Fig 1.
Increased IGF-1R expression in HCC cells.
(A) IGF-1R expression in various cancers. (B) IGF-1R protein levels in patients with HCC and normal individuals (*P < 0.05). (C) Survival analysis of IGF-1R. (D, E) IGF-1R expression in HCC and normal liver cells (***P < 0.001). (F, G) Differences in protein expression between HCC and normal liver cells via laser confocal microscopy (**P < 0.01, ***P < 0.001). (H) Protein expression in HCC and normal liver cells via immunohistochemical staining. All data are expressed as mean ± standard deviation from at least three independent experiments. Comparisons between two groups were analyzed using a two-tailed Student’s t-test. Comparisons among multiple groups were analyzed via one-way analysis of variance followed by Tukey’s post-hoc test. A P-value of <0.05 was considered statistically significant.
Fig 2.
IGF-1R promotes HCC cell proliferation.
(A, B) Western blotting assay revealing that siRNA2 and siRNA3 lentiviruses downregulated IGF-1R in HepG2 and Hep3B cells (**P < 0.01, ***P < 0.001). (C, D) Clone formation assay to detect the differences in the cell proliferation abilities of each cell group (*P < 0.05, **P < 0.01). (E, F) EdU assay to detect the differences in the cell proliferation abilities of each group (*P < 0.05, × 200). All data are expressed as mean ± standard deviation from at least three independent experiments. Comparisons between two groups were analyzed using a two-tailed Student’s t-test. Comparisons among multiple groups were analyzed via one-way analysis of variance followed by Tukey’s post-hoc test. A P-value of <0.05 was considered statistically significant. Data represent mean ± standard error of mean. n = 3.
Fig 3.
IGF-1R drives HCC cell migration and inhibits apoptosis.
(A, B) Scratch healing assay to detect the differences in the cell migratory ability of each group (*P < 0.05, **P < 0.01). (C, D) Transwell assay to detect the differences in the cell migratory ability of each group (*P < 0.05, **P < 0.01). (E, F) JC-1 assay to detect the differences in the apoptotic ability of each group (*P < 0.05, **P < 0.01, ***P < 0.001). All data are expressed as mean ± standard deviation from at least three independent experiments. Comparisons between two groups were analyzed using a two-tailed Student’s t-test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. A P-value of <0.05 was considered statistically significant. Data represent mean ± SEM. n = 3.
Fig 4.
IGF-1R drives HCC progression via the Akt/GSK-3β signaling pathway.
(A, B, C) Immunoblotting assay to detect the levels of p-IGF-1R, p-Akt, p-GSK-3β, and Snail in each cell group (*P < 0.05, **P < 0.01, ***P < 0.001). (D, E, F) Immunoblotting assay to detect the protein levels of E-cadherin and N-cadherin in each cell group (*P < 0.05, **P < 0.01, ***P < 0.001). Each immunoblotting experiment included three independent biological replicates. Band intensity was quantified using ImageJ software. Data were expressed as mean ± SEM. β-actin was used as the loading control. (G) Immunofluorescence staining assay to detect the protein levels of E-cadherin and N-cadherin in each cell group. Comparisons between two groups were analyzed using an unpaired t-test. Data were derived from three independent experiments (n = 3 biological replicates/group). Error bars represent SEM, and statistical significance is indicated in the figures.
Fig 5.
Reversal of IGF-1R’s Effects on HCC by suppressing Akt/GSK-3β (A, B,C).
The protein levels of p-Akt, p-GSK-3β, Snail, E-cadherin, and N-cadherin across experimental groups (*P < 0.05, **P < 0.01, ***P < 0.001). (D, E) Differences in the proliferation capacity of each group via the EdU assay (**P < 0.01, ***P < 0.001). (F, G) Cell proliferation ability of each cell group in the clone formation assay (*P < 0.05, **P < 0.01). (H, I) Transwell assay to determine the cell migration ability of each group (*P < 0.05, **P < 0.01). (J, K) Cell migration ability of each group. (L, M) Apoptotic capacity of each group via the JC-1 assay (*P < 0.05, **P < 0.01). All data are expressed as mean ± standard deviation from at least three independent experiments. Comparisons between two groups were analyzed using a two-tailed Student’s t-test. Comparisons among multiple groups were analyzed via one-way analysis of variance followed by Tukey’s post-hoc test. A P-value of <0.05 was considered statistically significant. Error bars represent SEM, and statistical significance is indicated in the figures.
Fig 6.
Effect of IGF-1R on HCC growth in vivo.
(A, B, C) Tumor weight and volume formed by each group of cells in vivo (**P < 0.01, ***P < 0.001). All data are expressed as mean ± standard deviation (SD) from at least three independent experiments. Comparisons between two groups were analyzed using a two-tailed Student’s t-test. In contrast, comparisons among multiple groups were analyzed using one-way analysis of variance, followed by Tukey’s post hoc test. A P-value of <0.05 was considered statistically significant.