Fig 1: EGFR/MET participated in regulation of phosphorylation of hetero-RTKs.A Western Blot was used to observe phosphorylation of RTKs in HCCLM3, MHCC97H and PLC/PRF/5 cell lines with Lenvatinib or Sorafenib added. B Western blot was used to determine the effect of down-regulation of EGFR/MET on hetero-RTKs, including: Phosphorylation of EGFR, MET, Her3, RON, IR/IGF-1R and RET in HCC cell lines with EGFR/MET inhibitor added. C Phosphorylation of EGFR, Her3, RON, IR/IGF-1R and RET in HCC cell lines with MET knock-down. D Phosphorylation of FGFR1, VEGFR2 and PDGFRβ with ligand stimulated in HCC cell lines with EGFR/MET inhibitor added. E Phosphorylation of FGFR1, VEGFR2 and PDGFRβ with ligand stimulated in HCC cell lines with MET knock-down. F Phosphorylation of RTKs in PLC/PRF/5 cell line with EGFR or MET knock-down.
Fig 2: miR-592 mimic abolishes the effect of ERBB3 overexpression on migration, invasion and colony formation in EOC cells. (A) Kaplan–Meier survival curves of patients with EOC at high and low ERBB3 expression in the Kaplan–Meier Plotter database. (B) SKOV3 cells were transfected with ERBB3 or/and miR-592 mimic. CCK-8 assay was used to measure cell viability in SKOV3. **P < .01. (C and D) Colony formation assay was conducted to assess SKOV3 cell proliferation. *P < .05; ***P < .001. Transwell assay was performed to assess cell migration (E and F) and invasion (G and H) in SKOV3 cells transfected with ERBB3 or/and miR-592 mimic. *P < .05; **P < .01; ****P < .0001. Values represent the means ± SD for three separate experiments.
Fig 3: ERBB3 interaction network and the mechanism underlying the miR-592/ERBB3 axis during EOC development. (A) Protein–protein interactions identified by STRING software. (B) The molecular mechanism underlying the miR-592/ERBB3 axis during EOC tumorigenesis.
Fig 4: miR-592 targets on PI3K/AKT pathway by negatively regulating ERBB3. (A) The interactions between the miR-592: target mRNAs were predicted by using bioinformatics tools such as miRTargetlink (https://ccb-web.cs.uni-saarland.de/mirtargetlink/network.php?type=miRNA&qval=hsa-miR-592). (B) Schematic illustration of the predicted ERBB3 3′-untranslated region (UTR)-binding site of miR-592. (http://www.targetscan.org). (C) SKOV3 cells were co-transfected with miRNA precursor (miR-592 or NC), a luciferase reporter vector containing the wildtype or mutant 3′-UTR of ERBB3, and the Renilla luciferase control vector. After 24 hours of incubation, the luciferase activity level was measured (normalized to Renilla activity). Data are represented as mean ± standard deviation and were obtained from three independent experiments. **P < .01. Values represent the means ± SD for three separate experiments. (D) Linear correlation between miR-592 and ERBB3 expression was analyzed by Spearman's correlation in EOC tissues from 12 patients. Spearman r = -0.6154; P = .0373. (E) Relative analysis of human miR-592 and ERBB3 expression in EOC (n = 6) and normal (n = 6) individuals. *P < .05. (F) Western blot results for p-PI3K/PI3K, p-AKT/AKT, and ERBB3 expressions in SKOV3 cells transfected with miR-592 or NC. The ERBB3 levels were quantified by scanning densitometry and normalized to the level of β-actin.
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