OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH TBRG4 Knockdown Suppresses Proliferation and Growth of Human Osteosarcoma Cell Lines MG63 Through PI3K/Akt Pathway This article was published in the following Dove Press journal: OncoTargets and Therapy Fei Huang1,* Background: The transforming growth factor β regulator 4 (TBRG4) has been proved to be Faxue Liao1,* involved in various types of tumor. However, its contribution in human osteosarcoma (OS) is Guangwen Ma1 still unclear. Yo n g H u 2 Patients and Methods: In the present study, immunohistochemistry and quantitative real- Chi Zhang1 time PCR were performed to investigate the expression of TBRG4 in OS tissues obtained from patients and three types of cell lines. The effect of TBRG4 knockdown using lentivirus Pengfei Xu1 1 on tumorigenesis was detected by CCK8, high-content screening analysis, colony formation Tangbing Xu fl 3 assay and ow cytometric analysis. Bioinformatics analysis was operated to investigate Jun Chang related signaling pathways following TBRG4 knockdown. 1Department of Orthopaedics, The Results: The results showed that the expression of TBRG4 increased significantly in OS fi Fourth Af liated Hospital of Anhui tissues and MG63 cell line. TBRG4 knockdown inhibited cell proliferation, colony and Medical University, Hefei, Anhui, People’s Republic of China; 2Department of tumor formation, while activating cell apoptosis. Ingenuity Pathway Analysis and Western Orthopaedics, The First Affiliated blot assay further indicated that TBRG4 knockdown may regulate the proliferation of human Hospital of Anhui Medical University, MG63 cells through PI3K/Akt signaling pathway. Hefei, Anhui, People’s Republic of China; 3Clinical Research Centre, Zhujiang Conclusion: Our results suggest that TBRG4 may become a promising therapeutic target Hospital, Southern Medical University, for the treatment of human OS. Guangzhou, Guangdong, People’s Republic of China Keywords: TBRG4, osteosarcoma, proliferation, PI3K/Akt, MG63 cells *These authors contributed equally to this work Introduction Osteosarcoma (OS) is the most frequent malignant primary bone tumor commonly found in children and adolescents.1–3 It is characterized by high malignancy, early metastasis, poor prognosis and low survival rate, with an annual incidence rate of 4.4 cases per million in the US.2 Although OS accounts for less than 1% of all diagnosed cancers in the US, it is the most common primary malignancy with the 5-year survival rate of <40%.4–6 In addition, treatments with poor prognosis, drug resistance and toxic side effects are still crucial obstacles for radical therapies.7,8 Therefore, novel treatments are required urgently. In recent years, targeted therapy has shown great potential in the treatment of OS, thus, based on the implicated pathogenesis of OS, identification of new effective prognosis markers and key Correspondence: Jun Chang molecular targets for OS will facilitate the accurate diagnosis and therapy for OS Clinical Research Centre, Zhujiang patients. Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Multiple studies have focused on novel molecular targets to improve OS therapeutic Republic of China efficacy including RTKs,9 steroid receptor co-activator,10 mTOR,11 and aurora Tel/Fax + 86 551 66331115 12 Email [email protected] kinases. Recently, there has been interest in developing better understanding of submit your manuscript | www.dovepress.com OncoTargets and Therapy 2020:13 7271–7281 7271 DovePress © 2020 Huang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. – http://doi.org/10.2147/OTT.S249477 php and incorporate the Creative Commons Attribution Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Huang et al Dovepress tumor genesis mechanisms, especially about transforming written informed consent before tissue specimens were growth factorβ (TGF-β). TGF-β is a growth factor which collected, and the study was conducted in accordance stimulates tumor growth in microenvironment of OS, the with the Declaration of Helsinki. OS tissues and para- expression of TGF-β is higher in patients with OS than that carcinoma tissue which come from pre-chemotherapy of in healthy individuals.13 Latest evidence has reported that treatment (referred to as normal tissues, at least 1.0 cm TGF-β is a potential target regulator in the metastatic apart from the visible cancerous tissues) were collected dissemination.14 However, whether TGF-β could be from distal femur for detecting the levels of TBRG4 a potential treatment target for OS remains unclear. expression by immunohistochemistry (IHC). The transforming growth factor β regulator 4 (TBRG4), also termed Fas-activated serine/threonine kinase domain- Cell Culture containing protein4, FASTKD4 or cell cycle progression Three human OS cells lines, MG63, U2OS, Saos-2 were 15–17 restoration protein2, CPR2, encodes a regulator for purchased from the Cell Bank of Chinese Academy of 18 TGF-β. It is therefore conceivable that TBRG4 could be Sciences (Shanghai, People's Republic of China). Cells a key molecular target in OS because of its regulation for were cultured in Roswell Park Memorial Institute 1640 med- TGF-β. TBRG4 is located on the 7p14-p13 chromosomal ium (Thermo Fisher Scientific, Waltham, MA, USA) with region, and participates in the development process of var- 10% fetal bovine serum and 1% antibiotics (Sangon Biotech, ious diseases, especially cancer. Previous studies have Shanghai, People's Republic of China) in a humidified atmo- demonstrated that TBRG4 is a novel oncogene in breast sphere at 37°C under 5% CO2. cancer, with its expression level correlating with breast tumor malignancy. In addition, deficiency of TBRG4 could Construction and Infection of shTBRG4 significantly inhibit tumor cell proliferation and migration 19 Lentivirus through promoting apoptosis. Thus, we speculated that A lentiviral system containing short hairpin RNA TBRG4 may have the same effect on human OS. Notably, (shRNA) was used to manipulate TBRG4 expression. Sevcikova et al,18 collected nine samples of bone marrow A candidate RNAi for human TBRG4 was designed to plasma cells from multiple myeloma (MM) patients to eval- target the sequence 5ʹ- GTTCTTCAGCCTGGTACAT-3ʹ. uate the related genes associated with the high risk signature Based on selected target sequences, shRNA interference of MM patients. They found that TBRG4 may serve as sequence was designed to synthesize DNA oligo. TBRG4 a crucial gene and is highly relatied with MM. Therefore, RNAi lentivirus was generated via ligating Stem-loop the hypothesis has been put forward that the altered TBRG4 DNA oligonucleotides into the lentiviral pGV115-GFP expression would affect human OS growth and development. vector (GeneChem, Shanghai, People's Republic of However, the effect of TBRG4 on human OS are still poorly China). The envelope plasmid was infected into MG63 understood. cells, and the scramble shRNA was used as the negative In the present study, we first evaluated the expression shRNA control. After 72 hours of transfection, the infected of TBRG4 in human osteosarcoma tissues and cell lines. cells with GFP proteins were observed using fluorescence A lentiviral system was used to determine the role of microscopy (Olympus XI-71, Tokyo, Japan) with TBRG4 silencing on tumor proliferation, apoptosis, and Lentivector Expression System (GeneChem) and fluores- colony formation. The effect of TBRG4 on differentially cent images were taken. After 72 hours of infection, expressed genes patterns in human osteosarcoma tissues MG63 cells were harvested and qRT-PCR and Western and cell lines was also evaluated. blot analysis were performed to determine knockdown efficiency. Patients and Methods Patients’ Tissues IHC Staining In the current study, 40 patients diagnosed with OS at the The patients’ OS tissue sections (5 μm thick) were First Affiliated Hospital of Anhui Medical University dewaxed in xylene for 5 minutes, rehydrated in alcohol between 2013 and 2016 were enrolled. The study was gradient and rinsed in deionized water. After incubating in approved by the Medical Ethics Committee of the Anhui 10 mM citrate buffer at 95°C for 12 minutes, the slides Medical University (2013–76). All the patients provided were fixed in fixative and blocked with 5% bovine serum 7272 submit your manuscript | www.dovepress.com OncoTargets and Therapy 2020:13 DovePress Dovepress Huang et al albumin for 60 minutes. Then the slides were incubated a density of 2000 cells per well. After transfection, the plate with TBRG4 antibody (1:1000, CST) at 4°C overnight, was imaged and analyzed once a day for 4 days continu- followed by incubation of secondary antibody at room ously using a Celigo Cell Imaging Cytometer (Nexcelom temperature for 1 hour. After extensive washing, the slides Bioscience, Boston, MA, USA). Images were processed were stained with DAB and counterstained with hematox- and quantified using the Cell Analyzer software. ylin. Finally, the slides were examined with a fluorescence The effect of shTBRG4 on MG63 cells proliferation microscope and the positive cells were brown in color. was also evaluated by CCK8 assay using the cell viability assay kit. First, 2000 cells per well were seeded in 96-well RNA Extraction and qRT-PCR plates and incubated for 24 hours. The cell-free group was Total RNA was extracted from MG63 cells using Trizol set as the blank control group, and three repeating pipettes reagent (Life Technologies, USA), and reverse transcribed were set in each group. Then, 10 µL CCK8 reagent was into cDNA using cDNA Synthesis Kit (Sangon, Biotech) added into each well and incubated for 4 hours at 37°C.