br In this study we found that A mediated
In this study, we found that A20-mediated deubiquitination of ERα in a tumor microenvironment rich with CD163+ macrophages played an important role in estrogen-driven endometrial carcinogenesis. Our study revealed that A20 may act as an oncogene for pathological ERα activation. In this way antagonizing A20 might be a potential ther-apeutic strategy to prevent estrogen-dependent EC.
2. Materials and methods
2.1. Ethics statement
This study complied with the tenets of the Helsinki Declaration and the National Guidelines for Animal Use in Research (China). This study was approved by the Medical Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University (Shanghai, People's Republic of China, hereafter referred to as 'Ob&Gyn Hospital').
2.2. Endometrial sample collection
Endometrial tissues in normal proliferative phase (PP), or with hy-perplasia, atypia hyperplasia (EAH), and EEC (including endometrioid adenocarcinoma grade 1, grade 2, and graded 3) were obtained from patients who underwent hysterectomy at Ob&Gyn Hospital from Cancer Letters 442 (2019) 137–147
January 2016 to November 2017. None of the patients received hor-mone therapy within 3 months before surgery. Normal endometrium was obtained from patients receiving hysterectomy because of leio-myoma, cervical high-grade squamous intraepithelial lesion (HSIL) or cervical cancer. Pathological diagnosis of endometrial samples was in-dependently confirmed by at least two pathologists.
Twenty-two fresh endometrial tissues were collected for flow cyto-metric analysis and patient information was listed in Supplementary Table S1. A further 29 cases were collected for IHC staining and patient information was listed in Supplementary Table S2.
2.3. Fresh endometrium preparation and flow cytometric analysis
Fresh endometrium was prepared as previously described  and seen in Supplementary materials and methods. Cells from fresh en-dometrium were labeled with fluorescent-tagged Bortezomib (PS-341) and then detected by flow cytometry (Beckman). Anti-Hu CD45-BV510, anti-Hu IFNγ-Alexa 647, anti-Hu TGFβ-BV421, anti-mIgG2b Kpa-BV510, anti-mIgG1 Kpa-Alexa 647, and anti-mIgG1 Kpa-BV421 antibodies were purchased from BD Pharmingen, while anti-Hu CD14-PerCP/Cy5.5, anti-Hu CD86-PE/Cy7, anti-Hu CD163-PE, anti-mIgG1 κ Isotype-PerCP/ Cy5.5, anti-mIgG1 κ Isotype-PE/Cy7, and anti-mIgG1 κ Isotype-PE an-tibodies were purchased from Biolegend.
2.4. Immunohistochemical (IHC) staining
IHC staining was performed as previously described . Primary antibodies used in IHC staining included CD163 (Abcam, ab182422), A20 (Cell Signaling Technology, D13H3), and ERα (Cell Signaling Technology, D8H8). Semi-quantitative optical analysis was performed as previously described (6).
2.5. Establishment of mouse model and microarray analysis
Female C57BL/6 mice received bilateral oophorectomy at 6–8 weeks and then were separated randomly into two groups. The mice in the control group were fed with normal chow diet while the other group received a high-fat diet (Research Diets, D12492) and subcutaneously implantation of an estrogen-releasing pellet (Innovative Research of America, NE-121, 0.36 mg/pellet). After 12 weeks of intervention, the endometrial samples were collected and total RNAs were extracted for microarray analysis using the Aﬀymetrix GeneChip Mouse Transcriptome Array 1.0 b y GMINIX (Shanghai, China). Microarray data was uploaded to the Gene Expression Omnibus repository, with the GEO accession number GSE102103 (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE102103).
EC cell lines RL95-2, Ishikawa, and SPEC2 were kindly provided by Dr. Yu Yinhua (MD Anderson Cancer Center, Houston, TX) and HEC-1A was kindly provided by Dr. Wei Lihui (Peking People Hospital, Beijing, China). EC cell line KLE, human acute monocytic leukemia cell line THP-1, and human embryonic kidney (HEK) 293T cells were purchased from ATCC (USA). RL95-2, Ishikawa, SPEC2, and HEC-1A cells were cultured in DMEM/F12 medium, and KLE and HEK-293T in DMEM medium, while THP1 cells were cultured in RPMI 1640 medium sup-plemented with 10% fetal bovine serum (FBS).