Knockdown of TBRG4 suppresses proliferation, invasion and promotes apoptosis of osteosarcoma cells by downregulating TGF-β1 expression and PI3K/AKT signaling pathway.
敲除 TBRG4 通过下调 tgf-β 1 表达和 PI3K/AKT 信号通路抑制骨肉瘤细胞的增殖、侵袭和促进凋亡。
- 作者列表："Huang F","Zhou P","Wang Z","Zhang XL","Liao FX","Hu Y","Chang J
: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 调节因子 4 (TBRG4) 是几种肿瘤致瘤进展中的新型调节因子。然而，迄今为止，TBRG4 在骨肉瘤中的表达和功能尚不清楚。本研究的目的是探讨 TBRG4 在骨肉瘤中的潜在生物学功能。定量实时聚合酶链反应 (qRT-PCR) 检测 TBRG4 在骨肉瘤组织和细胞系中的表达。通过免疫组化评估骨肉瘤组织中 TBRG4 蛋白的水平。采用慢病毒介导的短发夹 (sh) RNA 敲除骨肉瘤细胞 TBRG4，qRT-PCR 和 Western blot 分别检测 TBRG4 mRNA 和蛋白的表达。随后，分别采用高含量筛选分析和 CCK8 试验、肿瘤球形成试验、流式细胞术和 Transwell 侵袭试验测定骨肉瘤细胞的增殖、克隆形成能力、凋亡和侵袭能力。此外，检测骨肉瘤细胞在体内的生长和转移情况，以及 TBRG4 对转化生长因子 β 1 (tgf-β 1) 的影响。通过 qRT-PCR 和 Western blot 实验分别探索 PI3K/AKT 信号通路。结果表明，TBRG4 在骨肉瘤组织和细胞系中过表达，证实 TBRG4 的高表达与肿瘤晚期、肿瘤大小和淋巴结转移有关。功能实验表明，敲低 TBRG4 可以在体外抑制骨肉瘤细胞的增殖、侵袭和诱导凋亡，也可以在体内抑制骨肉瘤的生长和转移。通过检测 tgf-β 1 、 p-PI3K 、 PI3K 、 p-AKT 和 AKT 的表达水平，表明抑制 TBRG4 会降低 tgf-β 1 的表达，并使 PI3K/AKT 信号通路失活。这些结果首次显示 TBRG4 敲除可抑制骨肉瘤进展，提示 TBRG4 可能是骨肉瘤治疗的一个有前途的治疗靶点。
METHODS:BACKGROUND:Osteosarcoma is the most common primary bone malignancy in children and adolescents. In order to find factors related to its recurrence, and thus improve recovery prospects, a powerful clinical signature is needed. Long noncoding RNAs (lncRNAs) are essential in osteosarcoma processes and development, and here we report significant lncRNAs to aid in earlier diagnosis of osteosarcoma. METHODS:A univariate Cox proportional hazards regression analysis and a multivariate Cox regression analysis were used to analyze osteosarcoma patients' lncRNA expression data from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET), a public database. RESULTS:A lncRNA signature consisting of three lncRNAs (RP1-261G23.7, RP11-69E11.4 and SATB2-AS1) was selected. The signature was used to sort patients into high-risk and low-risk groups with meaningful recurrence rates (median recurrence time 16.80 vs. >128.22 months, log-rank test, P143.80 months, log-rank test, P=0.006). A multivariate Cox regression analysis showed that the significant lncRNA was an independent prognostic factor for osteosarcoma patients. Functional analysis suggests that these lncRNAs were related to the PI3K-Akt signaling pathway, the Wnt signaling pathway, and the G-protein coupled receptor signaling pathway, all of which have various, important roles in osteosarcoma development. The significant 3-lncRNA set could be a novel prediction biomarker that could aid in treatment and also predict the likelihood of recurrence of osteosarcoma in patients.
METHODS::Long non-coding RNAs (lncRNAs) regulates the initiation and progression of osteosarcoma (OS), specifically lncRNA RP11-361F15.2 has been shown to play prominent roles in tumorigenesis. Previously, M2-Like polarization of tumor-associated macrophages (TAMs) has been identified to play a key role in cancer migration/invasion. Hence, it is essential to understand the role of RP11-361F15.2 in tumorigenesis and its association with M2-Like polarization of TAMs. The results indicate that RP11-361F15.2 is significantly increased in OS tissues, and its expression is positively correlated with cytoplasmic polyadenylation element binding protein 4 (CPEB4) expression and negatively associated with miR-30c-5p expression. Further, overexpression of RP11-361F15.2 increased OS cell migration/invasion and M2-Like polarization of TAMs in vitro, as well as promoted xenograft tumor growth in vivo. Mechanistically, luciferase reporter assays indicated that RP11-361F15.2 upregulated CPEB4 expression by competitively binding to miR-30c-5p. Further, we have identified that RP11-361F15.2 promotes CPEB4-mediated tumorigenesis and M2-Like polarization of TAMs through miR-30c-5p in OS. We also identified that RP11-361F15.2 acts as competitive endogenous RNA (ceRNA) against miR-30c-5p thereby binding and activating CPEB4. This RP11-361F15.2/miR-30c-5p/CPEB4 loop could be used as a potential therapeutic strategy for the treatment of OS.
METHODS:PURPOSE:The objective of this study was to investigate potential correlations between pathologic fractures (PFs) and prognosis of patients with primary central high-grade osteosarcoma of the extremities. METHODS:We retrospectively analyzed 2,847 patients registered in the Consecutive Cooperative Osteosarcoma Study Group database with primary central high-grade osteosarcoma of the extremities, treated between 1980 and 2010. Intended treatment included pre- and postoperative chemotherapy and surgery. Univariable and multivariable survival analyses were performed for all patients and then differentiated for adult and pediatric (≤ 18 years at time of diagnosis) patients. RESULTS:A total of 2,193 patients were ≤ 18 years of age; 11.3% of all patients had PFs. In the overall cohort, presence of PF correlated significantly with tumor site, histologic subtype, relative tumor size, and primary metastases, but not with body mass index or local surgical remission. In univariable analysis, 5-year overall survival (OAS) of patients with and without PF was 63% versus 71%, respectively (P = .007), and 5-year event-free survival (EFS) was 51% versus 58% (P = .026). In pediatric patients, OAS and EFS did not differ significantly between patients with and without PF. In adults, 5-year OAS in patients with and without PF was 46% versus 69% (P < .001), and 5-year EFS was 36% versus 56% (P < .001). In multivariable analysis, PF was not a statistically significant factor for OAS or EFS in the total cohort or in pediatric patients. In adult patients, PF remained an independent prognostic factor for OAS (P = .013; hazard ratio [HR], 1.893). It was not a significant prognostic factor for EFS (P = .263; HR, 1.312). CONCLUSION:In this largest study to date with extremity osteosarcomas, we observed the occurrence of PF to correlate with inferior OAS expectancies in adult but not in pediatric patients.