Department of Orthopaedics, The Fourth Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Department of Orthopaedics, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Arch Biochem Biophys. 2020 Jun 15;686:108351. doi: 10.1016/j.abb.2020.108351. Epub 2020 Mar 30.
Transforming growth factor beta regulator 4 (TBRG4) is a novel regulator in tumorigenic progression of several tumors. However, so far, the expression and functions of TBRG4 in osteosarcoma are unknown. The aim of this study was to investigate the potential biological functions of TBRG4 in osteosarcoma. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of TBRG4 in osteosarcoma tissues and cell lines. The levels of TBRG4 protein in osteosarcoma tissues were assessed by immunohistochemistry. Lentivirus-mediated short hairpin (sh) RNA was employed to knock down TBRG4 in osteosarcoma cells, and the expressions of TBRG4 mRNA and protein were determined by qRT-PCR and Western blot assay, respectively. Subsequently, the proliferation, clonogenic ability, apoptosis and invasion of osteosarcoma cells were measured using high content screening analysis and CCK8 assay, tumor sphere formation assay, flow cytometry and Transwell invasion assays, respectively. Furthermore, the osteosarcoma cells growth and metastasis in vivo were detected, and the effect of TBRG4 on the transforming growth factor β1 (TGF-β1) and PI3K/AKT signaling pathway was explored by qRT-PCR and Western blot assay, respectively. The results showed the levels of TBRG4 were overexpressed in osteosarcoma tissues and cell lines, confirming that the high TBRG4 expression was related to advanced tumor stages, large tumor size, and lymph node metastasis. Functional assays showed knockdown of TBRG4 could inhibit proliferation, invasion and induce apoptosis of osteosarcoma cells in vitro, and could also suppress osteosarcoma growth and metastasis in vivo. By examining the expression levels of TGF-β1, p-PI3K, PI3K, p-AKT and AKT, it showed that the suppression of TBRG4 would reduce TGF-β1 expression and inactivate the PI3K/AKT signaling pathway. These results showed for the first time that TBRG4 knockdown could suppress osteosarcoma progression, suggesting TBRG4 might be a promising therapeutic target for osteosarcoma treatment.
转化生长因子β调节剂 4（TBRG4）是几种肿瘤发生进展中的新型调节剂。然而，到目前为止，TBRG4 在骨肉瘤中的表达和功能尚不清楚。本研究旨在探讨 TBRG4 在骨肉瘤中的潜在生物学功能。采用实时定量聚合酶链反应（qRT-PCR）检测骨肉瘤组织和细胞系中 TBRG4 的表达。采用免疫组织化学法检测骨肉瘤组织中 TBRG4 蛋白水平。采用慢病毒介导的短发夹（sh）RNA 敲低骨肉瘤细胞中的 TBRG4，通过 qRT-PCR 和 Western blot 检测 TBRG4 mRNA 和蛋白的表达。随后，通过高内涵筛选分析和 CCK8 检测、肿瘤球形成检测、流式细胞术和 Transwell 侵袭检测分别测量骨肉瘤细胞的增殖、克隆形成能力、凋亡和侵袭。此外，通过 qRT-PCR 和 Western blot 检测分别检测 TBRG4 对转化生长因子β1（TGF-β1）和 PI3K/AKT 信号通路的影响，检测 TBRG4 对骨肉瘤细胞体内生长和转移的影响。结果表明，TBRG4 在骨肉瘤组织和细胞系中过表达，证实高 TBRG4 表达与肿瘤晚期、肿瘤体积大、淋巴结转移有关。功能研究表明，TBRG4 敲低可抑制骨肉瘤细胞的体外增殖、侵袭并诱导其凋亡，还可抑制骨肉瘤的体内生长和转移。通过检测 TGF-β1、p-PI3K、PI3K、p-AKT 和 AKT 的表达水平，结果表明 TBRG4 的抑制可降低 TGF-β1 的表达并使 PI3K/AKT 信号通路失活。这些结果首次表明，TBRG4 敲低可抑制骨肉瘤的进展，提示 TBRG4 可能是骨肉瘤治疗的有前途的治疗靶点。
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