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

摘要：

目的　探讨调控转化生长因子-β1（transforming growth factor-β1，TGF-β1）基因对骨肉瘤细胞的恶性增殖和干细胞样特性的影响以及可能的机制。方法　本文采用试验对照研究。研究时间为2021年6月至12月。选取对数生长期的人骨肉瘤细胞，采用TGF-β1-siRNA及TGF-β1重组腺病毒分别下调、过表达TGF-β1基因；反转录聚合酶链反应（qRT-PCR）和蛋白免疫印迹（Western blotting）来检测TGF-β1-siRNA干扰效率；通过四甲基偶氮唑盐（methyl thiazolyl tetrazolium，MTT）法和平板克隆形成实验检测细胞的增殖特性；流式细胞术分选CD133阳性表达的肿瘤干细胞（cancer stem cells，CSCs），微球体形成实验检测细胞自我更新能力；Western blotting检测细胞凋亡和干细胞特性相关蛋白。组间比较采用t检验。结果　与人正常成骨hFOB1.19细胞相比，TGF-β1在骨肉瘤细胞系均有不同程度的增高；其中U2OS细胞中上调程度最高（t=8.76，P<0.05），MG-63细胞中上调程度最少（t=4.33，P<0.05）。因此，U2OS细胞用于基因沉默实验，MG-63细胞用于基因过表达实验。qRT-PCR和Western blotting实验显示，si-TGF-β1#1的基因沉默效率最高（t=10.88，P<0.05），因此，后续实验采用si-TGF-β1#1。MTT法、流式细胞术结果显示，抑制TGF-β1表达后骨肉瘤细胞的细胞活力明显降低，细胞凋亡增加；而TGF-β1过表达则提高细胞活力，细胞凋亡减少。悬浮成球、CD133阳性细胞筛选实验结果显示，抑制TGF-β1表达后明显削弱骨肉瘤细胞的微球体形成能力（t=6.88，P<0.05）和CD133阳性细胞比例（t=9.37，P<0.05）；反之，过表达TGF-β1会使骨肉瘤干细胞干性表达增强。同时，裸鼠体内实验亦证实Lv-TGF-β1可扩大肿瘤的体积和质量；反之si-TGF-β1预处理后瘤体的体积和质量有所减轻（t=9.27，P<0.05）。结论　TGF-β1在骨肉瘤中发挥促癌基因角色，TGF-β1增强骨肉瘤细胞的恶性增殖和干细胞特性，其作用机制可能与磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）信号通路有关。

关键词:

骨肉瘤, TGF-β1, PI3K/AKT信号通路, 干细胞特性