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.

中文翻译：

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TBRG4的抑制通过下调TGF-β1表达和PI3K / AKT信号通路来抑制骨肉瘤细胞的增殖，侵袭并促进其凋亡。

转化生长因子β调节剂4（TBRG4）是几种肿瘤的致瘤进展中的新型调节剂。但是，到目前为止，TBRG4在骨肉瘤中的表达和功能尚不清楚。这项研究的目的是调查TBRG4在骨肉瘤中的潜在生物学功能。实时定量聚合酶链反应（qRT-PCR）用于检测TBRG4在骨肉瘤组织和细胞系中的表达。通过免疫组织化学评估骨肉瘤组织中TBRG4蛋白的水平。用慢病毒介导的短发夹（sh）RNA敲除骨肉瘤细胞中的TBRG4，并分别通过qRT-PCR和Western blot法检测TBRG4 mRNA和蛋白的表达。随后，增殖，克隆形成能力，分别使用高含量筛选分析和CCK8分析，肿瘤球形成分析，流式细胞术和Transwell入侵分析测量骨肉瘤细胞的凋亡和侵袭。此外，检测了体内的骨肉瘤细胞的生长和转移，并分别通过qRT-PCR和Western blot方法研究了TBRG4对转化生长因子β1（TGF-β1）和PI3K / AKT信号通路的影响。结果表明，TBRG4在骨肉瘤组织和细胞系中过表达，证实了高TBRG4的表达与晚期肿瘤，大肿瘤和淋巴结转移有关。功能分析表明，敲除TBRG4可以在体外抑制骨肉瘤细胞的增殖，侵袭并诱导其凋亡，并且还可以在体内抑制骨肉瘤的生长和转移。通过检查TGF-β1，p-PI3K，PI3K，p-AKT和AKT的表达水平，表明抑制TBRG4会降低TGF-β1的表达并使PI3K / AKT信号通路失活。这些结果首次表明，TBRG4基因敲低可以抑制骨肉瘤的进展，表明TBRG4可能是有希望的骨肉瘤治疗靶标。