关键词:

骨肉瘤, TGF-β1, PI3K/AKT信号通路, 干细胞特性

Abstract:

Objective　To investigate the role of transforming growth factor-β1 (TGF-β1) in malignant proliferation and stemness features of osteosarcoma MG-63 cells and possible mechanism. Methods　The study ran from June to December 2021. Overexpression and knockdown of TGF-β1 in osteosarcoma cell lines was performed using lentiviral vector- TGF-β1 (Lv-TGF-β1) and siRNA- TGF-β1 (si- TGF-β1), respectively. Reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting were used to defect the siRNA interference efficiency. The clone formation assay and methyl thiazolyl tetrazolium (MTT) assay were used to investigate the proliferation of osteosarcoma MG-63 cells. The flow cytometry was used to detect the ratio of CD133 positive cancer stem cells (CSCs). Mammosphere-formation assay was used to determine the self-renewal ability. Apoptosis and stemness-related proteins were detected by Western blotting. t test was used for comparison between groups. Results　Compared with normal human hFOB1.19 osteoblasts, TGF-β1 increased in the osteosarcoma cell lines to varying degrees. Among which, the U2OS cells had the highest degree of upregulation (t=8.76, P<0.05), and the MG-63 cells had the least degree of upregulation (t=4.33, P<0.05). Therefore, the U2OS cells were used for gene silencing experiments, and the MG-63 cells were used for gene overexpression experiments. qRT-PCR and Western blotting experiments showed that si-TGF-β1#1 had the highest gene silencing efficiency (t=10.88, P<0.05). Therefore, si-TGF-β1#1 was used in subsequent experiments. The results of MTT assay and flow cytometry showed that the cell viability of osteosarcoma cells decreased significantly and the apoptosis increased after TGF-β1 expression was inhibited; overexpression of TGF-β1 increased the cell viability and decreased the apoptosis. Inhibition of TGF-β1 expression significantly reduced the microsphere-forming ability of osteosarcoma cells (t=6.88, P<0.05) and the proportion of CD133-positive cells (t=9.37, P<0.05). Conversely, overexpression of TGF-β1 enhanced the stemness expression of osteosarcoma stem cells. Meanwhile, in vivo experiments in nude mice also confirmed that Lv-TGF-β1 could expand the volume and weight of tumors. On the contrary, after si-TGF-β1 pretreatment, the tumor volume and weight of tumors were decreased (t=9.27, P<0.05). Conclusions　TGF-β1 plays an oncogenic role in osteosarcoma. TGF-β1 enhances the malignant proliferation and stemness features of osteosarcoma cells, and the mechanism of action may be related to phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway.

Key words:

Osteosarcoma, TGF-β1, PI3K/AKT signaling pathway, Stemness features