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TRAF4 Promotes Endometrial Cancer Cell Growth and Migration by Activation of PI3K/AKT/Oct4 Signaling T ⁎ Pengmu Xie, Xiuling Wang, Min Kong, Xiuyu Bai, Tao Jiang

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TRAF4 Promotes Endometrial Cancer Cell Growth and Migration by Activation of PI3K/AKT/Oct4 Signaling T ⁎ Pengmu Xie, Xiuling Wang, Min Kong, Xiuyu Bai, Tao Jiang Experimental and Molecular Pathology 108 (2019) 9–16 Contents lists available at ScienceDirect Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp TRAF4 promotes endometrial cancer cell growth and migration by activation of PI3K/AKT/Oct4 signaling T ⁎ Pengmu Xie, Xiuling Wang, Min Kong, Xiuyu Bai, Tao Jiang Department of Gynecology, Jining No.1 People's Hospital, Jining 272011, China ARTICLE INFO ABSTRACT Keywords: Objective: Endometrial cancer (EC) is ranked as the most common gynecologic malignancy of the female genital TRAF4 tract and the fourth most common neoplasia in women. Accumulated evidences reveal that TRAF4 plays a Endometrial carcinoma critical role in the progress of various cancers, but its functions in EC remains unclear. This study aimed to Cell growth explore the role and mechanism of TRAF4 in EC progress. Migration Methods: TRAF4 expression in EC tissues were assessed by qRT-PCR, IHC and western blot. TRAF4 expression in PI3K/AKT/Otc4 signaling Ishikawa and primary EC cells was inhibited and overexpressed by transient transfections. Thereafter, cell proliferation was identified by combination of clone formation assay and Ki67 staining assay. Cell viability, apoptosis and migration were respectively measured by MTT assay, flow cytometry assay and transwell mi- gration assay. Furthermore, qRT-PCR and western blot analysis were mainly performed to assess the expression levels of apoptosis-related proteins and PI3K/AKT/Oct4 pathway proteins. Results: TRAF4 was up-regulated in EC tissues. Knockdown of TRAF4 induced cell apoptosis and inhibited cell proliferation and migration. However, TRAF4 overexpression increased cell proliferation and migration. Furthermore, we found TRAF4 down-regulation repressed the activation of PI3K/AKT signaling pathway in Ishikawa and primary EC cells. We also found that Oct4 was a downstream factor of PI3K/AKT pathway and was positively regulated by TRAF4. TRAF4 might increase cell viability through PI3K/AKT/Oct4 pathway. Conclusion: The present study demonstrated that TRAF4 might exert an oncogenic role in EC progress via its regulation of PI3K/AKT/Oct4 pathway. 1. Introduction indirectly influence the function of many signaling molecules (Bradley and Pober, 2001). TRAF4, an atypical member of the TRAF protein Endometrial carcinoma (EC), one of the most common gynaecolo- family, was initially identified differential screening of human meta- gical malignancys of the female reproductive system throughout the static lymph nodes in breast carcinoma (Kedinger and Rio, 2007). world, is diagnosed most frequently in women between 45 and 65 years Previous studies suggested that TRAF4 was amplified and over- old and its incidence has increased in recent years (MM et al., 2016; expressed and was involved in the initiation and progression of primary Morice et al., 2016). EC is a heterogeneous disease characterized by breast cancers and metastases (Kornegoor et al., 2012; Régnier et al., dysregulation of cell proliferation and metastasis (Ihira et al., 2017). It 1995). Emerging evidence indicated that TRAF4 was highly expressed could be initiated and promoted by molecular pathways alterations, in various human malignancies, including breast cancer, lung cancer, such as proto-oncogene activation, tumor suppressor gene inactivation, colon adenocarcinomas, melanomas, neurogenic tumors, and lym- aberrant DNA methylation, and noncoding microRNA (miRNA) dysre- phomas (Camilleribroët et al., 2007; Yao et al., 2014). Besides, TRAF4 gulation (Dong et al., 2013). However, the genetic and epigenetic basis has been identified as an oncogene and plays a critical role in cell of EC is not yet fully understood. proliferation, apoptosis, migration and invasion in breast cancer and Tumor necrosis factor receptor-associated factors (TRAFs) belong to other kinds of cancer (Wang et al., 2013; Zhang et al., 2013a; Zhang a family of adapter proteins that are involved in tumor necrosis factor et al., 2014b). However, few studies have focused on the function of receptor superfamily signaling and share a common structural domain TRAF4 in EC, and the mechanism regarding TRAF4's role in tumor- at their C-terminus (Bradley and Pober, 2001; Lu et al., 2003). TRAFs igenesis remains unclear. have been identified to regulate cell life and death through directly or The present study aimed to assess the expression of TRAF4 in EC ⁎ Corresponding author at: Department of Gynecology, Jining No.1 People's Hospital, No. 6, Jiankang Road, Jining 272011, China. E-mail address: [email protected] (T. Jiang). https://doi.org/10.1016/j.yexmp.2019.03.003 Received 5 September 2018; Received in revised form 8 January 2019; Accepted 5 March 2019 Available online 07 March 2019 0014-4800/ © 2019 Published by Elsevier Inc. P. Xie, et al. Experimental and Molecular Pathology 108 (2019) 9–16 and explored the role and the underlying mechanism of TRAF4 in EC TRAF4 and sh-TRAF4. The Lipofectamine 3000 reagent (Invitrogen) cell growth and metastasis. We found that the expression of TRAF4 was was used for the cell transfection according to the manufacturer's in- up-regulated in EC in vitro. Additionally, TRAF4 inhibition repressed structions. A non-targeting sequence of plasmid and an empty plasmid cell growth and migration in EC cells, while TRAF4 overexpression were respectively used as negative controls (NCs) of pc-TRAF4 and sh- exerted an opposite effect. Moreover, we found underlying mechanism TRAF4, and referred as to pcDNA3.1 and sh-NC. The stably transfected of its regulation may be through regulation of PI3K/AKT/Oct4 cells were selected by the culture medium containing 0.5 mg/ml G418 pathway. This data may provide new insight into the function of TRAF4 (Sigma-Aldrich). After approximately 4 weeks, G418-resistant cell as well as its regulatory mechanisms in the pathogenesis of EC. clones were established. 2. Materials and methods 2.4. siRNA transfection 2.1. The collection of paired tumor and non-tumor tissues and cell culture Oct4 siRNA and control siRNA (si-NC) were purchased from Cell Signaling Technology (Beverly, MA, USA). Cells infected with pc- Twenty pairs of EC and their corresponding adjacent non-tumor TRAF4 were incubated with a mixture of siRNA and Lipofectamine endometrial tissues used in this study were obtained from patients who 2000 reagent (Invitrogen) in 100 ml of serum-free OPTI-MEM underwent surgery at Jining No.1 People's Hospital from Feb 2011 to (Invitrogen) according to the manufacturer's instructions. May 2015. All female patients aged from 30 to 65 years, and the average age was 48 years. The study protocol was approved by the 2.5. Quantitative real time RT-PCR analysis ethics committee of Jining No.1 People's Hospital. All patients had no preoperative neoadjuvant treatment. Tissue specimens were im- Total RNA was extracted using TRIzol reagent (Invitrogen), and was mediately frozen in liquid nitrogen after surgery and preserved at reverse transcribed into cDNA using a Reverse Transcription Kit −80 °C for subsequent testing. The specimen collection procedure was (Takara, Dalian, China) according to the manufacturer's instructions. performed with written informed patient consent and the approval of The expression of TRAF4 was conducted by qRT-PCR using the SYBR the Medical Ethics Committee. Green Master Mix (Takara) according to manufacturer's instruction. The The human EC cell lines, Ishikawa, was obtained from Japanese mRNA of GAPDH level was used as an internal control and relative ΔΔ Cancer Research Resources Bank (JCRB, Osaka, Japan) and were expression changes were calculated using the 2- Ct method. maintained in Dulbecco's modified Eagle Medium F12 (DMEM/F12) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bo- 2.6. Western blot vine serum (FBS, Hyclone, Logan, UT, USA), and 1% penicillin-strep- tomycin solution (Gibco, Life Technologies, Grand Island, NY) at 37 °C The proteins used for western blot were extracted from tissues and in an humidified atmosphere containing 5% CO2. cells using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) Primary EC cells culture was operated according to previous study supplemented with protease inhibitors (Roche). Equal amount of de- (Genç et al., 2007). Endometrium tissue was minced and dissociated natured protein (30 μg) was loaded and separated by sodium dodecyl with collagenase B (1 mg/ml) and DNAase (2 mg/ml) (Roche, Basel, sulfate-polyacrylamide gel (SDS-PAGE), and then transferred to poly- Switzerland) at 37 °C for 30 min. Then cells were separated and cen- vinylidene fluoride membranes (Millipore, Bedford, MA, USA). trifugated, washed twice with DMEM/F12 (Sigma-Aldrich) containing Membranes were blocked with 5% BSA in TBST at 37 °C for 60 min and 1% penicillin-streptomycin solution (Gibco) and 10% FBS (Hyclone), incubated respectively with the primary antibodies at 4 °C overnight. and transferred into plastic culture flasks (25 cm2, TPP, St. Louis, MO, The primary antibodies: anti-TRAF4 (#18527), anti-Bid (#8762), anti- USA). Endometrial cells were plated in DMEM-F12 medium and Bax (#5023), anti-Cleaved caspase 3 (#9662), anti-GAPDH (#2118), maintained at 37 °C in a humidified chamber containing 5% CO2, and anti-p-AKT (#4060), anti-AKT (#4685), anti-p-PI3K (#4228), anti-PI3K allowed to replicate until confluent in monolayer. Culture medium was (#4249), anti-Oct4 (#2750) were purchased from Cell Signaling replaced twice a week. Technology and were prepared in 5% BSA at a dilution of 1:1000. After washing with TBST buffer for three times, the membranes were in- 2.2. Immunohistochemical (IHC) analysis cubated with HRP-conjugated secondary antibodies (Sigma-Aldrich) at a 1:5000 dilution for 2 h at room temperature. ECL reagent (Beyotime Specimens were confirmed by hematoxylin and eosin-stained sec- Biotechnology) was used for detection. The bolts were visualized by tions. Formalin-fixed, paraffin-embedded sections (4 μm) were depar- using Image Lab™ Software (Bio-Rad, Hercules, CA, USA). affinized in xylene, rehydrated in graded alcohol, and rinsed in phos- phate-buffered saline (PBS). Endogenous peroxidase activity was 2.7.
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