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. 2023 Oct 3;29(1):133.
doi: 10.1186/s10020-023-00722-6.

RILP inhibits tumor progression in osteosarcoma via Grb10-mediated inhibition of the PI3K/AKT/mTOR pathway

Zhun Wei #  1 , Kezhou Xia #  1 , Di Zheng #  1 , Changtian Gong  1 , Weichun Guo  2
Affiliations

RILP inhibits tumor progression in osteosarcoma via Grb10-mediated inhibition of the PI3K/AKT/mTOR pathway

Zhun Wei et al. Mol Med. .

Abstract

Background: Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism.

Methods: Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed.

Results: Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial-mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway.

Conclusion: Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment.

Keywords: Autophagy; Epithelial–mesenchymal transition; Osteosarcoma; RILP.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
RILP is downregulated in osteosarcoma and its lower level correlates with poor prognosis in osteosarcoma patients. A Expression profile of RILP in various tumors based on TIMER database. B The relative mRNA level of RILP in tumor and adjacent tissues based on clinical samples. C The protein expression level of RILP in tumor and adjacent tissues on clinical samples was detected by western blotting. GAPDH was used as a loading control. D Quantitative analysis of the protein expression level of RILP. E The protein expression level of RILP in human osteoblasts (hFOB1.19) and osteosarcoma cell lines (HOS, MG63, U2OS and 143B) were subjected to western blotting analysis. GAPDH was used as a loading control. F The relative mRNA level of RILP in human osteoblasts (hFOB1.19) and osteosarcoma cell lines (HOS, MG63, U2OS and 143B). G–I Kaplan–Meier survival analysis of osteosarcoma patients with high and low expression of RILP in TCGA and GEO database. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
Overexpression of RILP inhibits proliferation, migration and invasion of osteosarcoma cells. A, B Overexpression of RILP was determined by western blot assays. GAPDH was used as a loading control. C, D The proliferation level of osteosarcoma after transfected with LV-RILP or LV-Control was evaluated by CCK-8 assay. E, F The colony formation ability of osteosarcoma cells stably overexpressing RILP or not was measured by colony formation assay. G Immunofluorescence staining assay of Ki-67 in osteosarcoma cells was performed the to measure the proliferation level, scale bar: 200 μm. H–J Representative images and quantitative analysis of osteosarcoma cell migration based on wound healing assay, scale bar: 200 μm. K, L Increased expression of RILP restrained osteosarcoma cell invasion ability based on transwell assay, scale bar: 400 μm. M–O Western blot and quantitative analyses of E-cadherin, N-cadherin and Vimentin. GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
Silencing of RILP promotes proliferation, migration and invasion of osteosarcoma cells. A, B The protein expression level of RILP after transfected with shRILP or not were detected by western blot assays. GAPDH was used as a loading control. C, D CCK-8 assay was applied to measure the proliferation level of osteosarcoma cells after transfected with shRILP or not. E, F Colony formation assay was performed to evaluate the colony formation ability of osteosarcoma cells after transfected with shRILP or not. G Immunofluorescence staining assay of Ki-67 in osteosarcoma cells was performed the to measure the proliferation level, scale bar: 200 μm. H–J The migration ability of osteosarcoma cells stably knocking down of RILP or not was detected by wound healing assay, scale bar: 200 μm. K, L The invasion ability of osteosarcoma cells stably knocking down of RILP or not was detected by transwell assay, scale bar: 400 μm. M–O Western blot and quantitative analyses of EMT-related protein including N-cadherin, E-cadherin and Vimentin. GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
RILP negatively regulates PI3K/AKT/mTOR pathway to restrain the proliferation, migration and invasion of osteosarcoma cells. A Heatmap illustrating RILP-related genes based on difference analysis. B KEGG enrichment analysis revealed that RILP-associated genes are mainly enriched in PI3K-AKT signaling pathway. C–E Western blot assay was performed to detect the protein expression level of PI3K, p-PI3K, AKT, p-AKT, mTOR and p-m-TOR in osteosarcoma cells stably overexpressing RILP. GAPDH was used as a loading control. F–H Western blot assay was performed to detect the protein expression level of PI3K, p-PI3K, AKT, p-AKT, mTOR and p-m-TOR in osteosarcoma cells stably knocking down RILP. GAPDH was used as a loading control. *P < 0.05, **P < 0.01
Fig. 5
Fig. 5
RILP induces OS cell autophagy by inhibiting PI3K/AKT/mTOR pathway. A, B Representative transmission electron micrographs autophagic vesicles in 143B and U2OS cells stably transfected with shNC/shRILP and LV-Control/LV-RILP. C–E The protein expression level of Beclin1 and p62 in osteosarcoma cells was detected after treated with 740Y-P or not. GAPDH was used as a loading control. F, G Representative images and quantitative analysis of Beclin1 immunofluorescence intensity in osteosarcoma cells stably transfected with RILP or not, scale bar: 200 μm. *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
The effect of RILP expression can affect migration and invasion of osteosarcoma cells that is mediated by autophagy. A–C Wound healing assay comparing the motility of osteosarcoma cells after treated with 3-methyladenine or not, scale bar: 200 μm. D, E Transwell invasion assay was performed to compare the invasion ability of osteosarcoma cells after treated with 3-methyladenine or not, scale bar: 400 μm. F–H Western blot analysis was applied to measure the protein expression level of E-cadherin, N-cadherin and Vimentin. GAPDH was used as a loading control. *P < 0.05, **P < 0.01
Fig. 7
Fig. 7
RILP restrains osteosarcoma progression via Grb10-mediated inhibition of PI3K/AKT/mTOR pathway. A Molecular docking techniques show that RILP can interact with Grb10. B RILP and Grb10 bind endogenously in 143B cells. C RILP and Grb10 bind exogenously in 293T cells transfected with flag-RILP and HA-Grb10 plasmid. D, E Western blot and quantitative analyses of Grb10 and p-Grb10 in RILP-upregulated cells. F, G Western blot and quantitative analyses of Grb10 and p-Grb10 in RILP-silenced cells. H–J Evaluation of p-PI3K and p-AKT in RILP-overexpressing cells after transfection of siGrb10 or siNeg. K–M Evaluation of p-PI3K and p-AKT in RILP-silenced after transfected with Grb10 overexpression plasmid or empty vector. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 8
Fig. 8
Increased expression of RILP inhibits tumor growth in vivo. A Tumor volume was monitored every 7 days to measure the tumor growth in vivo. B Comparison of xenograft tumor size in nude mice after injecting 143B osteosarcoma cells stably transfected with LV-Control/LV-RILP. C Tumor weight was compared between two groups. D, E Western blot and quantitative analyses of RILP, Grb10, p-Grb10, PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR in xenograft tumor. F IHC analysis of RILP, p-Grb10, Ki-67, p-AKT, p-mTOR for tissues of xenograft tumors. Scale bar: 200 μm. G, H Representative H&E staining images of lung tissue in two groups, the red arrows represent lung metastasis. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 9
Fig. 9
Working model of RILP regulating proliferation and autophagy in osteosarcoma
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