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Mir-369-3P Participates in Endometrioid Adenocarcinoma Via

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Mir-369-3P Participates in Endometrioid Adenocarcinoma Via Liu et al. Cancer Cell Int (2019) 19:178 https://doi.org/10.1186/s12935-019-0897-8 Cancer Cell International PRIMARY RESEARCH Open Access MiR-369-3p participates in endometrioid adenocarcinoma via the regulation of autophagy Ping Liu1,2, Chengbin Ma2, Qiongwei Wu2, Wenying Zhang2, Cao Wang2, Li Yuan3* and Xiaowei Xi1* Abstract Background: The aim of this study is to examine miRNA profling and miR-369-3p participates in endometrioid adenocarcinoma (EEC) via the regulation of autophagy. Methods: EEC and its adjacent normal tissues were obtained from 20 clinical patients after surgery. MiRNA profling was performed using next generation sequencing (NGS) and was validated with quantitative real time PCR (qRT-PCR). qRT-PCR was also employed to measure miR-369-3p and autophagy-related protein 10 (ATG10) expression levels. Western blotting assay was performed to measure the expressions of ATG10 and LC3B. Luciferase reporter assay was conducted to confrm the direct targeting of ATG10 by miR-369-3p. Cell proliferation and migration assays were uti- lized to analyze the role of miR-369-3p in HEC-1-A cells. Results: We found that miR-369-3p expression levels were down-regulated in EEC compared to the control tissues. The overexpression of miR-369-3p inhibited cell proliferation and migration in EEC; furthermore, ATG10 expression increased in EEC tissues. ATG10 was found to be a potential target of miR-369-3p via a dual-luciferase reporter assay, and ATG10 was shown to be down-regulated by miR-369-3p in protein levels. Conclusions: This study revealed that miR-369-3p inhibited cell proliferation and migration by targeting ATG10 via autophagy in EEC. Keywords: Endometrioid adenocarcinoma, MicroRNA, Autophagy, ATG10 Background Autophagy is an important catabolic process in which Endometrial adenocarcinoma (EEC) is a common and damaged organelles or protein recycling undergoes nutri- highly malignant gynecological tumor [1]. In recent ent starvation or stress [3], and it is required for glucose years, its incidence rate in China and abroad has shown homeostasis and lung tumor maintenance [4]. Autophagy an upward trend, with the prognosis of EEC being poor participates in a critical pathway that maintains intracel- and the improvement of prognosis depending on early lular balance in regard to both the initiation and preven- diagnosis [2]. However, the early diagnosis of EEC is chal- tion of cancer [5]. It also supports the survival of tumor lenging. Specifcally, identifying highly sensitive and spe- cells under metabolic and therapeutic stress [6]. Further- cifc tumor markers is a challenge that needs to be solved more, the inhibition of autophagy can increase the chemo for the early diagnosis of this cancer. sensitivity of cancer cells [7, 8]. However, autophagy is a double-edged sword when regarding health and disease [9]. Autophagy begins with autophagosome formation and concludes with a completed autophagolysosome [10]. *Correspondence: [email protected]; [email protected] In addition, the synthesis of the autophagosome complex 1 Gynecology Department, Shanghai General Hospital of Nanjing Medical University, Shanghai 200080, China is regulated by autophagy-related genes (ATG) [11–13]. 3 School of Basic Medical Sciences of Nanjing Medical University, Several important genes are involved in the autophagy Nanjing 211166, China process, including ATG3, ATG5, ATG7, ATG10, ATG12, Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Cancer Cell Int (2019) 19:178 Page 2 of 10 and LC310. More evidence has also highlighted the consent forms. Tis study’s protocol conformed to the importance of these aforementioned autophagy-related ethical guidelines of the 1975 Declaration of Helsinki. In genes in cancer maintenance, clinical therapy, and patho- this study, EEC and their adjacent normal tissues were genesis 8. ATG10 is an E2-like enzyme involved in two obtained from 20 clinical patients. Four pairs were used ubiquitin-like modifcations essential to autophagosome for next generation sequencing (NGS) detection, and the formation [14]. Increased expression of ATG10 in colo- others were used for qRT-PCR or Western blot (WB) rectal cancer is associated with lymphovascular invasion detection. Diagnosis of EEC was based upon clinical, and lymph node metastasis [15]. Trough the genetically imaging, and histopathological fndings based on explant engineered mouse models, ATG7 ablation was found to evaluation according to the revised WHO classifcation block tumor growth and induce tumor cell death in lung of tumors. Patients were aged 40–45 years, with a mean cancer, indicating that autophagy inhibition might serve age of 43.8 years. Te EEC stage, in accordance with the as a promising strategy for lung cancer therapy [12]. surgery-pathological staging criteria issued in 1988 by However, the mechanisms and clinical value of these the International Federation of Gynecology and Obstet- critical regulatory autophagy-related genes have not been rics (FIGO), was identifed as III–IV. Pathological records explored in regard to EEC. and tissue samples were collected during surgery and MicroRNAs (miRNAs or miRs) are classifed as small, immediately snap-frozen in liquid nitrogen. Te clinical endogenous, non-coding, single-stranded RNAs that reg- characteristics of the patients are presented in Table 1. ulate target genes post-transcriptionally by specifcally binding to the 3′ untranslated region (3′-UTR) of the Cell culture target mRNA [16]. It was reported that miRNAs play an HEC-1-A cells (human EEC cells-1-A) are human EEC important role in various biological processes, including cell lines and were obtained from the American Type cell growth, cell death, apoptosis, and tumorigenesis [17, Culture Collection (Manassas, VA, USA) and cultured 18]. Several studies using miRNA microarray analyses or in McCoy’s 5A modifed medium supplemented with miRNA-Seq have revealed that the expression of miR- 10% fetal bovine serum and 1% penicillin/streptomy- 369-3p increases in the skin tissues of psoriasis patients cin (Sigma-Aldrich, St Louis, MO, USA). HEC-1-A cells [19] and that the aberrant expression of miR-369-3p cor- are used as an accepted model for studying endometrial relates with disease severity in hepatocellular carcinoma cancer [22]. MiR-369-3p, a mimic, inhibitor, agomir [20]. Hu et al. [21] reported that miR-369 induced cellu- or antagomir, and an agomir negative-treated control lar reprogramming and regulated malignant phenotypes (NTC) (fnal concentration, 2.5 nmol/L) were purchased of human colorectal cancer. Li et al. found that miR- from RiboBio Inc. (GuangZhou, China). An adenovirus 369-3p was diferently expressed in Hirschsprung disease (Ad)-expressing system, used for the overexpression the and may play a crucial role in its pathology [22]. Agarwal ATG10 protein (Ad-expression ATG10), was purchased et al. [23] indicated that the upregulation of miR-369-3p from Hanbio, Shanghai, China, with an Ad-expressing suppresses cell migration and proliferation by targeting green fuorescent protein (GFP) serving as a control. SOX4 in Hirschsprung’s disease. However, the detailed Cells were treated with miR-369-3p agomir, antagomir, mechanism of miR-369-3p and its potential target gene or NTC in six-well plates following manufacture proto- involved in the pathogenesis of endometrial adenocarci- col. For the rescue experiments, cells were treated with noma is still not widely understood. both miR-369-3p agomir and Ad-expression ATG10. Terefore, we sought to determine whether miR- 369-3p is involved in the pathology of EEC and whether MicroRNA sequencing miR-369-3p contributes to EEC by regulating autophagy. MiRNAs were isolated using the miRNA kit (Qiagen, Hilden, Germany) according to the manufacturer instruc- Methods tions. Te quantity and quality of RNA were assessed Experimental subjects using the NanoDrop ND-1000 instrument (Nanodrop Tis is a retrospective study of EEC tissues and their adja- cent non-cancerous counterparts regarding four patients treated by surgical resection. Te EEC patients were Table 1 Clinical characteristics of the patients with EEC admitted to the Shanghai First People’s Hospital from Sex (female) 20 July 2017 to July 2018. None of the patients underwent Mean age 43.8 1.3 ± chemotherapy, radiotherapy, or steroid treatment. Tis Stage (FIGO) 1a 1b + study was approved by the Review Board of the Shang- TNM Classifcation I/II hai First People’s Hospital under the supervision of the Nodules metastases No Ethics Committee, and all patients signed the informed Liu et al. Cancer Cell Int (2019) 19:178 Page 3 of 10 Technologies, Wilmington, USA) and the Bioanalyser target genes into all three programs. Gene ontology (GO) 2100 system (Agilent Technologies, CA, USA) using the (TopGO v4.2) grouping categories (Biological Process, Agilent RNA 6000 Nano according to the manufacturer Cellular Components, and Molecular
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