Fig 2: miR-146a decreases SIRT1 activity by directly targeting Nampt mRNA. a–g miR-146a directly targets the Nampt 3′-UTR. The NAMPT protein level in NIH3T3 cells transfected with miR-146a mimics (a) or inhibitors (b), with relative fold changes are shown (n = 3). c The mouse miR-146a sequence and the predicted miR-146a target site in the 3′-UTR of WT and MUT Nampt (upper). Schematic of the luciferase reporter plasmids with the WT or MUT Nampt 3′-UTRs (lower). d, e Relative luciferase activity in HEK293T cells transfected with the indicated luciferase reporter plasmids along with miR-146a mimics or inhibitors, respectively (n = 3). f, g Endogenous Nampt mRNA levels in NIH3T3 cells transfected with miR-146a mimics or inhibitor (n = 3). h–m miR-146a decreases SIRT1 activity by directly targeting the Nampt 3′-UTR to inhibit NAMPT expression and NAD+ level. h Schematic of plasmids Nampt 3′-UTR containing wild-type or mutated miR-146a targeting site. i NAMPT protein level in NIH3T3 cells transfected with the indicated plasmids. j, k NAMPT and SIRT1 expression in NIH3T3 cells transfected with miR-146a mimics and the indicated plasmids, with relative fold changes shown (n = 3). l, m NAD+ level and SIRT1 activity in NIH3T3 cells transfected with miR-146a mimics and the indicated plasmids (n = 3). **p < 0.01, ***p < 0.001. ns means no significance

Fig 3: Metformin regulates miR-146a transcription in an AMPK-NF-κB-dependent manner. a, b Inhibition of AMPK promotes pri-miR-146a and miR-146a expression in senescent cells. NIH3T3 cells were treated with H2O2 and incubated in complete medium with or without treatment with the AMPK activators metformin (10 μM), AICAR (10 μM), alone or plus AMPK inhibitor Compound C (CC, 10 μM) for 3 days, then the levels of pri-miR-146a (a) and miR-146a (b) were tested (n = 3). c–e Overexpression of AMPK reduces pri-miR-146a and miR-146a expression in NIH3T3 cells. c Overexpression of AMPK in NIH3T3 cells. Immunoblots were performed to evaluate levels of p-AMPKα1, p-ACC, ACC, and β-actin in NIH3T3 cells transfected with pAMPKα1-flag or a control plasmid (pCtrl). d, e NIH3T3 cells were transfected with pAMPKα1-flag or pCtrl for 24 h, followed by treatment with H2O2 and incubation in a complete medium for 3 days. Levels of pri-miR-146a (d) and miR-146a (e) were evaluated (n = 3). f–h AMPK inhibits miR-146a expression by reducing p65 binding to the miR-146a promoter. f ChIP assays were performed to analyze p65 binding to the miR-146a promoter in NIH3T3 cells treated with H2O2 and metformin (n = 3). Following transfection with sip65 or negative control siRNA (si-NC), NIH3T3 cells were treated with H2O2 and metformin for 3 days, and pri-miR-146a (g) and miR-146a (h) levels were evaluated (n = 3). i–k AMPK inhibits miR-146a expression via IKKβ-mediated suppression of the NF-κB pathway. i Levels of p-ACC, p-AMPK, and p-IKKβ (Ser177) in NIH3T3 cells were tested by immunoblot. j Levels of p-IKKβ, IKKβ, p-p65 (Ser536), and p65 in NIH3T3 cells transfected with the pIKKβ-WT or pIKKβ-S177A (pIKKβ-MUT) plasmid. k Metformin regulates miR-146a level in an IKKβ-dependent manner. NIH3T3 cells were transfected with pIKKβ-WT and pIKKβ-S177A (pIKKβ-MUT) and then treated with H2O2 and metformin, followed by evaluation of miR-146a expression (n = 3). l Schematic depicting the miRNA-specific mechanism related to metformin-mediated prevention of senescence. Oxidative stress increased the expression of miR-146a, which targets the Nampt 3’-UTR to reduce NAMPT and NAD+ levels and SIRT1 activity, ultimately leading to cell senescence. Metformin induces AMPK activation to repress miR-146a transcription in an NF-κB-dependent manner, which increases NAMPT/NAD+ levels and SIRT1 activity and consequently suppresses cell senescence and mouse aging. *p < 0.05, **p < 0.01, ***p < 0.001. ns means no significance

Fig 4: Schematic representation of the mechanism underlying sorafenib-induced hyper-keratosis.Vascular endothelial cells release s-HBEGF upon sorafenib stimulation. s-HBEGF binds to EGFR and leads to JNK2 phosphorylation in keratinocytes. The activated JNK2 subsequently stabilizes SIRT1, which eventually results in keratinization. The classic SIRT1 inhibitor nicotinamide could effectively reverse sorafenib-induced HFSR.

Fig 5: SIRT1 is involved in sorafenib-induced hyper-keratosis.a HaCaT cells were transfected with non-targeting siRNA or siRNA targeting SIRT1. SIRT1 transcription level was detected by RT-qPCR (upper panel, n = 3) and the expression level of SIRT1 was determined by western blot (lower panel). b–e HaCaT cells were transfected with non-targeting siRNA or siRNA targeting SIRT1, followed by treatment with CdMCTRL or CdMSORA for 24 h (b, d) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h (c, e). The transcription levels of KRT1, KRT10, LORICRIN and IVL were measured by RT-qPCR (n = 3) (b, c). The cell survival rates were measured by SRB assay (n = 3) (d, e). f–i Mice were randomly divided into 4 groups. After injection of AAV1-shSirt1 adeno virus into the paws for 2 weeks, mice were treated with CMC-Na or sorafenib (100 mg/kg) daily by i.g. for 30 days (n = 5/group). f Representative western blot indicated the expression of SIRT1 in stratum corneum of mice in each group (n = 3/group). g Representative H&E staining and KRT5, KRT1, LORICRIN, SIRT1 immunohistochemistry staining were performed on the paws of mice. Scale bar, 50 μm. h Quantitative analysis of epidermal hyper-keratosis assessed by measuring the stratum corneum thickness. i s-HBEGF concentrations in the serum of each mouse were measured by ELISA (n = 5/group). Densitometric values are shown as optical density after GAPDH or ACTB normalization using Image J. Horizontal bars in (h) and (i) represent mean values. The results in (a), (b), (c), (d) and (e) are presented as the mean ± SD. Statistical analyses were performed using one-way ANOVA with LSD post hoc test in (b), (c), (h) and (i) and with Dunn’s post hoc test in (a). n.s. no significance; *P < 0.05; **P < 0.01; ***P < 0.001. SORA sorafenib, CdM HUVECs conditional medium, CTRL control.