Fig 2: In vivo therapeutic effects of miR21/DA3 polyplexes. (A) Western blot analysis with day 5 wound tissue samples showing PDCD4, TIMP3, and MMP2 expression levels. (B) Real-time MMP2 activity was visualized with an MMP2 probe after 5 and 10 days from miR21 treatment.

Fig 3: Anandamide downregulates TIMP3 expression and promotes MICA/B shedding. (a) The chemical structure of anandamide. (b) qRT-PCR analysis of TIMP3 mRNA levels in HepG2 cells with control DMSO or anandamide treatment. Data are means ± SD from three independent experiments. *P = 0.0021 (two-sided Welch’s t-test). (c) qRT-PCR analysis of the MICA/B mRNA levels in HepG2 cells with control DMSO or anandamide treatment. Data are means ± SD from three independent experiments. P-value of 0.20 (two-sided Welch’s t-test) was not considered to indicate significance. (d) Flow cytometry of surface MICA/B protein levels in HepG2 cells with control DMSO (red line) and anandamide (blue line) treatment. Gray-shaded histograms represent background staining with the isotype IgG. Representative results from three independent experiments are shown. Relative MFI data (n = 3) are shown in the right panel. Data are means ± SD. *P = 0.0012 (two-sided Welch’s t-test). (e) Soluble MICA/B quantification by NanoLuc assay in the culture medium of HepG2 cells with control DMSO or anandamide treatment. Data are means ± SD from three independent experiments. *P = 0.010 (two-sided Welch’s t-test).

Fig 4: Upregulation of TIMP3 expression by URB597 is responsible for the inhibition of MICA/B shedding. (a) A plot of the distribution of data from a cDNA microarray. The X- and Y-axes represent the average values of the log2-converted expression levels and the differences of log2-converted expression levels, respectively. SERPINA3, SERPINA7, SERPIND1, and TIMP3 (red) were identified as candidate inhibitors of MICA/B shedding. (b) qRT-PCR analysis of the TIMP3 mRNA level in HepG2 cells after treatment with URB597 or DMSO (control). Data are means ± SD from three independent experiments. A P-value of 0.44 (two-sided Welch’s t-test) was not considered to indicate significance *P = 7.4 × 10−5; *P = 2.0 × 10−4; ***P = 3.3 × 10−5 (two-sided Welch’s t-test). (c) Western blots of lysates from HepG2 cells without or with stable TIMP3-specific shRNA expression (HepG2shTIMP3). Representative images of three independent experiments are shown. (d) qRT-PCR analysis of the MICA/B mRNA levels in HepG2 and HepG2shTIMP3 cells. Data are means ± SD from three independent experiments. A P-value of 0.22 (two-sided Welch’s t-test) was not considered to indicate significance. (e) Flow cytometry of surface MICA protein levels in HepG2 cells treated with DMSO (black line) and HepG2shTIMP3 cells treated with DMSO (blue line) or URB597 (blue line). Gray-shaded histograms represent background staining with the isotype IgG. Representative results from three independent experiments are shown. Relative MFI data (n = 3) are shown in the right panel. Data are means ± SD. A P-value of 0.44 (two-sided Welch’s t-test) was not considered to indicate significance. *P = 0.027 (two-sided Welch’s t-test). (f) Soluble MICA/B quantification by NanoLuc assay in the culture medium of HepG2 or HepG2shTIMP3 cells treated with DMSO or URB597. Data are means ± SD from three independent experiments. A P-value of 0.40 (two-sided Welch’s t-test) was not considered to indicate significance. *P = 0.0020 (two-sided Welch’s t-test).

Fig 5: Rimonabant reverses the effect of URB597 on TIMP3 expression and MICA/B shedding. (a) The chemical structure of rimonabant, a cannabinoid CB1 receptor antagonist. (b) Time course of the experiment. Three groups (control DMSO treatment followed by the addition of control DMSO; URB597 treatment followed by the addition of control DMSO; and URB597 treatment followed by the addition of rimonabant) were analyzed. (c) qRT-PCR analysis of TIMP3 mRNA levels in the three experimental groups. Data are means ± SD from three independent experiments. *P = 9.0 × 10−4; **P = 0.016 (two-sided Welch’s t-test). (d) qRT-PCR analysis of the MICA/B mRNA levels in the three experimental groups. Data are means ± SD from three independent experiments. P-values of 0.22 and 0.15 (two-sided Welch’s t-test) were not considered to indicate significance. (e) Flow cytometry of surface MICA/B protein levels in the three experimental groups. Gray-shaded histograms represent background staining with the isotype IgG. Representative results from three independent experiments are shown. Relative MFI data (n = 3) are shown in the right panel. Data are means ± SD. *P = 0.0089; **P = 0.031 (two-sided Welch’s t-test). (f) Soluble MICA/B quantification by NanoLuc assay in the culture medium of the three experimental groups. Data are means ± SD from three independent experiments. *P = 0.0054; **P = 0.029 (two-sided Welch’s t-test).