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FRAT1 Enhances the Proliferation and Tumorigenesis of CD133+Nestin+

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FRAT1 Enhances the Proliferation and Tumorigenesis of CD133+Nestin+ Journal of Cancer 2020, Vol. 11 2421 Ivyspring International Publisher Journal of Cancer 2020; 11(9): 2421-2430. doi: 10.7150/jca.37622 Research Paper FRAT1 Enhances the Proliferation and Tumorigenesis of CD133+Nestin+ Glioma Stem Cells In Vitro and In Vivo Geng Guo1,*,, Jing Liu1,*, Yeqing Ren1,*, Xinggang Mao2,*, Yining Hao1, Chengliang Zhong3, Xiaolin Chen4, Xiaogang Wang1, Yongqiang Wu1, Shizhong Lian1, Lin Mei5, Yuanli Zhao4, 1. Department of Neurosurgery, The First Hospital, Shanxi Medical University, No.85 Jiefangnan Road, Taiyuan, Shanxi 030001, People’s Republic of China 2. Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, No.15 Changlexi Road, Xi'an, Shaanxi 710032, People's Republic of China 3. GCP Center, The First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, No.88 Changling Road, Tianjin 300192, People's Republic of China 4. Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, No.6 Tiantan Xili, Beijing 100050, People’s Republic of China 5. Department of Orthopedics, The First Hospital, Shanxi Medical University, No.85 Jiefangnan Road, Taiyuan, Shanxi 030001, People’s Republic of China * These authors contributed equally to this work and should be regarded as co-first authors. Corresponding authors: Geng Guo, Department of Neurosurgery, The First Hospital, Shanxi Medical University, No.85 Jiefangnan Road, Taiyuan, Shanxi 030001, People’s Republic of China. E-mail: [email protected]. Yuanli Zhao, Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, No.6 Tiantan Xili, Beijing 100050, People’s Republic of China. E-mail: [email protected] © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2019.06.15; Accepted: 2020.01.18; Published: 2020.02.10 Abstract Glioma stem cells (GSCs) are considered the source for development, recurrence, and poor prognosis of glioma, so treatment targeted GSCs is of great interest. The frequently rearranged in advanced T cell lymphomas-1 (FRAT1) gene is an important member of the Wnt/β-catenin signaling transduction pathway, and aberrantly activation of Wnt signaling has been identified to contribute to the tumorigenesis, proliferation, invasion of a variety kinds of cancer stem cells. However, correlations between FRAT1 and GSCs and the specific mechanisms remain unclear. In this study, we aimed to investigate the effect of FRAT1 on GSCs proliferation, colony formation, sphere formation and tumorigenesity in vitro and in vivo and its underlying mechanism. Lentiviral transfection was used to construct GSCs with low FRAT1 expression. The expression of FRAT1 on GSCs proliferation in vitro was assessed by cell counting kit-8(CCK-8). Colony formation and sphere formation assays were conducted to assess the colony and sphere formation ability of GSCs. Then, an intracranial glioma nude mouse model was built to measure the effect of low FRAT1 expression on GSCs proliferation and tumorigenesity in vivo. Real-time PCR, Western blot, and Immunohistochemistry were processed to detect the mRNA and protein expressions of FRAT1, β-catenin in the glioma tissue of xenograft mice to study their correlations. The functional assays verifed that low FRAT1 expression inhibited CD133+Nestin+ GSCs proliferation, colony formation, sphere formation ability in vitro. In vivo GSCs xenograft mice model showed that low FRAT1 expression suppressed the proliferation and tumorigenesity of CD133+Nestin+ GSCs and reduced β-catenin mRNA and protein expression. Furthermore, the expression of FRAT1 and β-catenin were positively correlated. Altogether, results indicate that FRAT1 enhances the proliferation, colony formation, sphere formation and tumorigenesity of CD133+Nestin+ glioma stem cells in vitro and in vivo as well as the expression of β-catenin. Therefore, inhibiting proliferation of GSCs and FRAT1 may be a molecular target to GSCs in treating human glioma in the future. Key words: Glioma stem cells (GSCs); Proliferation; The frequently rearranged in advanced T cell lymphomas-1(FRAT1); Wnt/β-catenin pathway Introduction Glioma is the most common primary malignant existing glioma treatments (surgery, radiotherapy, tumor of the adult central nervous system. Although and chemotherapy) have been improved, therapeutic http://www.jcancer.org Journal of Cancer 2020, Vol. 11 2422 efficacies have not been significantly enhanced [1]. expression occurs in human glioma tissue and human Increasing studies have confirmed the existence of a glioma cell lines, and its expression is significantly small subpopulation of cancer stem cells (CSCs) in associated with glioma malignancy. Given the high hierarchically organized solid tumors, which are expression and protumorigenic role of FRAT1 in considered responsible for tumor initiation, glioma, we investigated its function and molecular proliferation, progression and metastasis [2, 3]. mechanism in GSCs biology. Here, we report that Previous work has indicated the presence of CSCs in FRAT1 acts as a key regulator of β-catenin/Wnt numerous human malignancies including glioma, signaling to modulate GSCs proliferation in vitro and which are known as glioma stem cells (GSCs) [4-10], in vivo. Our results are the first demonstration that and stem cell features found in GSCs, like the FRAT1 has the ability to activate the Wnt pathway self-renewing, unlimited proliferating, and in glioma stem cells to promote stemness and tumor multi-directional differentiated tumor primitive cells progression. [11], are thought to contribute to the incidence, recurrence, and poor prognosis of glioma [12]. Materials and Methods Recently, molecular targeted therapy has become an Glioma cell culture important adjunct to glioma treatment and targeted therapy of GSCs is of interest. U251, U87 and SHG44 human glioma cell lines Specific cell surface markers have been used in (purchased from the Cell Bank of Peking Union collectively identifying and isolating GSCs. CD133, a Medical College, Beijing, China) was cultured in stem cell-specific marker found in several types of DMEM complete medium (containing 10% fetal tumor, was also identified in increasing the bovine serum, 100 U/ml penicillin, and 100 U/ml tumorigenic ability and resistance to chemotherapy streptomycin) (Invitrogen, Carlsbad, CA) in a 37°C and radiotherapy of GSCs [13]. Nestin, another incubator with 5% CO2. Medium was changed daily important cell surface marker for GSCs, was found in and cells were passaged once every 2–3 days. PBS increasing robust CSC properties, for instance, (Invitrogen) was used to wash cells, followed by tumorsphere-forming ability and tumorsphere size enzymatic cell dissociation with 0.25% trypsin [14]. CD133 and nestin expressions in glioma have (Invitrogen). A slightly impaired brain tissue been found correlate increasingly with malignancy fragment of a patient who underwent intracerebral grades, and Nestin/CD133 co-expression has been hemorrhage was used to culture primary astrocytes to thought to be an important feature of GSCs [13, 15, be a control. Written informed consent was obtained 16]. from the patient. The grey matter of the brain tissue The Wnt/β-catenin signal transduction pathway was dissociated, washed in PBS and dispersed is a highly conservative signaling pathway, and its repeatedly and then the resulting cell suspension was abnormal activation is closely related to filtered and cultured in DMEM with 10% fetal bovine over-proliferation of glioma. Our studies previously serum. indicated that an aberrantly activated Wnt signaling Lentiviral transfection and collecting of stably pathway existed in glioma [17, 18]. Researches have transfected cells delineated multiple transcription regulation Related sequence synthesis and plasmid mechanisms of downstream genes of Wnt pathway, packaging were supported by Genechem, Ltd., which explained the tumorigenesis, proliferation, Shanghai, China, with the FRAT1 interference invasion and other stem cell properties maintained in sequence of 5’-ACGCGGGTCCCAACCAGAA-3’ and CSCs through Wnt signaling [19-22], and recently, independent sequence of 5’-TTCTCCGAACGTGTC vital roles of Wnt pathway in modulating GSCs has ACGT-3’. First, 5×105 cells/ml were seeded into also been identified [23]. 6-well plates and labeled as follows: U251-FRAT1-KD The frequently rearranged in advanced T cell indicated U251 cells transfected with lentiviral vector lymphomas-1 (FRAT1) proto-oncogene is an containing FRAT1-interference to knock down important member of the Wnt/β-catenin signaling FRAT1, U251-vector indicated U251 cells transfected transduction pathway. FRAT1 downregulation with lentiviral vector containing an unrelated inhibits the proliferation, migration, and invasiveness sequence and U251-control indicated normal U251 of glioma cells. High FRAT1 expression in human cells with PBS (equal volume of transfection reagents glioma upregulates intracellular accumulation of in U251-FRAT1-KD and U251-vector). Cells were β-catenin, suggesting that FRAT1 may promote the incubated in a 37°C incubator with 5% CO2 for 18-24 h incidence and development of glioma through the until 70-80% confluency, followed by transfection in Wnt/β-catenin signal transduction pathway [24, 25]. accordance with the manufacturer’s protocol Our previous study confirmed that high FRAT1 http://www.jcancer.org Journal
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