Fig 2: Trehangelins (THG) suppress the expression of CCN1 in keratinocytes by inhibiting the translocation of Yes-associated protein (YAP) to the nucleus. Structure of THG A (a). Estimated LogP of THG, Trehalose derivatives and mono/disaccharides was calculated using ChemDraw. Detailed list is shown in Supplementary Information (Table S1) (b). Effects of THG on CCN1 expression. Keratinocytes were pretreated with THG for 24 h and then were stimulated with or without 10 U plasmin, after which they were incubated for another 24 h. The expression of cellular CCN1 from the lysate (c) and secreted CCN1 from the supernatant (d) was determined by Western Blotting and was quantified. Values reported are means ± SD of n = 3 replicates, Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.001 (c, d). Immunocytochemistry of YAP and analysis of nuclear YAP-positive cells 0–3 h after Ca or THG treatment (e). Values reported are means of n = 10,000 cells, Bar = 100 µm (e).

Fig 3: Overexpression of miR-582-5p attenuates YAP/TAZ signaling activity. (A–D) Western blot (A,C) and Image J quantification (B,D) were employed to measure the changes in protein levels of YAP, TAZ, and their phosphorylated counterparts following the overexpression of miR-582-5p in H1299 (A,B) and H661 (C,D) cell lines. GAPDH served as a loading control. (E–H) Representative images of immunofluorescence staining of YAP, TAZ, and DAPI, and the corresponding quantification for the subcellular localization of TAZ and YAP post-transfection of miR-582-5p in H1299 (E,F) and H661 cells (G,H). Scale bar = 10 µm. (I,J) RT-qPCR analysis was conducted upon overexpression of miR-582-5p in H1299 (I) and H661 (J) cells to examine relative mRNA levels of YAP, TAZ and the YAP/TAZ transcriptional targets, ANKRD1, CTGF and CYR61. The ?CT of examined genes were normalized against that of 18SrRNA in order to determine relative expression. (K) Western blot analysis of TAZ protein levels upon concomitant overexpression of GFP/TAZ S89A and miR-582-5p mimics in H1299 cells. (L) RT-qPCR analysis of YAP, TAZ, and the YAP/TAZ transcriptional targets, ANKRD1, CTGF, and CYR61, upon concomitant overexpression of GFP/TAZ S89A and miR-582-5p mimics in H1299 cells. Results are expressed as the mean of triplicate experiments with error bars representing standard deviation. Statistical analysis was conducted using Student’s t-test with * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: Treatment with THG ameliorates the structure of the basement membrane in ex vivo human skin explants. Effects of topically applied THG on CCN1 expression in plasmin-treated ex vivo skin explants. On day 1, 0–1% THG was applied on the stratum corneum and 340 U plasmin was applied to the medium. clCCN1 in the supernatant on day 5 was determined by Western Blots. Values reported are means ± SD of n = 3 replicates, Tukey’s test, *p < 0.05 (a). CCN1 expression and localization determined by immunohistochemistry, Bar = 100 µm (b). Effects of topically applied THG on the expression of CCN1 and COL7 in UVB irradiated ex vivo skin explants. 1% THG was topically treated from day 1. On days 1, 2 and 3, the inserts were exposed to 200 mJ/cm2 UVB. The expression of CCN1 and COL7 was determined by immunohistochemistry of skin sections collected on day 5, Bar = 50 µm (c). The area of CCN1 and COL7 around the basement membrane was quantified. Values reported are means ± SD of 9 fields from n = 3 replicates, Tukey’s test, *p < 0.05, ***p < 0.001 (d). The volume of COL7 and ELN was determined by immunohistochemistry of decolorized skin explants, Bar = 50 µm (e).

Fig 5: MiR-582-5p directly targets the 3’UTRs of NCKAP1 and PIP5K1C. (A) Venn diagram shows an integrated analysis of two gene lists, namely predicted miR-582-5p targets and regulators of actin cytoskeleton, which were derived from TargetScan and Wikipathways, respectively. Nine candidate genes were identified. (B,C) RT-qPCR analysis was performed to examine the changes in relative mRNA levels of the nine candidate genes post-transfection of miR-582-5p mimics in H1299 (B) and H661 (C) cells. The ?CT of examined genes were normalized to that of 18SrRNA in order to determine the relative expression. (D) Western blot reflects the changes in protein levels of NCKAP1 and PIP5K1C in H1299 and H661 cells upon overexpression of miR-582-5p. (E) Dual luciferase assays were performed to evaluate the predicted binding sites of miR-582-5p to eight sites on NCKAP1 3’ UTR (NCKAP1-1—NCKAP1-8) and to two sites on PIP5K1C 3’UTR (PIP5K1C-1—PIP5K1C-2). Firefly luciferase readouts were normalized to the corresponding Renilla luciferase readouts to evaluate relative luciferase activity. (F–H) The wildtype (WT) and mutant (Mut) miR-582-5p binding sites on the 3’UTRs of NCKAP1 (F,G) and PIP5K1C (H) were constructed into a pmiR-Glo reporter plasmid followed by co-transfection of the aforementioned plasmids and microRNA mimics. Firefly luciferase readout was normalised to the corresponding Renilla luciferase readout. (I,J) Cell proliferation kinetics was quantified following the siRNA-mediated knockdown of NCKAP1 (I) and PIP5K1C (J). (K,L) Cell apoptosis was measured using Caspase-3/7 Glo upon the knockdowns of NCKAP1 (K) and PIP5K1C (L) in H1299 cells. The readouts of Caspase-3/7 Glo were normalized against that of CellTiter Glo. (M,N) RT-qPCR analysis was conducted upon siRNA-mediated knockdowns of NCKAP1 (M) and PIP5K1C (N) to examine the relative mRNA levels of NCKAP1, PIP4K1C, and the YAP/TAZ transcriptional targets, ANKRD1, CTGF, and CYR61 in H1299 cells. (O) A schematic working model illustrates the potential negative feedback loop between miR-582-5p and YAP/TAZ. YAP/TAZ represses pri-miR-582 gene expression but is eventually required for the maintenance of mature miR-582-5p expression. MiR-582-5p directly inhibits the expression of NCKAP1 and PIP5K1C and attenuates YAP/TAZ activity, potentially by compromising F-actin. Results are presented as the mean of three or five replicates as indicated with error bars representing standard deviation. Statistical analysis was conducted using Student’s t-test with * p < 0.05, ** p < 0.01, *** p < 0.001.