Fig 1: Targeting IL-6R could reverse tamoxifen (TAM) resistance in BQ overexpressing ER+ breast cancer. (A) Knockdown of IL-6R could enhance the efficacy of TAM in control cells and reverse TAM resistance in BQ overexpressing cells. Cell lines with stable IL-6R knockdown were used. The cells were treated with 5 µM of TAM for 96 h. MTT was used to determine cell viability. (B) Tocilizumab (TCZ) could reduce cell viability in BQ overexpressing cells in the presence of 5 µM of TAM. The effect of TCZ on cell viability was compromised by IL-6R knockdown. Two-hundred-and-fifty ng/mL of TCZ or BSA was used. MTT assay was performed after 96 h of the treatment. (C) TCZ could reverse TAM resistance in BQ-overexpressing cells. The cells were treated with 250 ng/mL of TCZ and 5 µM of TAM for 2 weeks. Clonogenic assay was performed. (D) Statistical analysis of (C). (E) TCZ could reduce TAM resistant in vivo. TAM-resistance cell lines LCC2 shCtrl and LCC2 shIL-6R (shIL-6R.1) were used for xenograft establishment. The mice were randomized into different groups. The mice received 0.5 mg of TAM and 2 mg/Kg of TCZ through subcutaneous injection. The mice were treated twice per week for 8 weeks. (F) The graph showed the volume change of tumors during the treatment period. Results were shown as mean ± SD from at least three independent experiments. Students’ t test was used to determine the statistical significance between treatment and control groups. *** represents p < 0.001.
Fig 2: The expression of IL6R and IL6 is regulated by NFAT1 in glioma cells. a Expression data of IL6R, IL6 and NFAT1 mRNA in gliomas were downloaded from TCGA dataset and analysed. The expression of IL6R and IL6 were significantly correlated with that of NFAT1. b The expression of NFAT1 in different glioma cells was examined using western blotting. c Effect of NFAT1 knockdown on the expression of IL6R and IL6 was examined by western blotting in M1 and U87 cells. d Effect of NFAT1 overexpression on the expression of IL6R and IL6 was examined by western blotting in T98G and N2 cells. e Double-labelled immunofluorescence staining showing that the downregulation of NFAT1 is accompanied decreased IL6R expression in M1 cells, while upregulation of NFAT1 is accompanied increased IL6R expression in N2 cells. Scale bar =10 µm. f The levels of IL-6 secretion were examined by ELISA in cell-free supernatants from glioma cells after NFAT1 silencing or overexpression. g-h Effect of NAFT1 on IL6R (g) and IL6 (h) promoter activities. Silencing NFAT1 in both M1 and U87 cells significantly reduced the luciferase activity driven by the wildtype IL6R and IL6 promoters compared with control-shRNA. Mutating NFAT1 binding sites resulted in reduced promoter activity compared with the wildtype promoter. NFAT1 overexpression in T98G and N2 cells increased luciferase promoter activities. i Binding of NFAT1 to IL6R and IL6 promoters. Binding of NFAT1 was decreased when NFAT1 was silenced in M1 and U87 cells, while binding was increased when NFAT1 was overexpressed in T98G and N2 cells. j In M1 cells, NFAT1 knockdown significantly inhibited subcutaneous tumour growth. *P < 0.05 and **P < 0.01
Fig 3: SNHG12 facilitates the expression of IL-6R by NF-?B1. (A) Transcription factor for upregulation of IL-6R by SNHG12 predicted by the LNCmap database. (B) sub-localization of SNHG12 predicted by the LNCmap database. (C) sub-localization of SNHG12 visualized by FISH (D) expression of SNHG12 detected by RT- qPCR. (E) the binding of SNHG12 and NF-?B1 determined by RIP. (F) effects of SNHG12 and NF-?B1 on IL-6R promoter activity assessed by dual luciferase assay. (G) The binding of NF-?B1 to IL-6R promoter determined by ChIP. (H) the expression of IL-6R after silencing or overexpressing SNHG12 determined by Western blotting analysis. * p < 0.05 vs. control or IL-6R-WT + NC. # p < 0.05 vs. IgG. Statistical comparisons were performed using unpaired t test when only two groups were compared or by Tukey’s test-corrected one-way ANOVA with when more than two groups were compared.
Fig 4: miR-451a expression is downregulated in patients with MM. (A) Relative expression levels of miR-451a in normal controls and patients with MM. (B) Relative expression levels of miR-451a in patients with MM based on R-ISS stage. (C) Relative expression levels of IL-6 in normal controls and patients with MM. (D) Correlation between miR-451a and IL-6 levels. (E) Western blot showing the levels of IL-6R expression in plasma cells from bone marrow. (F) Intensity of IL-6R expression as determined by flow cytometry. (G) Immunohistochemical analysis of tumor cells. Tumor cells were positive for CD138 and IL-6R. Bar: 100 µm in H&E. Bar:50 µm in immunohistochemical analysis. (H) Correlation between miR-451a and IL-6R levels. ***P<0.0001. miR, microRNA; MM, multiple myeloma; IL-6, interleukin-6; IL-6R, IL-6 receptor; H&E, hematoxylin and eosin.
Fig 5: IL6R knockdown inhibits the growth and invasion of glioma cells. a IL6R protein levels in M1 and U87 cells that were transfected with IL6R-shRNA1, IL6R-shRNA2 or control-shRNA, respectively, were tested by western blot analyses, and IL6R knockdown was confirmed. b Targeting of IL6R via specific shRNAs significantly decreased the proliferation of M1 and U87 cells as assessed with MTS assay. c Representative microphotographs showing the detection of apoptosis using the TUNEL assay in control and IL6R silenced M1 cells. Scale bar = 25 µm. Histogram showing the quantification of the apoptotic rate. d Representative microphotographs showing the invasion of M1 and U87 cells in the presence of IL6R-shRNA or control-shRNA using the matrigel assay. Scale bar = 50 µm. Histogram showing the quantification of invasive cells. e The migration of M1 and U87 cells in the presence of IL6R-shRNA or control-shRNA was tested using the wound healing assay. Scale bar = 500 µm. Histogram showing the quantification of the migration rate. f IL6R knockdown decreased the colony formation rate of M1 and U87 cells. Scale bar = 50 µm. g Representative images of the effect of IL6R knockdown on M1 and U87 GSC neurospheres. Scale bars = 50 µm. Histograms showing the quantification of relative sizes and numbers of indicated neurospheres. Results are presented as mean ± SEM of triplicate samples from three independent experiments. *P < 0.05 and **P < 0.01
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