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RSC Advances View Article Online PAPER View Journal | View Issue TRIM22 functions as an oncogene in gliomas through regulating the Wnt/b-catenin signaling Cite this: RSC Adv.,2018,8, 30894 pathway Shi-lei Tang,†a Yuan-lin Gao†b and Wen-zhong Hu *a The tripartite motif-containing (TRIM) family is a group of proteins that are implicated in a plethora of pathological conditions. TRIM22 has been found to be involved in various cancers; however, the role of TRIM22 in gliomas has not been reported. The present study aimed to evaluate the expression pattern of TRIM22 and its function in gliomas. TRIM22 expressions in glioma tissues and cell lines were measured by RT-PCR and western blot analysis. To knockdown TRIM22 by small hairpin RNAs (shTRIM22), the U118 cells were transfected with pLKO.1-shTRIM22 plasmid or pLKO.1 plasmid. Cell proliferation was measured using CCK-8 assay. Transwell assays were performed to evaluate the migration and invasion. The epithelial–mesenchymal transition (EMT) was assessed by detecting the expressions of E-cadherin, Creative Commons Attribution-NonCommercial 3.0 Unported Licence. N-cadherin and vimentin with western blot analysis. A xenograft mouse model was established to evaluate the effect of TRIM22 silencing on tumor growth in vivo. The expressions of b-catenin, cyclin D1, and c-Myc were analyzed by western blot analysis. TRIM22 was significantly overexpressed in glioma tissues and cell lines. In vitro studies demonstrated that TRIM22 knockdown inhibited cell proliferation, migration, and invasion. Additionally, TRIM22 silencing increased the expressions of E-cadherin, and decreased the expressions of N-cadherin and vimentin. Nude mouse xenograft assay showed that TRIM22 silencing inhibited tumor growth in vivo. Furthermore, silencing of TRIM22 inhibited the Received 3rd July 2018 activation of the Wnt/b-catenin pathway. Treatment with LiCl, an activator of the Wnt/b-catenin Accepted 20th August 2018 pathway, attenuated the effects of shTRIM22 on U118 cells. Silencing of TRIM22 inhibited proliferation, This article is licensed under a DOI: 10.1039/c8ra05684f migration and invasion, as well as repressing the EMT process in glioma cells. The Wnt/b-catenin rsc.li/rsc-advances pathway was involved in the effect of TRIM22. Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM. 1. Introduction The tripartite motif-containing (TRIM) family is a group of proteins that are characterized by a conserved structure with Glioma is a type of tumor originating in the glial cells in the a RING domain, one or two B-box domains, a predicted coiled brain.1 It accounts for about 33 percent of all brain tumors, and coil, and usually a C-terminal domain.5 Several lines of evidence 80 percent of all malignant brain tumors.2 It represents a rela- suggest that TRIM proteins have a broad range of functions in tively serious health burden.1 On the basis of their histopa- regulation of many cellular processes such as cell differentia- thology, gliomas can be subdivided into grades I–IV according tion, proliferation/cycle, and apoptosis.6 Consistently, the to the World Health Organisation (WHO) classication of alterations of these proteins are implicated in a plethora of tumors in the central nervous system.3 The treatment of gliomas pathological conditions.6,7 Some members are thought to be is a combined therapy with surgery, radiation therapy and important regulators of carcinogenesis.8,9 Among these chemotherapy, which depends on the location and grade of members, TRIM22 has been proved to be involved in various malignancy.4 However, the conventional treatment has an cancers, such as endometrial cancer,10 leukemia,11 non-small alarmingly dismal prognosis. Gliomas generally recur even aer cell lung cancer,12 and breast cancer.13 However, the role of optimal initial treatment. Therefore, novel approaches in the TRIM22 in glioma has not been investigated. The present study treatment of this disease are still needed. aimed to evaluate expression pattern of TRIM22 and its func- tion in glioma. a RETRACTED Department of Neurosurgery, Huaihe Hospital of Henan University, No. 8 Baobei Road, Kaifeng 475000, Henan Province, China. E-mail: [email protected]; Fax: 2. Materials and methods +86-0371-23906516; Tel: +86-0371-23906516 2.1 Patient samples bDepartment of Neurology, Kaifeng Central Hospital, Kaifeng 475000, Henan Province, China The clinical samples used in this study are consisted of 21 † These authors contributed equally to this work. glioma tissues and matched normal brain tissues were collected 30894 | RSC Adv.,2018,8,30894–30901 This journal is © The Royal Society of Chemistry 2018 View Article Online Paper RSC Advances from patients with a mean age of 44 Æ 5 years. The patients 2.5 Sh-TRIM22 transfection underwent surgical treatment at Huaihe Hospital of Henan Small hairpin RNAs (shRNA) targeting TRIM22 (sh-TRIM22) and University between May 2014 and September 2017. This study its negative control (sh-control) were purchased from Shanghai was performed in strict accordance with the NIH guidelines for Genechem Co., Ltd. (Shanghai, China). The shTRIM22 was the care and use of laboratory animals (NIH Publication No. 85- constructed into pLKO.1 plasmids (Sigma). The pLKO.1-sh- 23 Rev. 1985) and was approved by the Institutional Animal TRIM22 plasmid or pLKO.1-sh-control was transfected into Care and Use Committee of Huaihe Hospital of Henan U87 cells. Then the transfected cells were subjected to 14 days of À University (Kaifeng, China). All patients were informed and selection in DMEM medium containing 1.0 mgmL 1 puromycin have signed the informed consent. (Sigma). Finally, the individual puromycin-resistant cells were isolated and collected for further experiments as stable 2.2 Cell culture expressing cells. Normal microglia cell line HEB, and glioma cell lines including A172, LN229, U251, and U87 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells 2.6 Cell proliferation assay were grown in Dulbecco's modi ed Eagle's medium (DMEM; Cell counting-kit-8 (CCK-8) assay was used for the determina- Hyclone, Logan, UT, USA) containing 10% fetal bovine serum tion of cell proliferation. U87 cells were seeded in 96-well plates (FBS; Gibco Laboratories, Grand Island, NY, USA) and 1% at a density of 1 Â 104 cells per well. Then 10 ml CCK-8 solution penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at (Dojindo, Kumamoto, Japan) was added to each well at 0 h, 24 h, 37 C in an atmosphere with 5% CO2. 48 h, and 72 h, and incubated for 1 h at 37 C. Subsequently, the absorbance values were determined at 450 nm using a micro- 2.3 Quantitative real-time polymerase chain reaction (qRT- plate reader (Bio-Rad). PCR) analysis Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Total RNA was extracted from the tissues and cells by Trizol reagent (Invitrogen-Thermo Fisher Scientic, Waltham, MA, 2.7 Cell migration and invasion assays USA). Then the obtained RNA was used to synthesize cDNA Cell invasive and migratory abilities were assayed using using a cDNA Reverse Transcription Kit (Fermentas-Thermo transwell assays with 8 mm transwell plates (Corning Inc., Fisher Scientic, USA) following the manufacturer's protocol. Corning, NY, USA). Cells (100 ml) suspended in DMEM at The mRNA levels of TRIM22 were quantied using the SYBR® adensityof1Â 105 cells per ml were added to the upper Green PCR Master Mix (Thermo) on an ABI-7300 sequence chambers coated with matrigel (for invasion assay) or without detection system (Applied Biosystems, Foster, CA, USA). The matrigel (for migration assay). And DMEM containing 10% 0 This article is licensed under a primers used are as follows: TRIM22, forward 5 -GCAC GCTC FBS (600 ml) was added to the lower chambers. Aer incuba- 0 0 ATCT CAGA TCTC C-3 and reverse 5 -TTTT GGCT TTTC AATG tion for 24 h, the inserts were removed and xed with 4% 0 0 0 TCCA G-3 ; GAPDH, forward 5 -AATC CCAT CACC ATCT TC-3 methanol for 15 min, followed by staining with 0.1% crystal 0 0 and reverse 5 -AGGC TGTT GTCA TACT TC-3 . GAPDH was used violet for 15 min. Images of ve random elds were captured Open Access Article. Published on 03 September 2018. Downloaded 9/29/2021 1:08:09 AM. as a reference gene. The relative mRNA levels of TRIM22 to under a microscope (Olympus Corporation, Tokyo, Japan). The ÀDDC GAPDH were calculated by the 2 t method. numbers of the cells were counted, and the average numbers were calculated. 2.4 Western blot analysis Total proteins of tissues and cells were prepared using RIPA buffer (Invitrogen). Protein concentrations were determined 2.8 Nude mouse xenogra model using the BCA protein assay kit (Thermo). The proteins (30 mg) The animal experiments were approved by the Institutional were separated by 10% SDS-PAGE and then transferred to Animal Care and Use Committee of Huaihe Hospital of Henan nitrocellulose membranes (Millipore, Billerica, MA, USA) using University (China). The experiments were performed according semi-dry method. Subsequently, the membranes were blocked to the National Institute of Health guidelines. Twelve female 6 for 1 h with 5% non-fat milk at room temperature. Aer that, week-old athymic nude mice (HFKBio, Peking, China) were the membranes were subjected to incubation with primary randomly separated into two groups: control group (n ¼ 6) and antibodies against TRIM22 (Sigma; 1: 1000); E-cadherin, N- TRIM22 silencing group (n ¼ 6). The pLKO.1-shTRIM22 cadherin, vimentin (Thermo; 1:1000); b-catenin, cyclin D1, c- plasmid or pLKO.1 plasmid transfected cells (5 Â 106 cells per Myc or b-actin (Abcam, Cambridge, MA, USA; 1: 800) over- mouse) were subcutaneously inoculated into le right of the night at 4 C.