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Original Article Downregulation of ARG2 Inhibits Growth of Colorectal Cancer Cells and Increases Expression of the Cd3ζ Chain in Co-Cultured T-Cells

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Original Article Downregulation of ARG2 Inhibits Growth of Colorectal Cancer Cells and Increases Expression of the Cd3ζ Chain in Co-Cultured T-Cells Int J Clin Exp Med 2019;12(6):6946-6957 www.ijcem.com /ISSN:1940-5901/IJCEM0089594 Original Article Downregulation of ARG2 inhibits growth of colorectal cancer cells and increases expression of the CD3ζ chain in co-cultured T-cells Youjun Wu1,2*, Changzheng He1*, Shidong Hu1, Zilong Hu1, Yuxuan Li1, Xiaowei Xing1, Xiaohui Du1 1Department of General Surgery, The 1st Medical Center of Chinese People’s Liberation Army General Hospital, 28 Fuxing Road, Haidian District, Beijing 100853, P.R. China; 2Department of General Surgery, The 8th Medical Center of Chinese People’s Liberation Army General Hospital, Jia 17 Heishanhu Road, Haidian District, Beijing 100091, China. *Equal contributors. Received December 9, 2018; Accepted May 7, 2019; Epub June 15, 2019; Published June 30, 2019 Abstract: Arginase (ARG) 2 is upregulated in some malignancies and has been associated with poor prognosis in patients with these malignancies. The present study aimed to explore expression and function of ARG2 in colorectal cancer (CRC). Expression of ARG2 in CRC tumor tissues was analyzed using the public database Cancer RNA-seq Nexus (CRN). Using Western blotting and RT-qPCR, HT29 cells were selected as target cells for ex vivo experiments. Small interfering RNAs (siRNAs) were then used to silence ARG2 in HT29 cells. The siRNA with the highest interfer- ing efficiency was selected. Cell Counting Kit-8, would healing assays, and Transwell assays were subsequently per- formed, aiming to elucidate the proliferation and invasion of CRC cells. The impact of ARG2 on cell cycle/apoptosis was also investigated. In addition, HT29 cells were co-cultured with Jurkat T-cells in a Transwell system to mimic the tumor microenvironment. Expression of the CD3ζ chain in T-cells was examined via Western blotting. Results showed that expression of ARG2 was significantly higher in CRC tumor tissues than in adjacent normal mucosae. Knockdown of the ARG2 gene significantly attenuated proliferation, migration, and invasion abilities of CRC cells. It also affected the cell cycle and apoptosis of these cells. Expression of the CD3ζ chain in co-cultured T-cells was markedly increased. The current study highlights the important role that ARG2 plays in CRC, providing a potential novel target for diagnosis and treatment of CRC. Keywords: Arginase 2, colorectal cancer, CD3ζ Introduction als have shown that targeted therapy can ben- efit CRC patients [2, 3]. However, gene muta- Colorectal cancer (CRC) is one of the most com- tions and drug resistance have limited the mon and lethal malignancies worldwide, posing therapeutic effects of these drugs [4, 5]. Th- a huge threat to human health [1]. Despite erefore, a comprehensive exploration of the great advancements made in the treatment of pathogenesis of CRC is of primary concern for CRC in recent decades, surgery and radio-che- the identification of additional biomarkers and motherapy remain the primary therapeutic therapeutic targets that may facilitate prognos- strategies for treatment of CRC. A large propor- tic prediction and personalized treatment, ulti- tion of patients ultimately experience recur- mately improving survival rates of CRC patients. rence or metastasis as the major causes of tumor-related deaths. An important recent Arginase (ARG), an enzyme associated with development in CRC treatment is the applica- tumorigenesis, partly due to its contribution to tion of targeted drugs, such as cetuximab and the immunosuppressive tumor microenviron- bevacizumab. They exert anti-tumor effects by ment [6, 7], is overexpressed in several malig- blocking epidermal growth factor receptors and nancies. It has been associated with poor prog- vascular endothelial growth factor (VEGF) sig- nosis in these patients [8-10]. ARG is expressed naling pathways. Several large-scale clinical tri- as two subtypes, ARG1 and ARG2, which show Downregulation of ARG2 inhibits CRC cell growth different cellular and histological localization rent study investigated the possible relation- under physiological conditions. The former is ship between ARG2 and tumor immunity in localized in the cytoplasm and is produced CRC. It has been considered one of the most mainly in the liver, while the latter exists in the important features of ARG2. mitochondria and is mainly expressed by the kidneys. Ex vivo experiments have demonstrat- Materials and methods ed the pro-tumorigenic features of ARG in some cancers. In the context of prostate cancer, Public database androgens can upregulate expression of ARG1 The public database ‘Cancer RNA-seq Nex- and ARG2 in human LNCaP cells. This inhibits us (CRN, http://syslab4.nchu.edu.tw/index.jsp)’ the activation of tumor-specific T-cells and ulti- was used to analyze differences in ARG2 mately promotes tumor growth. In contrast, expression levels between CRC tumor tissues androgen-deprivation therapy can increase the and adjacent normal mucosae. The keyword infiltration of immune cells within tumor tissues ‘ARG2’ was used to search corresponding through the downregulation of ARG1/ARG2. In datasets. addition, knockdown of ARG1/ARG2 by small interfering (si) RNAs has been shown to limit Cell culturing and co-culturing the growth of LNCaP cells. A conditioned medi- um from these LNCaP cells activates peripheral Human CRC cell lines Caco2, HT29, and blood mononuclear cells [11]. ARG2 is also HCT116, as well as the leukemia cell line Jurkat, highly expressed in most human thyroid carci- were obtained from the ATCC (Manassas, VA, noma cases. However, it is seldom expressed USA). Caco2 and HCT116 cells were cultured in in normal thyroid tissues and thyroid adenoma Dulbecco’s Modified Eagle’s medium (Thermo cells. Silencing of the ARG2 gene in thyroid Fisher Scientific, Inc., Waltham, MA, USA). WRO cells by siRNAs promotes apoptosis and HT-29 cells were cultured in McCoy’s 5A medi- decreases expression of proliferation markers um (Sigma-Aldrich; Merck KGaA, Darmstadt, Ki67 and PCNA [8]. Tate et al. [12] reported Germany). Jurkat T-cells were cultured in RPMI that three murine cell lines, SIRCC-1.2 (CL-2), 1640 medium (Gibco; Thermo Fisher Scientific, SIRCC 1.19 (CL-19), and Renca, express ARG2 Inc.). All media were supplemented with 10% and that the addition of the ARG inhibitor nor- FBS (Gibco; Thermo Fisher Scientific, Inc) and NOHA (NG-hydroxy-L-arginine) into the culture 100 IU/mL penicillin/streptomycin (Invitrogen; medium of these cells significantly suppresses Thermo Fisher Scientific, Inc). The cells were proliferation. Similarly, nor-NOHA exerts inhibi- cultured at 37°C with 5% CO . tory effects on the growth of the human CRC 2 cell line Caco2, which displays robust ARG Co-culturing of CRC cells and T-cells was per- activity. Additionally, because nor-NOHA is an formed using a Transwell system (BD Biosci- intermediate product of nitric oxide synthase ences, Franklin Lakes, NJ, USA). Briefly, 6×105 (NOS), the growth of Caco2 cells is markedly CRC cells were plated in the lower chamber and reduced after co-culturing with rat aortic endo- 5×105 Jurkat T-cells were seeded in the upper thelial cells (rich in NOS). Furthermore, these chamber. Seventy-two hours after seeding, the effects may be blocked by the NOS inhibitor T-cells were harvested and analyzed via We- S-ethylisothiourea (EITU) [13]. stern blotting, determining expression levels of the CD3ζ chain. This experiment was repeated Previous studies have revealed that ARG activ- with L-arginine supplementation. In this experi- ity in the serum and/or tumor tissues of CRC ment, 50 μL of L-arginine (Beyotime Institute of patients is significantly increased, compared Biotechnology, Shanghai, China, 10 mM) was with that in healthy individuals and/or normal added to the lower chamber of the co-culture mucosae [14-17]. Moreover, ARG1 is seldom system every 24 hours. expressed in CRC [18-20]. Therefore, it would be interesting to determine whether ARG2 is Cellular transfection overexpressed in CRC, assessing its impact on proliferation, migration, and invasion of CRC Three small interfering (si) RNAs (siRNA_1, 2, 3) cells, as well as its effects on cell cycle and targeting ARG2 and one scrambled negative apoptosis of these cells. Additionally, the cur- control siRNA (siRNA_NC) were obtained from 6947 Int J Clin Exp Med 2019;12(6):6946-6957 Downregulation of ARG2 inhibits CRC cell growth Shanghai GenePharma Co., Ltd (Shanghai, Ch- Flow cytometry ina). Sequences were as follows: siRNA_1, 5’- CTGAAGAAATCCGTCCACT-3’; siRNA_2, 5’-GG- Regarding cell cycle analysis, the cells were CAATCGGTACCATTAGT-3’; siRNA_3, 5’-CATGG- centrifuged at 1,000 g for 5 minutes. They were AACGAACATTTGAT-3’; and siRNA_NC, 5’-TTCT- washed with cold PBS and re-centrifuged. After CCGAACGTGTCACGTTT-3’. Lipofectamine 2000 suspension in 300 μL of PBS containing 10% Reagent (Invitrogen; Thermo Fisher Scientific, FBS, the cells were fixed with 70% ethanol at Inc.) was used for cell transfection, according to 4°C for 24 hours. They were centrifuged, manufacturer protocol. CRC cells were divided washed twice with cold PBS, and incubated into three groups, including blank (cells treated with 100 μL of RNaseA (1 mg/mL) for 30 min- with Lipofectamine 2000 only), NC (cells tre- utes at 37°C. The cells were subsequently ated with negative control siRNA and Lipofe- stained with 400 μL of propidium iodide (PI, 50 ctamine 2000), and siRNA (cells treated with μg/mL) (Becton-Dickinson, San Jose, CA, USA) ARG2 siRNA and Lipofectamine 2000) groups. for 10 minutes and detected using a FACSC- All the experiments were repeated at least alibur flow cytometer (BD Bioscience, USA). three times. Data was assessed using FLOWJO v10 soft- ware. For cell apoptosis analysis, the cells were RT-qPCR centrifuged and washed twice with cold PBS. They were then stained with 5 μL of Annexin Total RNA from the cells was extracted using V-APC (BD Biosciences) for 15 minutes and 5 TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), μL of propidium iodide (PI, 50 μg/mL) (Becton- according to manufacturer instructions.
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