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Deltex3 Inhibits Epithelial Mesenchymal Transition In Journal of Cancer 2021, Vol. 12 860 Ivyspring International Publisher Journal of Cancer 2021; 12(3): 860-873. doi: 10.7150/jca.48141 Research Paper Deltex3 inhibits Epithelial Mesenchymal Transition in Papillary Thyroid Carcinoma via promoting ubiquitination of XRCC5 to regulate the AKT signal pathway Lidong Wang1, Yonglian Huang1, Chenxi Liu1, Mingyue Guo1, Zhennan Ma1, Jingni He1, Ailian Wang2, Xiaodan Sun3, Zhen Liu1 1. Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, China. 2. Department of Pathology, Shengjing Hospital of China Medical University, Shenyang, China. 3. Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, China. Corresponding author: Zhen Liu, PhD, Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, China. E-mail: [email protected]. Zip code: 110004. Telephone number: +86018940254591. © 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: 2020.05.13; Accepted: 2020.11.02; Published: 2021.01.01 Abstract Background: Papillary thyroid carcinoma (PTC) is one of the most common endocrine malignant tumors. Poor prognoses such as high recurrence rate always appear in PTC patients with cervical lymph node metastasis. The process of ubiquitination plays important roles in PTC. As ubiquitin E3 ligases, Deltex (DTX) family proteins were reported to associate with multiple cancers. However, functions and mechanisms of DTX3 in PTC are currently unknown. Methods: In this study, DTX3 expressions were examined in 114 PTC and paired paracancerous normal tissues through quantitative real-time polymerase chain reaction and western blot. The clinical significances of DTX3 expressions in PTC patients were also investigated. After stable transfection with either short hairpin RNA to knock down DTX3 expression or full-length complementary DNA to upregulate DTX3 expression, changes of malignant phenotypes in two PTC cell lines K1 and TPC-1 were observed using cell viability, flow cytometry, wound healing and transwell assays. Afterwards, altered expressions of epithelial-mesenchymal transition (EMT) and AKT signal pathway related proteins were measured by western blot. Immunoprecipitation and mass spectrometry (IP-MS), immunofluorescence and Co-IP were utilized to identify the possible DTX3 interacting proteins. Results: Both mRNA and protein expressions of DTX3 were lower in PTC tissues and correlated with the presence of cervical lymph node metastasis (P<0.05). DTX3 overexpression inhibited migration and invasion of PTC cells, decreased Vimentin and phosphorylated AKT expressions, but promoted E-cadherin expression (P<0.05). Moreover, knockdown of DTX3 led to opposite changes (P<0.05). Total 46 probable DTX3 interacting proteins were identified by IP-MS. Among them, X-ray repair cross-complementing protein 5 (XRCC5) and NADH: Ubiquinone Oxidoreductase Complex Assembly Factor 5 (NDUFAF5) were verified to be associated with DTX3. Moreover, DTX3 was proved to be co-localized with XRCC5 in nucleus and promote ubiquitination of XRCC5. Conclusions: DTX3 suppresses EMT by partially facilitating ubiquitination of XRCC5 to inhibit AKT signal pathway in PTC. Key words: papillary thyroid carcinoma; Deltex3; epithelial-mesenchymal transition; AKT signal pathway; XRCC5 Introduction Papillary thyroid carcinoma (PTC), with patients with PTC can be treated by properly applied increasing incidence in recent years, is the most surgery and postoperative regular thyroxine prevalent type of thyroid carcinoma [1-3]. As suppressive therapy alone or with postoperative treatments of PTC have been greatly improved, most radioactive iodine therapy [4-7]. However, a growing http://www.jcancer.org Journal of Cancer 2021, Vol. 12 861 body of evidence indicated that the presence of evaluated before surgery; Pathological diagnosis was cervical lymph node metastasis varied from 69.6% to PTC. The exclusion criteria included cases with 85% in PTC patients, which was considered as an incomplete clinicopathological data; patients who extremely high risk factor of PTC recurrence [8-10]. received radiotherapy or chemotherapy before So, it is very important to study and understand the surgery; and, patients with previous thyroid surgery underlying molecular mechanisms related to the history. metastasis of PTC. Paracancerous tissues of the same PTC patients Ubiquitination plays significant roles in tumor were collected at least 2 cm away from PTC areas. The cells [11]. Ubiquitin E3 ligase can recognize target age of patients, the tumor size and cervical lymph specificity and catalyze the combination of ubiquitin node metastasis were classified based on the with the substrate [12]. Deltex (DTX) family proteins Classification System for Differentiated Thyroid belong to ubiquitin E3 ligases [13]. The classic Carcinoma in the 8th edition of the American Joint structures of DTX family proteins are divided into Committee on Cancer (AJCC) [17]. Clinical data was three parts, including a C-terminal ubiquitin E3 ligase retrieved from patients’ clinical records and presented domain of RING finger, a motif with rich prolines and in Table 1. All tissue specimens and clinical data were the WWE domains at N-terminus [14]. Notably, the collected following protocols approved by the Ethics structure of DTX3 exists within a special truncated Committee of Shengjing Hospital of China Medical N-terminal domain lacking homology to other DTX University. proteins [15]. DTX1, as a member of DTX proteins, has been already reported to influence the progression of Table 1. The correlation analysis of DTX3 mRNA and protein tumor cells, which indicated that DTX family proteins expressions in patients with PTC played critical roles in tumor cells [16]. Parameters Total mRNA P-value Protein P-value To date, little is known about the roles of DTX3 Age (years) 0.610 0.415 in the progression of PTC. In the present study, we <55 76 0.510±0.272 0.566±0.267 evaluated DTX3 expressions in 114 PTC tissues and ≥55 38 0.483±0.259 0.521±0.296 Gender 0.438 0.498 paired paracancerous normal tissues. Meanwhile, we Male 32 0.532±0.280 0.579±0.294 compared the correlations between DTX3 expressions Female 82 0.489±0.262 0.540±0.271 and clinicopathological characteristics of patients with Tumor diameter 0.299 0.138 ≤ 2 cm 48 0.532±0.245 0.596±0.263 PTC. Then, we determined the effects and > 2 cm 66 0.479±0.281 0.518±0.284 mechanisms of DTX3 on cell proliferation, apoptosis, Cervical lymph node metastasis 0.002* 0.001* cell cycle, migration, invasion, and epithelial- No 40 0.607±0.261 0.663±0.282 Yes 74 0.444±0.254 0.490±0.255 mesenchymal transition (EMT) of PTC cells. In Multifocality 0.919 0.947 addition, we assessed the probable DTX3 interacting No 61 0.499±0.270 0.552±0.274 proteins in PTC cells. With these results, this study Yes 53 0.504±0.265 0.549±0.282 Extra-capsular invasion 0.137 0.281 will enhance our understanding of DTX3 functions in No 69 0.531±0.281 0.573±0.280 PTC. Yes 45 0.455±0.239 0.516±0.271 *P<0.05. Materials and methods Patients and clinicopathological data Cell culture The interactive web application of Gene Human thyroid epithelial cell line Nthy-ori3-1, Expression Profiling Interactive Analysis (GEPIA) PTC cell lines K1 and TPC-1 were cultured. was used to visualize the gene expressions of DTX3 in Nthy-ori3-1 and K1 were purchased from the multiple human carcinomas and normal tissues of European Collection of Cell Cultures and TPC-1 was The Cancer Genome Atlas (TCGA) by box plots. gifted by Professor Lei Yang (China Medical Total 114 samples of PTC patients who received University, China). K1 was cultured in a medium surgical therapy were selected from March 2018 to mixture of Dulbecco's modified Eagle's medium, March 2019 in Shengjing Hospital of China Medical Ham’s F12 (Corning Life Sciences, NY, USA) and University. Several exclusion and inclusion criteria MCDB105 (Sigma-Aldrich St.Louis, Missouri) at a were considered when selecting participants. The 1:2:1 ratio, which supplemented with 10% fetal bovine inclusion criteria were as follows: The age of patients serum (FBS; Gibco Co., Grand Island, NY, USA), 100 was 18-80 years; Patients with PTC were all found and U/mL penicillin, 100 mg/mL streptomycin, and 2 diagnosed for the first time; No treatment was mmol/L glutamine (Sigma-Aldrich). Nthy-ori3-1 and accepted before surgery; Neck Color Doppler TPC-1 were grown in Roswell Park Memorial Ultrasound or Computed Tomography was at least Institute-1640 medium (Corning Life Sciences, NY, http://www.jcancer.org Journal of Cancer 2021, Vol. 12 862 USA) and supplemented with 10% FBS, 100 U/mL protease inhibitor cocktail (K1011, APExBIO penicillin, 100 mg/mL streptomycin, and 2 mmol/L Technology LLC) were added into tissues and cells. glutamine in a humidified incubator with 5% CO2 at The homogenate was centrifuged at 14,000×g for 45 37°C. min at 4 °C. 40 μg total protein samples were separated by sodium dodecyl sulfate-polyacrylamide Nuclear and cytoplasmic protein extract gel electrophoresis (SDS-PAGE) and then transferred Nuclear and cytoplasmic protein extracts of K1 to a polyvinylidene fluoride (PVDF) membrane. 5% and TPC-1 cells were separated and collected with the bovine serum albumin (BSA) was used to block the Nuclear and Cytoplasmic Protein Extraction Kit membranes for 2 h at room temperature. Then, the (P0027, Beyotime Biotechnology, China), according to membranes were incubated with specific primary the manufacturer’s protocols. antibodies overnight at 4 °C. Then, the membranes Plasmid construction and transfection were incubated with secondary antibodies for 2 h at room temperature. Antibodies used in this study DTX3 short hairpin RNA (shRNA) and its included the following: rabbit polyclonal anti-DTX3 negative control (neg-shRNA) were designed and (1:1000, cat.
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