Table 1.
Chemicals and reagents with their catalog numbers and companies.
Table 2.
Sequences of the primers for qPCR.
Table 3.
Transfection plasmid combinations of dual luciferase.
Fig 1.
CCK8, QPCR, ELISA, and western blot detection in an acute inflammatory model of adipose stem cells.
(A) Results of CCK8 detection of cellular activity in an acute adipose stem cell inflammation model. (B-D) ELISA results of IL-6, IL-1β, TNF-α. (E-G) QPCR results of IL-6, IL-1β, and TNF-α. (H-I) QPCR results of TLR4 and TLR2. (J) Western blotting results of TLR4 and TLR2. Data are mean ± SD of three independent experiments (n = 3). *** p <0.001; ** p <0.01; * p <0.05.
Fig 2.
CCK8, QPCR, ELISA and Western-Blot detection in a chronic inflammatory model of adipose stem cells.
(A) Results of CCK8 detection of cellular activity in a model of chronic adipose stem cell inflammation. (B-D) ELISA results of IL-6, IL-1β, TNF-α. (E-G) QPCR results of IL-6, IL-1β, and TNF-α. (H-I) QPCR results of TLR4 and TLR2. (J) Western blotting results of TLR4 and TLR2. Data are mean ± SD of three independent experiments (n = 3). *** p <0.001; ** p <0.01; * p <0.05.
Table 4.
Significantly differentially expressed miRNAs in the acute model group and the normal control group.
Table 5.
Significantly differentially expressed miRNAs in the chronic model group and the normal control group.
Fig 3.
qPCR verification of miRNA microarray analysis results.
The relative expression of miR-96-5p (A), miR-126-3p (B), miR-126-5p (C), miR-133a-3p (D), miR-141-3p (E), miR-150-3p (F), miR-223-3p (G) and miR-2909 (H) in the acute inflammation model and the slow inflammation model group. All experiments were repeated in triplicate and analyzed by the 2-ΔΔCt method. *** p < 0.001; ** p < 0.01; * p < 0.05.
Fig 4.
The 11 miRNAs that were significantly differentially expressed in both acute and chronic inflammatory models were analyzed using four target gene prediction softwares for Venn diagram aggregation analysis.
miR-96-5p (A), miRNA-126-3P (B), miRNA-126-5P (C), miR-141-3p (D), miR-133a-3p (E), miR-150-3p (F), miR-223-3P (G), miR-2909 (H), miR-200c-3p (I), miR-3591-3p (J), miR-4483 (K).
Fig 5.
Heat map of differentially expressed miRNAs, and analysis of functions and pathways corresponding to possible target genes.
(A) Heat map analysis of differentially expressed miRNAs in three groups (normal group N: Normal, acute inflammation model group A: Acute, chronic inflammation model group C: Chronic). Columns and rows represent samples and specific miRNAs. The miRNA cluster tree is shown on the left. The code on the legend is the value after log2 conversion. The color scale indicates the relative expression level of the miRNA. Red indicates that the expression of miRNA in the treated group is higher than that in the control group, while green indicates that the expression of the miRNA in the treated group is lower than that in the control group. Black indicates that there is no difference in miRNA between the two groups. (B) GO analysis of target genes predicted by differentially expressed miRNAs in the normal group and the acute inflammation model group. (C) GO analysis of target genes predicted by differentially expressed miRNAs in the normal group and the chronic inflammation model group. The GO category is shown on the x-axis and the number of genes is shown on the y-axis. Biological processes (BP) are a major class of GO analysis, including various processes associated with inflammation. Molecular function (MF) is rich in protein binding, zinc ion binding, transcription factor activity, and so on. The cellular component (CC) is enriched in the nucleus and is indispensable for membrane, cytoplasm and so on. (D) Analysis of target gene pathways predicted by differentially expressed miRNAs in the normal group and the acute inflammation model group. (E) Analysis of target gene pathways predicted by differentially expressed miRNAs in the normal group and the acute inflammation model group. The 15 most abundant signal transduction pathways were selected based on the P value (P < 0.01). The x-axis shows the signal path term and the y-axis shows the degree of enrichment.
Fig 6.
Dual-luciferase assay for miR-223-3P targeting STAT3 and for IL-6;miR-2909 targeting KLF4.
(A) predicted binding sites for miR-223-3P and STAT3, (B) changes in luciferase activity of added STAT3 3'-UTR pMIR-TM luciferase vector and miR-223-3p mimic, (C) STAT3 molecular level of added miR-223-3p mimic, (D) STAT3 protein level of added miR-223-3p mimic, (E) miR-223-3P and IL-6 predicted binding site, (F) changes in luciferase activity of added IL- 6 3'-UTR psi-CHECK2 luciferase vector and miR-223-3p mimic, (G) IL-6 molecular level of added miR-223-3p mimic, (H) IL-6 protein level of added miR-223 -3p mimic, (I) miR-2909 and KLF4 predicted binding sites, (J) changes in luciferase activity of added KLF4 3'-UTR pMIR GFP luciferase vector and miR-2909 mimic, (K) the molecular level of added KLF4 and miR-2909, (L) the protein level of added KLF4 and miR-2909. *** p <0.001; ** p <0.01; * p <0.05.
Fig 7.
miR-223-3p mimic and control, miR-223-3p inhibitor and inhibitor control were transfected into adipose stem cells.
The molecular expression level and protein expression levels of IL-6, TNF-α, IL-1β, TLR4, TLR2, miR-223-3p and STAT3. (A-C) protein levels of IL-6, TNF-α, IL-1β, (D-J) molecular levels of IL-6, TNF-α, IL-1β, miR-223-3p, STAT3, TLR4, TLR2, (K) protein levels of TLR4, TLR2, STAT3, p-STAT3. *** p <0.001; ** p <0.01; * p <0.05.
Fig 8.
miR-223-3p directly regulates TLR2, TLR4, and secretion of inflammatory cytokines IL-6, TNF-α and IL-1β.
(A) STAT3 protein level after STAT3 siRNA transfection, (B-D) protein expression of IL-6, TNF-α, IL-1β after transfection of miR-223-3p and STAT3 siRNA, (E-I) molecular expression level of TLR2, TLR4, IL- 6. TNF-α and IL-1β, (J) protein expression of TLR2 and TLR4. *** p <0.001; ** p <0.01; * p <0.05.
Fig 9.
STAT3 and p-STAT3 inhibitor were transfected into adipose stem cells,respectively, and the molecular expression levels and protein expression levels of STAT3, TLR4, TLR2, IL-6, TNF-α, IL-1β were detected.
(A-C) IL-6, TNF-α, IL-1β protein levels, (D-I) IL-6, TNF-α, IL-1β, STAT3, TLR4, TLR2 molecular expression levels, (J) TLR4, TLR2, STAT3, p-STAT3 protein expression levels. *** p <0.001; ** p <0.01; * p <0.05.
Fig 10.
IL-6, siIL-6, TNF-α, and siTNF-α were transfected into adipose stem cells, respectively, to detect the expression of miR-223-3p, STAT3, and pSTAT3.
(A-B) mir-223-3p and STAT3 molecular expression after transfection of IL-6, siIL-6, (C) STAT3 protein expression after transfection of siIL-6, (D) STAT3 and pSTAT3 protein expression after transfection of IL-6, siIL-6, (E-F) mir-223-3p and STAT3 molecular expression after transfection of TNF-α, siTNF-α, (G) STAT3 protein expression after transfection of siTNF-α, (H) STAT3 and pSTAT3 protein expression after transfection of TNF-α and siTNF-α. *** p <0.001; ** p <0.01; * p <0.05.
Fig 11.
miR-2909 is dependent on NF-kB to target KLF4 to regulate IL-6, IL-1β, TNF-α.
The miR-2909 mimic and the control, miR-2909 inhibitor and inhibitor control, and NF-kB inhibitor were transfected into adipose stem cells, respectively, (A-C) protein expression levels of IL-6, IL-1β, and TNF-α, (D-I) molecular expression level of IL-6, IL-1β, TNF-α, miR-2909, KLF4, NF-kB, (J) protein expression level of KLF4, NF-kB, pNF-kB. *** p <0.001; ** p <0.01; * p <0.05.