Fig 1: HOXB5 transcriptionally regulated IL6 expression and regulated the proliferation of GSCs via JAK2/STAT3 signaling. a TCGA and CGGA datasets showed that higher HOXB5 expression was associated with enrichment of IL6-mediated JAK2/STAT3 signaling. b The relative expression correlation between HOXB5 and IL6 in 70 cases of glioma patients was detected by qRT-PCR. (Total: r = 0.6160, p < 0.0001; Grade II: r = 0.4548, p = 0.0439; Grade III: r = 0.4723, p = 0.0171; Grade IV: r = 0.5090, p = 0.0094; Pearson’s correlation analyses). c, d, l, m qRT-PCR (c), ELISA (d), and western blotting (l, m) showed the IL6 expression was altered after HOXB5 knockdown or overexpression in GSCs. (c: GSC406: p < 0.01; GSC201: p < 0.001; One-Way ANOVA; d: GSC406: p < 0.001; GSC201: p < 0.01; One-Way ANOVA). e Sequence motif representing the consensus HOXB5 binding motif (JASPAR database), and Schematic diagram of the putative HOXB5 binding site in the 3′-UTR of IL6. f The luciferase reporter assays showed that HOXB5 knockdown or overexpression affected the luciferase activities of IL6 in GSCs. (GSC406: p < 0.001; GSC201: p < 0.001; One-Way ANOVA). g The ChIP qRT-PCR showed that HOXB5 bound to the promoter of IL6. (GSC406: p < 0.01; GSC201: p < 0.001; One-Way ANOVA). h MTS assays showed that HOXB5 knockdown or overexpression affected the cell viability of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. (GSC406: p < 0.001; GSC201: p < 0.001; One-Way ANOVA). i The EDU assays showed that HOXB5 knockdown or overexpression affected the proliferation of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. Scale bar = 100 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). j The neurospheres formation assays showed that HOXB5 knockdown or overexpression affected the relative size of the neurospheres of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. Scale bar = 20 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). k Limiting dilution assays showed that HOXB5 knockdown or overexpression affected the neurosphere-forming capacity of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. (GSC406: p < 0.05; GSC201: p < 0.05; ELDA analysis; circles represent corresponding points, triangles mean the point is outside of the log fraction number wells). l and m Western blotting showed the expression of downstream targets of the IL6-mediated JAK2/STAT3 signaling pathway with HOXB5 knockdown or overexpression in GSCs. EV: empty vector, OE: overexpression, NC: negative control, KD: knockdown. All data were expressed as the mean ± SD (three independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001
Fig 2: Overexpression of HMBOX1 blunts the inflammation and apoptosis of LPS-induced hPDLSCs. (A) Expression of inflammation-related proteins IL-6, TNF-α and IL-1β measured by western blotting. (B) Secretion of inflammation-related proteins IL-6, TNF-α and IL-1β measured by ELISA. (C) Apoptosis was measured and quantified by TUNEL. (D) Expression of apoptosis-related proteins Bcl-1, Bax and caspase 3 was measured by western blotting. ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. LPS+Ov-NC. hPDLSCs, human periodontal ligament stem cells; HMBOX1, Homeobox containing 1; LPS, lipopolysaccharide; Ov, overexpression; NC, negative control.
Fig 3: HMBOX1 blunts the inflammation and apoptosis of LPS-induced hPDLSCs through the NF-κB/CXCL10 axis. After PMA treatment, (A) the expression of inflammatory factors IL-6, TNF-α and IL-1β was measured by western blotting, whilst (B) the secretion of inflammatory factors IL-6, TNF-α and IL-1β was measured by ELISA. After PMA treatment, (C) apoptosis was measured using TUNEL, whereas (D) the expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 in LPS-induced hPDLSCs transfected with Ov-HMBOX1 and si-CXCL10 was measured by western blotting. ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. LPS+Ov-NC+si-NC; ∆P<0.05, ∆∆P<0.01, ∆∆∆P<0.001 vs. LPS+Ov-HMBOX1+si-NC; @@P<0.01, @@@P<0.001 vs. LPS+Ov-HMBOX1+PMA+si-NC. hPDLSCs, human periodontal ligament stem cells; HMBOX1, Homeobox containing 1; LPS, lipopolysaccharide; Ov, overexpression; NC, negative control; CXCL10, C-X-C motif chemokine ligand 10; PMA, phorbol 12-myristate 13-acetate; si, small interfering.
Fig 4: The SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop regulated glioma tumorigenesis in vivo. a Representative images showed the size of intracranial tumors in the coronal location of eight groups (negative control, circATP5B knockdown, miR-185-5p mimic, HOXB5 overexpression, SRSF1 overexpression, circATP5B knockdown combined with HOXB5 overexpression, miR-185-5p mimic combined with HOXB5 overexpression, SRSF1 overexpression combined with circATP5B knockdown in GSC406). Scale bar = 10 mm. b The measured tumor volumes among eight GSC406 groups are indicated. (*p < 0.05 vs. the negative control group; ##p < 0.05 vs. the circATP5B knockdown group, $$p < 0.05 vs. the miR-185-5p group, &&p < 0.05 vs. the SRSF1 overexpression group; Student’s t-test). c Kaplan-Meier survival curves showed that HOXB5 overexpression, SRSF1 overexpression, circATP5B knockdown combined with HOXB5 overexpression, miR-185-5p mimic combined with HOXB5 overexpression in GSC406 shortened the survival times of nude mice. At the same time, it prolonged the survival times after miR-185-5p mimic was transfected, circATP5B knockdown, and SRSF1 overexpression combined with circATP5B knockdown in GSC406. For each group, n = 5. d Representative immunohistochemical staining showing the changes in HOXB5, SRSF1, IL6, Ki67, CD133 and nestin in the negative control, circATP5B knockdown, miR-185-5p mimic, HOXB5 overexpression, SRSF1 overexpression, circATP5B knockdown combined with HOXB5 overexpression, miR-185-5p mimic combined with HOXB5 overexpression, SRSF1 overexpression combined with circATP5B knockdown group in orthotopic xenograft models. Scale bar = 50 μm. e The German scoring of HOXB5 protein expression in eight groups. (the circATP5B knockdown group vs. the negative control group: p < 0.001; the miR-185-5p mimic group vs. the negative control group: p < 0.001; the HOXB5 overexpression group vs. the negative control group: p < 0.01; the SRSF1 overexpression group vs. the negative control group: p < 0.01; the circATP5B knockdown combined with HOXB5 overexpression group vs. the negative control group: p < 0.01; the miR-185-5p mimic combined with HOXB5 overexpression group: p < 0.01; the SRSF1 overexpression combined with circATP5B knockdown group vs. the negative control group: p < 0.001; Student’s t-test). f Schematic diagram showing that the SRSF1/circATP5B/miR-185-5p/HOXB5 axis promoted glioma proliferation through IL6-mediated JAK2/STAT3 signaling pathway. All data were expressed as the mean ± SD (three independent experiments). EV: empty vector, OE: overexpression, NC: negative control, KD: knockdown. All data were expressed as the mean ± SD (three independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001
Fig 5: CircGNB1 promoted GSCs tumorigenesis in vivo. a Hematoxylin and eosin staining of intracranial tumor plantation showed the tumor size in the coronal location of five groups. Scale bar = 10 mm. b The measured tumor volumes among each group are indicated. c Representative immunohistochemical staining showed the changes in XPR1, Ki-67, IL6, and IGF2BP3 in each orthotopic xenograft model. Scale bar = 50 µm. d Kaplan–Meier survival curves revealed nude mice’s survival times in each group (n = 10). e Schematic diagram demonstrated that IGF2BP3/circGNB1/miR-515-5p/miR-582-3p/XPR1 promoted malignant phenotype of GSCs via IL6/JAK2/STAT3 axis. Data represent mean ± SD (three independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001
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