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TRIB3 Promotes the Proliferation and Invasion of Renal Cell Carcinoma

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TRIB3 Promotes the Proliferation and Invasion of Renal Cell Carcinoma Int. J. Biol. Sci. 2019, Vol. 15 587 Ivyspring International Publisher International Journal of Biological Sciences 2019; 15(3): 587-597. doi: 10.7150/ijbs.29737 Research Paper TRIB3 Promotes the Proliferation and Invasion of Renal Cell Carcinoma Cells via Activating MAPK Signaling Pathway Baoan Hong1,2,3,4, Jingcheng Zhou1,2,3,4, Kaifang Ma1,2,3,4, Jiufeng Zhang1,2,3,4, Haibiao Xie1,2,3,4, Kenan Zhang1,2,3,4, Lei Li1,2,3,4, Lin Cai1,2,3,4, Ning Zhang5,6, Zhongyuan Zhang1,2,3,4, Kan Gong1,2,3,4 1. Department of Urology, Peking University First Hospital, Beijing 100034, P.R. China 2. Hereditary Kidney Cancer Research Center, Peking University First Hospital, Beijing 100034, P.R. China 3. Institute of Urology, Peking University, Beijing 100034, P.R. China 4. National Urological Cancer Center, Beijing 100034, P.R. China 5. Department of Urology, Beijing Cancer Hospital, Beijing 100142, P.R. China 6. Beijing Institute for Cancer Research, Beijing 100142, P.R. China Corresponding authors: Kan Gong, Zhongyuan Zhang. Address: No. 8, Xishiku Street, Xicheng District, Beijing 100034, China, Tel: +86-010-83575101, Fax: +86-010-66551122, E-mail: [email protected]; [email protected] © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2018.09.05; Accepted: 2018.11.15; Published: 2019.01.01 Abstract Tribbles pseudokinase 3 (TRIB3) is a member of the mammalian pseudokinase tribbles family and is involved in multiple biological processes. However, the role of TRIB3 in renal cell carcinoma (RCC) remains unclear. In this study, we aimed to elucidate the biological functions of TRIB3 in RCC and explore its underlying mechanisms. TRIB3 expression and its correlation with clinicopathological features was evaluated in 123 patients with RCC. A series of cytological experiments were performed to clarify the biological functions of TRIB3, and potential molecular regulatory mechanisms were explored using transcriptome sequencing. TRIB3 expression was significantly elevated in RCC tissues compared to that in paracancerous tissues, and high expression of TRIB3 was correlated with both advanced tumor stage and unfavorable prognosis. TRIB3 knockdown markedly inhibited RCC cell proliferation, migration and invasion. Furthermore, overexpression of TRIB3 promoted RCC cell proliferation, migration, invasion and xenograft tumor growth. Notably, TRIB3 expression was modulated by hypoxia-inducible factor-1α (HIF-1α), which enhanced cell viability and invasiveness via targeting the MAPK signaling pathway. This study reveals the potential oncogenic role of TRIB3 in RCC pathogenesis and illustrates the mechanisms underlying TRIB3-mediated tumor progression, providing new insight into the development of TRIB3 as a tumor biomarker and therapeutic target. Key words: Tribbles pseudokinase 3, renal cell carcinoma, cell proliferation, cell invasion, mitogen-activated protein kinases Introduction Tribbles pseudokinase 3 (TRIB3), a member of pathways. Interaction partners of TRIB3 include the tribbles-related family, contains the ubiquitin ligase, proteasomal degradation systems, substrate-binding domains but lacks the conserved autophagy, transcription factors, and members of the amino acid catalytic motifs essential for kinase MAPK-ERK, TGF-β, and JAG1/Notch signaling activity [1, 2]. Despite lacking these catalytic pathways [3-7]. Additionally, TRIB3 regulates a series structures, TRIB3 is a key regulator of a variety of of cellular processes, such as cellular stress response, cellular functions. TRIB3 can act as a scaffold or cell proliferation and differentiation, glucose and lipid adaptor to regulate multiple signal transduction metabolism, and epithelial-to-mesenchymal transition http://www.ijbs.com Int. J. Biol. Sci. 2019, Vol. 15 588 (EMT) [8-13]. As a sensor of cellular stress, TRIB3 time for these patients was 60 ± 15 months. We expression is upregulated in diverse examined the clinicopathological features, including microenvironments, such as during endoplasmic the gender, age, tumor size, pathology type, tumor reticulum stress, hypoxia and nutrient deprivation. stage, tumor grade and prognosis. Both the RCC Depending on the cell physiological status, TRIB3 tissues and paracancerous tissues were collected. maintains cell survival by blunting potentially Formalin-fixed, paraffin-embedded specimens for deleterious stress signals [14-16]. tissue microarray construction were reviewed by two Recently, emerging evidence suggests that TRIB3 professional pathologists (QH and YZ). Fresh RCC is a crucial modulator of tumorigenesis. Several and normal renal tissues were preserved at −80 °C. studies have demonstrated that TRIB3 is elevated in multiple cancer cell lines and primary tumors, Immunochemical staining and analyses including colorectal cancer, breast cancer and lung Immunochemical staining was performed as cancer [17-19]. It is involved in tumor progression and previously reported [25]. Primary antibodies included related to poor prognosis. Hypoxia and nutrient TRIB3 (rabbit monoclonal, 1:250 dilution, #ab137526, deficiency occur during the growth of solid tumors Abcam, Cambridge, UK), HIF-1α (rabbit polyclonal, [20-22], and these tumor microenvironments activate 1:50 dilution, #sc10790, Santa Cruz, Dallas, TX, USA), a variety of cellular stress response pathways, such as and HIF-2α (rabbit polyclonal, 1:40 dilution, #ab199, unfolded protein responses and endoplasmic Abcam). The secondary antibody was purchased from reticulum stress, leading to upregulation of TRIB3 OriGene (poly-peroxidase-conjugated anti-rabbit/ expression. Intriguingly, Wennemers et al. [23] mouse immunoglobulin G, #PV-9000, OriGene, reported that high expression of TRIB3 is related to a Beijing, China). The slides were independently good prognosis for breast cancer patients. Erazo et al. assessed by two pathologists. As previously reported, [24] found that upregulated TRIB3 inhibits the protein expression was semiquantitatively evaluated Akt/mTORC1 axis, exerting anti-tumor effects. These using an immunoreactive score (IRS) [26]. The studies suggested that TRIB3 may either promote or percentage of immunostaining was scored as follows: suppress tumor progression. Therefore, the biological 0 (0), 1 (< 10%), 2 (10-50%), 3 (51-80%) and 4 (> 75%). functions and interaction mechanisms of TRIB3 in The staining intensity was defined as 0: no color cancers remain controversial. reaction; 1: mild reaction; 2: moderate reaction; and 3: Renal cell carcinomas (RCCs) are solid tumors intense reaction. The two staining scores were accompanied by microenvironments of hypoxia and multiplied to calculate the expression intensity. IRSs nutrient deficiency. Currently, the expression of ranged from 0 to 12, which were divided into two TRIB3 in RCC and its association with grades: low expression (score ≤ 6) and high expression clinicopathological features and prognosis remain (score > 6). unclear. In this study, we evaluated the expression of Cell culture and lentivirus transduction TRIB3 to clarify the relationship between TRIB3 and clinicopathological features, further illuminating its Five human RCC cell lines (786-O, 769-P, ACHN, biological functions and regulatory mechanisms in A498 and Caki-1) were utilized in this study. HK-2, RCC. Our results found that TRIB3 expression is human normal proximal tubular cell line, was used as significantly elevated in RCC tissues compared to that the control cell line. They were obtained from the in paracancerous tissues. Furthermore, high Institute of Urology, Peking University (Beijing, expression of TRIB3 was correlated with an advanced China). The media (Gibco Invitrogen, Carlsbad, CA, tumor stage and unfavorable prognosis in RCC. USA) were supplemented with 10% fetal bovine Notably, TRIB3 expression was modulated by serum (FBS, PAN-Biotech GmbH, Adenbach, hypoxia-inducible factor-1α (HIF-1α), which Germany), penicillin G (100 U/ml) and streptomycin mediated RCC cell proliferation, migration and (100 μg/ml). Cells were incubated at 37 °C in a 5% invasion via targeting the MAPK signaling pathway. CO2 atmosphere. The TRIB3 overexpression lentivirus Materials and methods (LV-TRIB3) and its negative control lentivirus (LV-control), the TRIB3 knockdown lentivirus Patients and RCC specimens (LV-shTRIB3) and the nontargeting control lentivirus Ethical approval was obtained from the Ethics (LV-scrambled), and the HIF-1α overexpression Committee of Peking University First Hospital lentivirus (LV-HIF-1α) and its negative control (Beijing, China). In this study, we recruited 123 RCC lentivirus (LV-vector) were produced and verified by patients who underwent surgery at Peking University GeneChem Corporation (Shanghai, China) First Hospital from 2012 to 2013. The mean follow-up (Supplement 1). Cells were incubated and transfected http://www.ijbs.com Int. J. Biol. Sci. 2019, Vol. 15 589 with lentivirus and polybrene (5 μg/ml; GeneChem phosphate buffered saline (PBS). Then, serum-free Corporation). Puromycin (1.5 μg/ml) was used to medium was added to incubate the cells. Images were screen stable cell clones. The expression efficiency was acquired using an inverted microscope at 0 h, 12 h and verified by quantitative real-time PCR (qRT-PCR) and 24 h to assess cell migration. western blot analysis. Transwell assay RNA isolation and qRT-PCR The indicated
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