Fig. 2: miR-133a-3p suppressed the proliferation of nasopharyngeal carcinoma cells via the modulation of the epidermal growth factor receptor (EGFR)/phosphoinositide-3 kinase (PI3K)/AKT signaling pathway. | Cell Death & Disease

Fig. 2: miR-133a-3p suppressed the proliferation of nasopharyngeal carcinoma cells via the modulation of the epidermal growth factor receptor (EGFR)/phosphoinositide-3 kinase (PI3K)/AKT signaling pathway.

From: VPS33B interacts with NESG1 to modulate EGFR/PI3K/AKT/c-Myc/P53/miR-133a-3p signaling and induce 5-fluorouracil sensitivity in nasopharyngeal carcinoma

Fig. 2

a, b 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (a) and 5-ethynyl-2′-deoxyuridine incorporation (b) assays were performed to demonstrate the impact of miR-133a-3p on the proliferation of A549 and H1975 cells. Student’s t test, one-way analysis of variance (ANOVA), mean ± SD, **P < 0.01. Scale bar: 100 μm. c That miR-133a-3p directly targeted EGFR was confirmed by luciferase reporter assay, one-way ANOVA, and Dunnett’s multiple comparison test. Mean ± SD, *P < 0.05. d Changes in EGFR, PI3K/AKT, p-PI3K/p-AKT, c-Jun, c-Myc, p53, CCND1, and E2F1 expression were detected by western blot analysis in HONE1 and SUNE1 cells after transfection of miR-133a-3p mimics. e Bioinformatics analysis revealed the promoter regions of miR-133a-3p with the putative p53 binding site. f Quantitative polymerase chain reaction (qPCR) and Gel electrophoresis confirmed the amplification of p53-binding sites after chromatin immunoprecipitation (ChIP) using antibody against p53. IgG antibody was used as the negative control. Student’s t test, mean ± SD, **P < 0.01, ***P < 0.001. g Electrophoretic mobility shift assay (EMSA) and supershift assay of p53 binding to miR-133a-3p promoter in HONE1 and SUNE1 cells. Labeled wild-type probe was incubated without (lane 1) or with (lane 4) cell nuclear proteins in the absence or presence of unlabeled probe (lanes 2–3). Unlabeled wild-type probe (lane 2) and mutant p53 probe (lane 3) were used to compete with p53 binding, each at 100-fold excess. Supershift assay (lane 5) was performed using an anti-p53 antibody. h Bioinformatics analysis revealed the promoter regions of p53 with the putative c-Myc binding site. i qPCR and Gel electrophoresis confirmed the amplification of c-Myc-binding sites after ChIP using antibody against c-Myc. IgG antibody was used as the negative control. Student’s t test, mean ± SD, **P < 0.01. j EMSA and supershift assay of c-Myc binding to p53 promoter in HONE1 and SUNE1 cells. Labeled wild-type probe was incubated without (lane 1) or with (lane 4) cell nuclear proteins in the absence or presence of unlabeled probe (lanes 2–3). Unlabeled wild-type probe (lane 2) and mutant c-Myc probe (lane 3) were used to compete with c-Myc binding, each at 100-fold excess. Supershift assay (lane 5) was performed using an anti-c-Myc antibody

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