SLC25A22 Promotes Proliferation and Metastasis of Osteosarcoma Cells via the PTEN Signaling Pathway

Abstract

Osteosarcoma is a highly malignant bone tumor. However, due to the high complexity of the occurrence and metastasis of osteosarcoma, the exact mechanism promoting its development and progression remains to be elucidated. This study highlights the causal link between solute carrier family 25 member 22 (SLC25A22) and the development, progression, and metastasis of osteosarcoma. SLC25A22 is upregulated in human osteosarcoma and predicts a poor prognosis. The upregulation of SLC25A22 in osteosarcoma tissues was significantly associated with cell proliferation, invasion, and metastasis. Studies of functional gain (overexpression) and loss (knockdown) showed that SLC25A22 significantly increases the ability of osteosarcoma cells to proliferate, as well as invade and metastasize in vitro. At the same time, the expression of SLC25A22 promoted the progression of the cellcycle of osteosarcoma cell lines and inhibited the apoptosis of osteosarcoma cells. Analysis using a mouse xenograft model showed that xenografts of SLC25A22 stable overexpressing osteosarcoma cells had a significant increase in tumor volume and weight compared to the control group. Lung metastasis models in mice showed that expression of SLC25A22 promoted lung metastasis of osteosarcoma in vivo. Furthermore, SLC25A22 inhibited phosphatase and tensin homolog expression and increased phosphorylation of protein kinase b (Akt) and Focal Adhesion Kinase (FAK) in the phosphatase and tensin homolog signaling pathway. In summary, SLC25A22 is highly expressed in osteosarcoma, promoting osteosarcoma cell proliferation and invasion by inhibiting the phosphatase and tensin homolog signaling pathway.

Keywords: PTEN signaling pathway; SLC25A22; metastasis; osteosarcoma; proliferation.

Figure 1.

SLC25A22 is highly expressed in human osteosarcoma. A, The total RNA of 40 osteosarcoma tissues and their adjacent tissues was extracted, and SLC25A22 mRNA was detected by RT-PCR. B, The protein of 40 osteosarcoma tissues and their adjacent tissues was extracted and the protein level of SLC25A22 was detected by Western blot. C, The expression of SLC25A22 in osteosarcoma tissues and paracancerous tissues was detected by IHC. D, A typical IHC schematic showing the expression of SLC25A22 in osteosarcoma tissues and adjacent tissues. E, Survival curve of patients with osteosarcoma expressing SLC25A22 at high and low levels. IHC indicates immunohistochemical; RT-PCR, Reverse transcriptase-polymerase chain reaction.

Figure 2.

SLC25A22 increases proliferation of osteosarcoma cell lines in nude mice models. A, 293, SF-86, U2OS, Saos-2, 143B, MG63, HOS cells were lysed and SLC25A22 protein levels were detected by Western blot. B, SLC25A22 knockdown efficiency and overexpression were detected by Western blot. C, The knockdown and overexpression efficiency of SLC25A22 was examined by RT-PCR. D, SLC25A22 KD U2OS and Saos-2 cells and overexpressing HOS cells were cultured in 96-well plates, and the number and viability of the cells were respectively measured using CCK-8 after 1, 2, and 3 days of culture. E, SLC25A22 KD U2OS and Saos-2 cells and overexpressing HOS cells were subjected to colony formation experiments, and the number of clones was recorded. F, Saos-2 cells and HOS cells were infected with lentivirus containing SLC25A22-shRNA and SLC25A22 overexpression vector, respectively. Stably infected Saos-2 cells and HOS cells were implanted subcutaneously in nude mice. After 3 weeks, the tumor was removed and photographed. G, During tumor growth, the tumor volume was recorded. H, At the end of the experiment, the tumor weight of each group was measured. I, After the above-mentioned grouped tumor tissues were embedded in paraffin, the expression of PCNA was detected by immunohistochemistry. J, The tumor paraffin sections were subjected to the TUNEL assay, and green fluorescence represented tissue cells with DNA damage.

Figure 3.

SLC25A22 accelerates cell cycle progression and inhibits cell apoptosis of osteosarcoma cells. A, SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells were subjected to cell cycle detection by flow cytometry. The results were analyzed with Modifit software and the proportions for each period were calculated. B, SLC25A22, cyclin B1, cdc25c, and cyclin D1 protein levels were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells. C, Forty-eight hours after U2OS and Saos-2 cells were transfected with shRNA (SLC25A22 KD or control), cells were stained with FITC-Annexin V and PE-PI, and apoptotic cells were detected by flow cytometry and subsequently analyzed with Flowjo software. D, Cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bad were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells.

Figure 4.

A, SLC25A22 promotes the invasion and metastasis of osteosarcoma cells by altering the PTEN signaling pathway. Wound healing assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in 6-well plates for wound healing experiments, and healing was observed and photographed at 24 hours. B, Cell invasion assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in Transwells and the invaded cells were stained with crystal violet and photographed. C, E-cadherin, vimentin, and MMP-9 protein levels were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. D, PTEN, phosphorylated Akt, and phosphorylated FAK were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. E, Control and SLC25A22 knockdown Saos-2 cells were used to construct a lung metastasis model, and HE staining showed 2 groups of lung metastases. F, Comparison of the number of lung metastatic cells in the control and SLC25A22 knockdown groups. G, The number of mice with lung metastases in the two groups, control and SLC25A22 knockdown lung metastasis model mice.