 1(b)). |
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These data indicated that Sirt2 was likely a direct target of miR-339. |
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Overexpression of miRNA-339 Downregulates Sirt2 Expression in Human SH-SY5Y Cells |
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To further validate whether miRNA-339 could downregulate Sirt2 expression in human neurons, we tested the effect of miRNA-339 on Sirt2 expression level in SH-SY5Y cells transfected with miRNA-339 mimic. |
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Overexpression of miRNA-339 significantly decreased Sirt2 expression in a dose-dependent (Figure 2(a), P < 0.01 versus control) and time-dependent manner (Figure 2(b), P < 0.05 versus control). |
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Overexpression of miRNA-339 significantly decreased Sirt2 expression in a dose-dependent (Figure 3(a), P < 0.01 versus control) and time-dependent manner (Figure 3(b), P < 0.001 versus control). |
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Knockdown of miRNA-339 significantly increased Sirt2 expression in a dose-dependent (Figure 4(a), P < 0.01 versus control) and time-dependent manner (Figure 4(b), P < 0.001 versus control). |
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Our results showed that Sirt2 is the direct target of miR-339, indicating that miR-339 can contribute to an upregulation of the acetylated status of Sirt2 target, including NF-kappaB and FOXO1. |
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To test this hypothesis, we measured the acetylated status of Sirt2 target (NF-kappaB and FOXO1) in SH-SY5Y cells treated with miRNA-339 mimic. |
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Overexpression of miRNA-339 Downregulates Sirt2 Expression in Rat PC12 Cells |
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To further validate whether miRNA-339 can downregulate Sirt2 expression in rat neurons, we tested the effect of miRNA-339 on Sirt2 expression level in NGF-induced PC12 cells transfected with miRNA-339 mimic. |
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Knockdown of miRNA-339 Upregulates Sirt2 Expression in Rat PC12 Cells |
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To further validate whether knockdown of miRNA-339 can upregulate Sirt2 expression in rat neurons, we tested the effect of miRNA-339 on Sirt2 expression level in NGF-induced PC12 cells transfected with miRNA-339 inhibitor. |
