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Downloaded from the TCGA Data Portal Website ( Cell Proliferation Was Determined Using an MTT Assay ORIGINAL RESEARCH published: 28 August 2020 doi: 10.3389/fonc.2020.01468 Clinical Significance, Cellular Function, and Potential Molecular Pathways of CCT7 in Endometrial Cancer Liwen Wang 1,2†, Wei Zhou 1†, Hui Li 2,3, Hui Yang 1 and Nianchun Shan 1* 1 Department of Gynecology and Obstetrics, Xiangya Hospital, Central South University, Changsha, China, 2 Xiangya School of Medicine, Central South University, Changsha, China, 3 Department of Reproductive, Xiangya Hospital, Central South University, Changsha, China Objective: Endometrial cancer (EC) is a common gynecologic malignancy; myometrial Edited by: invasion (MI) is a typical approach of EC spreads and an important index to assess tumor Sarah M. Temkin, metastasis and outcome in EC patients. CCT7 is a member of the TCP1 chaperone Virginia Commonwealth University, family, involved in cytoskeletal protein folding and unfolding. In this study, the role of United States CCT7 in EC development was investigated. Reviewed by: Syed S. Islam, Methods: Clinical data for 87 EC cases and expression of CCT7 were analyzed. CCT7 King Faisal Specialist Hospital & Research Centre, Saudi Arabia was knocked out using siRNA-CCT7 in Ishikawa and RL95-2 cells, and their function Elena Gershtein, about proliferation, apoptosis, and invasion was further tested. Bioinformatics methods Russian Cancer Research Center NN were used to predict the potential pathways of CCT7 in EC development. Blokhin, Russia *Correspondence: Results: The rates of CCT7-positive cells in EC and adjacent normal endometrium Nianchun Shan tissues had a significant difference (67.8 vs. 51.4%, p = 0.035), and the expression [email protected] rate increased from low to high pathological stage (39.7% in the I/II stage, 71.4% in the †These authors have contributed III/IV stage, p = 0.029). A similar change was found in protein level. CCT7 expression equally to this work differed significantly between the deep MI group (>1/2) and the superficial MI group (≤1/2) (p = 0.039). However, there were no differences with respect to age, pathological Specialty section:RETRACTED This article was submitted to type, and histological grade. CCT7 suppression induced a function loss in both Ishikawa Women’s Cancer, and RL95-2 cells. Bioinformatics analysis demonstrated that EC patients with lower-level a section of the journal Frontiers in Oncology CCT7 expression had better overall survival (p = 0.0081). Gene ontology enrichment Received: 12 December 2019 indicated that “RNA binding,” “Mitochondrion,” “Translation,” and “Spliceosome” were Accepted: 09 July 2020 most significantly enriched potential pathways. Five hub genes, PSMA5, PSMD14, Published: 28 August 2020 SNRPB, SNRPG, and TXNL4A, were all significantly upregulated in EC and had a positive Citation: Wang L, Zhou W, Li H, Yang H and correlation with CCT7. Shan N (2020) Clinical Significance, Conclusions: CCT7 may be involved in EC development by excessively activating tumor Cellular Function, and Potential Molecular Pathways of CCT7 in cell function to promote MI or distant/nodal metastasis, which may contribute to the Endometrial Cancer. prognosis of EC patients. Front. Oncol. 10:1468. doi: 10.3389/fonc.2020.01468 Keywords: endometrial cancer, CCT7, myometrial invasion, FIGO stage, overall survival Frontiers in Oncology | www.frontiersin.org 1 August 2020 | Volume 10 | Article 1468 Wang et al. CCT7 and Endometrial Cancer INTRODUCTION MATERIALS AND METHODS Endometrial cancer (EC) is the fourth highest malignant tumor Tissue Sample Collection in women and the most common gynecologic cancer of the This retrospective study was approved by the Ethics Committees female reproductive system in the United States, with an overall of Xiangya Hospital, Central South University. Patients were incidence of 4.4% around the world in 2018 (1, 2). When fully informed of the study and provided consent before diagnosed at an early stage, EC is highly curable and has specimen collection. A total of 87 EC specimens from consecutive excellent overall 5-year survival rates (3). Although the overall patients (cancer and adjacent normal tissues) were confirmed prognosis is better than that of other cancer types, many by post-operative pathological diagnosis from March 2015 to women (especially young women) have aggressive neoplasms November 2015 at Xiangya Hospital Central South University. and require a hysterectomy, resulting in a loss of fertility and For all patients, relevant clinical data were collected and checked menstrual function and, accordingly, in physical and mental again for confirmation. The age of patients ranged from 31 to 70 disability (4). years, and mean age was 51.3 ± 12.2 years. A total of 75 cases The International Federation of Gynecology and Obstetrics were endometrioid adenocarcinoma, 5 were adenosquamous (FIGO) proposed that EC should be surgically staged, which carcinoma, 3 were serous papillary adenocarcinoma, 2 were clear comprises total hysterectomy, bilateral salpingo-oophorectomy, cell carcinoma, and 2 were mucinous carcinoma. According to cytologic washings, and pelvic and para-aortic lymphadenectomy 2009 FIGO guidelines (5) for the surgical staging of EC, 32 (5). A reliable risk stratification of EC could help to plan were Ia, 36 were Ib, 5 were II, 12 were III, and 2 were IV. a therapeutic schedule and predict the clinical outcomes Histopathological grading was as follows: 49 cases were G1, 33 for EC patients. The risk classification of EC is mainly cases were G2, and 5 cases were G3. according to tumor histology, tumor grade, and myometrial Immunohistochemistry (IHC) infiltration (MI) depth (6, 7). Tumor histology and histological Specimens were fixed in 10% formalin, embedded in paraffin, and grade may be evaluated preoperatively by endometrial biopsy. sectioned. Two experienced pathologists made the histological The MI depth of EC has a close relationship with lymph diagnosis, and CCT7 expression was evaluated using SP node involvement and distant metastasis and is the most Immunohistochemistry Kits (Abcam Ltd., Cambridge, UK). important risk factor in apparent early-stage EC, but it is Results were evaluated according to the following criteria (where difficult to determine preoperatively (8–10). Therefore, based on brown staining of the cytoplasm and cell membrane indicated comprehensive MI assessment preoperatively, it may assist in positive results) based on the degree of positive staining: weakly better planning of surgical procedures and avoiding unnecessary positive (+), 1 point; positive (+), 2 points; strongly positive lymph node dissections (11). Currently, magnetic resonance (+ + +), 3 points. Additionally, based on the percentage of imaging (MRI) and transvaginal ultrasound (TVS) are the positive cells, they were scored as follows: <25% positive cells commonest techniques for evaluating preoperatively the depth of (+), 1 point; 25–50% (+), 2 points; ≧ 50% (+ + +), 3 points; no MI. However, there are still some controversies and limitations positive cells indicated a negative result. Finally, a comprehensive on these techniques in the evaluation of the depth of MI indicator was calculated by summing the two scores, where ≦ by the EC (12, 13). Thus, it is necessary to develop new 3 points was defined as negative, 4–6 points was interpreted as methods to assist in comprehensively assessing the depth positive, and > 6 points was interpreted as strongly positive. of MI. The eukaryotic cytoplasmic chaperonin-containing TCP-1 Western Blotting Analysis (CCT), also called TRiC (TCP1 ring complex), is a complex Proteins were isolated from EC tissues via NP-40 lysis buffer composed of two back-to-back stacked hetero-octameric rings; (Abcam Ltd., Cambridge, UK) and then separated in 12% each ring is constituted by eight different types of subunits SDS-PAGE gels and blotted on nitrocellulose membranes. The (CCTα, β, γ, δ, ǫ, ζ, η, θ; CCT1 to CCT8 in yeast) (14, 15). filters were hybridized with polyclonal anti-CCT7 (Abcam Ltd., The eukaryotic chaperoninRETRACTED CCT is involved in the folding of Cambridge, UK) at 4◦C overnight, followed by incubation with cytoskeletal proteins (actins and tubulins) in an ATP dependent the secondary anti-rabbit (Abcam Ltd., Cambridge, UK) for 1 h approach (16, 17). In our previous study, we identified that at room temperature. Anti-Tubulin (Abcam Ltd., Cambridge, the CCT7 might be closely associated with EC by using UK) was used as the loading control. Gray-scale values were isobaric tags for relative and absolute quantitation (iTRAQ)- analyzed using the ImageJ software and were also described based proteomic analysis (p = 0.02). In the further experiment, previously (19). our results revealed that the mRNA expression of CCT7 was significantly higher in EC tissues than normal pericarcinous Cell Culture and Transfection tissue (p < 0.001, n = 10) (18). Therefore, in this study, we Endometrial cancer (Shanghai Zhongqiaoxinzhou Biotech, aimed to detect the CCT7 expression in EC tissues and further ZQ0472) and RL95-2 (Shanghai Zhongqiaoxinzhou Biotech, explore the developmental mechanism of EC at the cellular level. ZQ0362) cell lines were cultured in Dulbecco’s Modified Eagle’s Bioinformatics methods were also used to insight into potential Medium (Hyclone) supplemented with 5% fetal bovine serum molecular pathways. (FBS, Gibco), 300 mmol/L L-glutamine (Hyclone), 5 µg/mL Frontiers in Oncology | www.frontiersin.org 2 August 2020 | Volume 10 | Article 1468 Wang et al. CCT7 and Endometrial Cancer bovine insulin (Hyclone), 10,000 units/mL penicillin (Hyclone),
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