TRIM17 promotes the progression of osteosarcoma by regulating PDK1 m6A modification-mediated AKT/mTOR pathway activation through ubiquitination of FTO

Figures

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  Fig. 1. TRIM17 is highly expressed in osteosarcoma tissues and cells and is negatively correlated with patient prognosis. A Volcanic maps show TRIM family genes associated with prognosis in patients with osteosarcoma. B RNA-Seq analysis of TRIM genes differentially expressed in osteosarcoma tissues and adjacent normal tissues. C Venn diagram of differentially expressed TRIM family genes. D, E TRIM17 expression levels in osteosarcoma tissues and adjacent normal tissues were detected by IHC. F, G TRIM17 expression levels in osteosarcoma tissues and adjacent normal tissues were detected by Western blot. H–J TRIM17 expression levels in normal osteoblasts and osteosarcoma cell lines. K Kaplan–Meier survival analysis of TRIM17 gene. L ROC curve was used to analyze the prognosis accuracy of TRIM17. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001.

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  Fig. 2. TRIM17 knockdown inhibited clonability and survival potential, migration, and invasion of osteosarcoma cells. A–C The expression level of TRIM17 in knockdown stable cells was verified. D–G The effect of TRIM17 knockdown on cell clonability and survival potential was detected by CCK-8 and colony formation assay, scale bar: 200 μm. H, I Transwell invasion assay was used to detect the effect of TRIM17 knockdown on cell invasion ability, scale bar: 200 μm. J, K The effect of TRIM17 knockdown on cell migration was detected by wound healing assay, scale bar: 200 μm. L–N Effect of TRIM17 knockdown on expression of EMT-related proteins. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001.

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  Fig. 3. Overexpression of TRIM17 promotes clonability and survival potential, migration, and invasion of osteosarcoma cells. A–C The expression level of TRIM17 in overexpressed stable cells was verified. D–G The effect of overexpression of TRIM17 on cell clonability and survival potential was detected by CCK-8 and colony formation assay, scale bar: 200 μm. H, I Transwell invasion assay was used to detect the effect of overexpression of TRIM17 on cell invasion ability, scale bar: 200 μm. J, K The effect of overexpression of TRIM17 on cell migration was detected by wound healing assay, scale bar: 200 μm. L–N Effect of overexpression of TRIM17 on expression of EMT-related proteins. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). ** P < 0.01, *** P < 0.001.

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  Fig. 4. TRIM17 knockdown inhibits AKT/mTOR signaling pathway activity. A, B Differentially expressed genes after silencing TRIM17 gene expression. C KEGG enrichment analysis. D Effect of silencing TRIM17 on AKT/mTOR pathway gene expression. E–G Effect of silencing TRIM17 on AKT/mTOR pathway protein phosphorylation. H–J Effects of overexpression of TRIM17 on AKT/mTOR pathway protein phosphorylation. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). ** P < 0.01, *** P < 0.001.

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  Fig. 5. TRIM17 interacts with FTO and FTO regulates the AKT/mTOR signaling pathway. A Overexpression efficiency of 293T cells transfected with plasmid. B The interacting proteins of TRIM17 were identified by IP-MS. C Molecular docking predicts the interaction between TRIM17 and FTO. D Endogenous Co-IP of TRIM17 and FTO in untransfected 143B and U2OS cells. IgG was used as a negative control. E The interaction between TRIM17 and FTO in 293T cells co-transfected with TRIM17 and FTO overexpressing plasmids was detected by Co-IP assay. F The co-localization of TRIM17 and FTO was detected by cellular immunofluorescence, scale bar: 10 μm. G Kaplan–Meier survival analysis of FTO gene. H Correlation between TRIM17 and FTO in TCGA osteosarcoma dataset. I–K Effect of silencing FTO on AKT/mTOR pathway protein phosphorylation. L–N Effects of overexpression of FTO on AKT/mTOR pathway protein phosphorylation. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). ** P < 0.01, *** P < 0.001.

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  Fig. 6. TRIM17 promotes ubiquitination and degradation of FTO protein. A, B The effect of overexpression of TRIM17 on FTO mRNA was detected by qRT-PCR. C, D Effect of overexpression of TRIM17 on the level of FTO protein. E, F Effect of silencing TRIM17 on FTO protein levels. G–J Cells were treated with CHX (100 μg/mL) and collected at 0, 2, 4, and 8 h. The FTO protein level was detected and the kinetics of protein degradation was analyzed. K, L Effect of TRIM17 on ubiquitination level of FTO protein. M–O Overexpression of FTO regulates the effects of TRIM17 on AKT/mTOR signaling pathway. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). ** P < 0.01, *** P < 0.001.

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  Fig. 7. FTO-mediated m6A demethylation regulates PDK1 mRNA stability. A, B Differential gene expression in si-NC group and si-FTO group. C The number of m6A enriched regions in MeRIP-seq sequencing. D m6A modifies the motif region. E Venn map of differential genes. F Kaplan–Meier survival analysis of PDK1 gene. G–I The effect of FTO expression on PDK1 expression level. J MeRIP assay verified the existence of m6A modification in PDK1. K Effect of FTO on PDK1 m6A modification level. L Influence of Dual-Luciferase Reporter Assay on plasmid luciferase activity. The PDK1-MUT is a c.301 A → G mutation (corresponding to p.Lys101 →Glu) in the PH domain. M Effect of FTO on the half-life of PDK1 mRNA. N The levels of PDK1 mRNA were detected by qRT-PCR. O Western blot was used to detect the protein expression in each group. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). ** P < 0.01, *** P < 0.001.

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  Fig. 8. TRIM17 influences the progression of osteosarcoma in vivo via the FTO/PDK1/AKT/mTOR signaling axis. A–C Silencing TRIM17 can inhibit the volume and weight of osteosarcoma in vivo. D, E The expression levels of TRIM17, FTO, PDK1, EMT-related proteins, and AKT/mTOR related proteins in subcutaneous tumor tissues were detected by western blot. F mRNA levels of TRIM17, FTO, and PDK1 in subcutaneous tumor tissues were detected by qRT-PCR. G The expression levels of TRIM17, FTO, PDK1, EMT-related proteins, and AKT/mTOR related proteins in subcutaneous tumor tissue samples were analyzed by immunohistochemistry (IHC). Scale: 400 μm. H, I HE staining and in vivo imaging showed the number of lung metastases in nude mice in the shNeg-Luc and shTRIM17-Luc groups. Scale: 200 μm. Student’s t-test (two groups) and one-way analysis of variance (more than two groups) were used to analyze the differences between groups. All data are expressed as mean ± standard deviation (SD). ** P < 0.01, *** P < 0.001.