Journal name: OncoTargets and Therapy Article Designation: Original Research Year: 2018 Volume: 11 OncoTargets and Therapy Dovepress Running head verso: Dong et al Running head recto: The effect of TRIM37 on lung cancer cells open access to scientific and medical research DOI: 183303 Open Access Full Text Article ORIGINAL RESEARCH TRIM37 targets AKT in the growth of lung cancer cells Shumin Dong1 Background: TRIM37 is an ubiquitin E3 ligase. Growing evidence has demonstrated the Xueqin Pang2 high value of TRIM37 as a potential biomarker for diagnosis of certain cancers. However, the Haijun Sun3 biological function of TRIM37 in lung cancer is still unknown. Chunluan Yuan4 Materials and methods: In order to gain a deep insight into the function of TRIM37 in Chuanyong Mu5 lung cancer cells, in the present study lentiviral vector was used to mediate RNA interference Shiying Zheng6 and overexpression of TRIM37 in lung cancer cells (H292, H358, and H1299). In addition, a specific AKT inhibitor LY294002 was utilized to examine the correlation between the expres- 1 Department of Thoracic Surgery, sion of TRIM37 and AKT. The First Affiliated Hospital of Soochow University, The First Results: TRIM37 acts as a positive regulator of cell proliferation in lung cancer cells. More- People’s Hospital of Lianyungang City, over, cell apoptosis analyses showed the antiapoptosis function of TRIM37, which was mainly Lianyungang, Jiangsu 222000, People’s dependent on the regulation of BCL2 and BAX. Our results also indicated that AKT might be Republic of China; 2Department of Gastroenterology, The First Affiliated a target of TRIM37 in lung cancer cells. Hospital of Soochow University, Conclusion: This research not only helps in understanding the molecular mechanisms of TRIM37 Suzhou, Jiangsu 215006, People’s Republic of China; 3Department of in detail but also provides evidence to develop novel biomarkers for lung cancer diagnosis. Thoracic Surgery, The First People’s Keywords: TRIM37, cell proliferation, apoptosis, BCL2, BAX, AKT Hospital of Lianyungang City, Lianyungang, Jiangsu 222000, People’s Republic of China; 4Department of Introduction Oncology, The First People’s Hospital of Lianyungang City, Lianyungang, Lung cancer is one of the most commonly diagnosed cancers worldwide and has been ranked Jiangsu 222000, People’s Republic of as the top killer cancer all over the world.1,2 Though the traditional treatment (resection and China; 5Department of Respiratory Medicine, The First Affiliated Hospital radiotherapy) can contribute to fighting against the recurrence of lung cancer, the outcome of Soochow University, Suzhou, usually remains poor. Therefore, gaining a deep insight into the molecular mechanism Jiangsu 215006, People’s Republic of China; 6Department of Thoracic of lung cancer would help to develop new biomarkers and therapy for this disease. Surgery, The First Affiliated Hospital TRIM37 is one of the members of TRIM family, which is mapped on chromo- of Soochow University, Suzhou, some 17q22-23.3 TRIM37 serves as an ubiquitin E3 ligase and plays an essential role Jiangsu 215006, People’s Republic of China in oncogenesis. A previous report has shown that TRIM37 is upregulated in human colorectal cancer (CRC) and contributes to the development of CRC.4 Moreover, the Correspondence: Chuanyong Mu Department of Respiratory Medicine, expression of TRIM37 is significantly upregulated in pancreatic cancerous tissues, The First Affiliated Hospital of Soochow which indicates the oncogenic role of TRIM37 in pancreatic cancer.5 In addition, high University, Shizi Street No 188, Suzhou, 6 Jiangsu 215006, People’s Republic expression of TRIM37 is observed in osteosarcoma. Growing evidence has indicated of China that TRIM37 participates in the tumorigenesis of several cancers. However, the bio- Email [email protected] logical function of TRIM37 in lung cancer is less well understood. Shiying Zheng BCL2 mediates the intrinsic apoptosis pathway. A previous report has shown that Department of Thoracic Surgery, The BCL2 is also essential for cell cycle progression.7 The dysregulation of BCL2 not only First Affiliated Hospital of Soochow University, Shizi Street No 188, Suzhou, damages normal development but also contributes to tumor progression.8 The high Jiangsu 215006, People’s Republic level of BCL2 suppresses apoptosis in human lung cancer cells, which serves as the of China Email [email protected] antiapoptotic factor in this cancer.9 In addition, BAX belongs to the BCL2 family, which submit your manuscript | www.dovepress.com OncoTargets and Therapy 2018:11 7935–7945 7935 Dovepress © 2018 Dong et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/OTT.S183303 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Dong et al Dovepress functions as proapoptotic marker.10 Therefore, the occurrence alcohols were used to deparaffinize and rehydrate samples. of cell apoptosis depends on the ratio of BAX/BCL2. Dysreg- Antigen retrieval was carried out through steam heating for ulation of the ratio of BAX/BCL2 may lead to carcinogenesis. 15 minutes in 0.01 M citrate buffer at pH 6.0, and slides were Taken together, BCL2 family members have potential value rinsed three times with PBS (0.02 M) after natural cooling. as critical targets in lung cancer treatment.11,12 Then, the activity of endogenous peroxidase was suppressed The AKT pathway plays an essential role in various using Tris-buffered saline with Tween 20 containing 3% biological functions, which is commonly abnormal in human hydrogen peroxide for 10 minutes. The sections were incu- cancer.13 The activity of AKT protein relies on its phospho- bated with a primary antibody (TRIM37; Novus, St Charles, rylation. A previous report has indicated that the AKT phos- MO, USA) overnight at 4°C followed by biotin-labeled phorylation (p-AKT) on Thr308 correlates with the activity secondary antibody (Longislandbio, Shanghai, People’s of AKT protein in human non-small-cell lung cancer.14 The Republic of China) at room temperature for half an hour. PI3K/AKT pathway is involved in cell cycle and apoptosis of Then, hematoxylin was used for nuclear counterstaining after lung cancer cells.15 Some evidence has demonstrated that the slides were reacted with diaminobenzidine substrate. major function of TRIM37 in glioma cells is the inactivation of PI3K/AKT signaling pathway.16 Therefore, further studies RNA isolation and real-time PCR are needed to examine the relationship between TRIM37 and Total RNA from all samples was extracted using TRIzol AKT in lung cancer cells. reagent (Thermo Fisher Scientific). After that, cDNA syn- In the present study, we systematically analyzed the func- thesis kit (Thermo Fisher Scientific) was used to synthesize tion of TRIM37 and its possible targets in lung cancer cells. cDNA according to the instruction of the manufacturer. The RNA interference was used to knockdown the expression of conditions of real-time PCR were as follows: 95°C for 10 TRIM37 in lung cancer cells, while lentiviral vector was used to minutes followed by 40 cycles of 95°C for 15 seconds and mediate overexpression of TRIM37. Our data provide not only 60°C for 45 seconds. The expression was normalized to that a deep insight into the molecular mechanisms of TRIM37 but of GAPDH. The relative gene expression was calculated by also evidence to develop novel biomarkers for lung cancer. the 2-ΔΔCt method. All data represented the average of three replicates. The primer sequences used in this study are listed Materials and methods in the Supplementary materials. Tissue specimens and cell culture A total of 30 pairs of lung cancer tissues and adjacent non- Lentiviral vector-mediated RNA cancerous tissues were obtained from patients at the First Affiliated Hospital of Soochow University. Samples were interference and overexpression snap-frozen in liquid nitrogen and stored at -80°C for further of TRIM37 analysis. All patients provided written informed consent. This Lentiviral plasmids (pLKO.1) containing three shRNAs research was approved by the independent ethics committee of directed to various regions of human TRIM37 (NM_015294.5) the First Affiliated Hospital of Soochow University and was and a negative control shRNA (siNC) were synthesized carried out in accordance with the Declaration of Helsinki. (Major, Shanghai, People’s Republic of China), respectively. All the cell lines (A549, H1299, H1975, H358, H292, Lentiviral plasmid (pLVX-puro) containing the full length and 16HBE) used in this study were obtained from the cell of human TRIM37 cDNA sequence and the mock plasmid bank of Shanghai Institute for Biological Sciences (Shanghai, served as the negative control (oeNC). All of them were tran- People’s Republic of China). FBS (10%; Thermo Fisher siently transfected into lung cancer cells as indicated above Scientific, Waltham, MA, USA) was mixed into all culture using Lipofectamine 2000 (Thermo Fisher Scientific) accord- media along with 2 mM L-glutamine and 1%