circ-Amotl1 in extracellular vesicles derived from ADSCs improves wound healing by upregulating SPARC translation

Figures

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  Fig. 1. SPARC regulates ECM remodeling to activate Notch signal pathway to promote wound healing process A, Overlaps of differential expressed genes in both GSE168760 and GSE181022 datasets; B, differential expressed genes in GSE168760; C, differential expressed genes in GSE181022; D, SPARC expression detected by western blot; E, wound healing area in each group; F, wound healing rate in each group; G, H&E staining on epithelial tissues; H, Immunohistochemistry detected the positive expression of Ki67; I, PathCards supported the implication of SPARC in ECM remodeling and Notch signal pathway; J, western blot detecting the expressions of MMP2, MMP9, Notch1, Jagged1 and Hes1. Data were expressed as mean ± standard deviation (SD). Comparison between two groups was analyzed using independent sample t test, n = 10. Cellular experiment was conducted for 3 times. ∗, compared with oe-NC group, P < 0.05. ECM, extracellular matrix.

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  Fig. 2. IGF2BP2 increases SPARC expression by elevating SPARC mRNA stability A, SRAMP database predicted m6A modification sites of SPARC; B, Starbase predicted the binding sites of IGF2BP2 and SPARC mRNA; C, qRT-PCR detected the mRNA expressions of IGF2BP2 and SPARC; D, western blot detected the protein expressions of IGF2BP2 and SPARC; E, RNA pull down detected the SPARC mRNA level pulled down by IGF2BP2; F, Actinomycin D detected SPARC mRNA stability. Data were expressed as mean ± standard deviation (SD). Comparison between two groups was analyzed using independent sample t test, n = 10. Cellular experiment was conducted for 3 times. ∗, compared with oe-NC group, P < 0.05.

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  Fig. 3. IGF2BP2 can regulate SPARC expression to increase the proliferation and migration of HSFs. A, qRT-PCR detected the mRNA expressions of IGF2BP2 and SPARC; B, western blot detected the protein expressions of IGF2BP2 and SPARC; C, CCK8 assay measured cell proliferation ability; D, cell scratch measured cell migration ability; E, Transwell measured cell migration ability; F, qRT-PCR detected the expressions of Collagen I and Collagen III; G, western blot detected the protein expressions of Collagen I and Collagen III. Data were expressed as mean ± standard deviation (SD). Comparison among three groups was analyzed using one-way ANOVA. Analysis on data among multiple time points were conducted by repeated ANOVA, with Bonferroni as post hoc analysis. Cellular experiment was conducted for 3 times. ∗, compared with oe-NC + sh-NC group, P < 0.05. #, compared with oe-IGF2BP2+sh-NC group, P < 0.05.

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  Fig. 4. circ-Amotl1 derived from ADSCs can bind IGF2BP2 to increase SPARC expression A, inverted microscope observed the morphology of ADSCs derived from adipocytes; B, ADSCs were identified by flow cytometry; C-D, TEM and NTA analyzed Evs; E, Western Blot detected the biomarkers of Evs; F, PKH67 fluorescence detected ADSC-Evs; G, Starbase predicted the binding of circ-Amotl1 and IGFBP2; H, RNA pull down verified the binding of circ-Amotl1 with IGFBP2; I, Immunofluorescence co-localization analysis detected the expression of circ-Amotl1; J, western blot detected the expressions of Evs biomarkers; K, qRT-PCR detected the expression of circ-Amotl1 in cultured HSFs; L, western blot detected the expressions of IGF2BP2 and SPARC; Data were expressed as mean ± standard deviation (SD). Comparison between two groups was analyzed using independent sample T test. Comparison among three groups was analyzed using one-way ANOVA, n = 10. Cellular experiment was conducted for 3 times.∗, compared with Control group, P < 0.05 (CM: culture medium; del: depletion); ADSCs, adipose-derived stromal cells; Evs, extracellular vesicle; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis.

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  Fig. 5. circ-Amotl1 in ADSC-Evs increases SPARC expression through binding IGF2BP2 to enhance proliferation and migration of HSFs. A, qRT-PCR detected the expression circ-Amotl1 in ADSCs and ADSC-Evs; B, qRT-PCR detected the expression of circ-Amotl1 in HSFs; C, western blot detected the expression of IGF2BP2 and SPARC; D, CCK8 detected cell proliferation ability; E, cell scratch assay detected cell migration ability; F:Transwell assay detected cell migration ability; G, qRT-PCR detected the expressions of Collagen I and Collagen III; H, western blot detected the expressions of Collagen I and Collagen III. Data were expressed as mean ± standard deviation (SD). Comparison among three groups was analyzed using one-way ANOVA. Analysis on data among multiple time points were conducted by repeated ANOVA, with Bonferroni as post hoc analysis. Cellular experiment was conducted for 3 times.∗, compared with Control group or sh-NC group, P < 0.05; #, compared with Evs-shNC group, P < 0.05.

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  Fig. 6. circ-Amotl1 in ADSC-Evs can promote wound healing in mice through regulating IGF2BP2/SPARC axis. A, qRT-PCR detected the expression of circ-Amotl1; B, western blot detected the protein expressions of IGF2BP2 and SPARC; C, wound healing process in each group; D, comparison on wound healing rate in each group; E, H&E staining to observe the scar width; F, immunohistochemistry to detect the positive expression of Ki67. Data were expressed as mean ± standard deviation (SD). Comparison between two groups was analyzed using independent sample t test. Comparison among three groups was analyzed using one-way ANOVA. Analysis on data among multiple time points was conducted by one-way ANOVA, n = 10. Cellular experiment was conducted for 3 times. Experiment was conducted for 3 times.∗, compared with Control group, P < 0.05; #, compared with Evs-shNC group, P < 0.05.