Tumor Biol. DOI 10.1007/s13277-014-1763-x
RESEARCH ARTICLE
TRIM31 is downregulated in non-small cell lung cancer and serves as a potential tumor suppressor Hui Li & Yi Zhang & Yue Zhang & Xue Bai & Yang Peng & Ping He
Received: 4 December 2013 / Accepted: 13 February 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The present study aims to investigate expression pattern and biological roles of TRIM31 in human non-small cell lung cancer (NSCLC). We examined TRIM31 expression in 116 NSCLC tissues and 20 corresponding normal lung tissues by immumohistochemistry. We found TRIM31 downregulation in 47 out of 116 (40.5 %) cancer samples, which correlated with tumor status (p=0.0132), advanced p-TNM stage (p=0.001), and nodal metastasis (p=0.0382). TRIM31 expression was lower in lung cancer cell lines than normal bronchial cell line HBE. Transfection of TRIM31 plasmid was performed in H157 and H1299 cells. TRIM31 overexpression inhibited cell growth rate and colony formation ability in both cell lines. In addition, expression of cell cycle regulator cyclin D1 and cyclin E were decreased after TRIM31 transfection. In conclusion, TRIM31 might serve as a tumor suppressor in non-small cell lung cancer.
Keywords TRIM31 . Immunohistochemistry . NSCLC . Cyclin D1 . Cyclin E
direct role in tumor progression is an important task for developing a novel therapeutic strategy [4–6]. TRIM31 was a member of RBCC protein, which was composed of RING finger, B-box and coiled-coil domains. TRIM31 expression was found to be upregulated in patients with chronic gastritis and stomach cancer [7]. Transfection of TRIM31 suppresses colony formation while its knockdown enhances cancer cell growth, making it a negative regulator of cell proliferation [7]. TRIM31 was reported to interact with p52 (Shc) and its overexpression suppressed c-Src induced anchorage-independent growth [8]. However, the expression pattern of TRIM31 protein and its clinical significance in human NSCLC have not been well characterized to date. The biological roles of TRIM31 in NSCLC have not been explored. In order to address these questions, we examined TRIM31 expression in NSCLC by immunohistochemistry and analyzed its clinical significance. Also, the potential molecular mechanism for and biological significance of TRIM31 were investigated in vitro.
Materials and methods Introduction
Patients and specimens
Lung cancer is one of the leading causes of all cancer-related deaths worldwide [1]. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. The development and progression of NSCLC is a complex process. Tumor proliferation and invasion is the main cause of malignant lung cancer progression [2, 3]. Understanding the molecular basis of its development and identification of tumor suppressor playing a
This study was conducted with the approval of the local institutional review board at the Shengjing Hospital. One hundred sixteen cases of NSCLC samples and 20 tumor adjacent normal lung tissues were obtained from the Shengjing Hospital of China Medical University during the period of 2008 to 2012. The histological diagnosis and grade of differentiation of the tumors were defined by evaluation of the hematoxylin and eosin-stained tissue sections, according to the World Health Organization guidelines of classification (2004). All 116 specimens were reevaluated with respect to their histological subtypes, differentiation status, and tumor
H. Li : Y. Zhang : Y. Zhang : X. Bai : Y. Peng : P. He (*) Department of Geriatrics, Shengjing Hospital of China Medical University, 36 Sanhao Road, Shenyang 110004, China e-mail: [email protected]
Tumor Biol.
stages. The p-TNM staging system of the International Union Against Cancer (seventh edition) was used to classify specimens. Immunohistochemistry Surgically excised tumor specimens were fixed with 10 % neutral formalin and embedded in paraffin, and 4-μm-thick sections were prepared. Immunostaining was performed using the avidin–biotin–peroxidase complex method (Ultrasensitive™, MaiXin, Fuzhou, China). The sections were deparaffinized in xylene, rehydrated with graded alcohol, and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min in an autoclave. Hydrogen peroxide (0.3 %) was applied to block endogenous peroxide activity, and the sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with TRIM31 antibody (1:150, Prteintech, USA). Antibody was diluted in PBS and antibody incubation was performed at 37 °C in humidity box for 2 h. Biotinylated goat anti-rabbit serum IgG was used as a secondary antibody. After washing, the sections were incubated with streptavidin–biotin conjugated with horseradish peroxidase, and the peroxidase reaction was developed with 3,3′diaminobenzidine tetrahydrochloride. Counterstaining with hematoxylin was performed, and the sections were dehydrated in ethanol before mounting. Two independent investigators examined all tumor slides randomly. Five views were examined per slide, and 100 cells were observed per view at ×400 magnification. Immunostaining of TRIM31 was scored following a semiquantitative scale by evaluating in representative tumor areas, the intensity and percentage of cells. Cytoplasmic staining of the tumor cells was considered as positive immunostaining. The intensity of TRIM31 cytoplasmic staining was also scored as 0 (no staining), 1 (weak), and 2 (marked). Percentage scores were assigned as 1(1–25 %), 2 (26–50 %), 3 (51–75 %), and 4 (76–100 %). The scores of each tumor sample were multiplied to give a final score of 0 to 8, and the total expression of TRIM31 was determined as either negative or low expression (−): score
RESEARCH ARTICLE
TRIM31 is downregulated in non-small cell lung cancer and serves as a potential tumor suppressor Hui Li & Yi Zhang & Yue Zhang & Xue Bai & Yang Peng & Ping He
Received: 4 December 2013 / Accepted: 13 February 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The present study aims to investigate expression pattern and biological roles of TRIM31 in human non-small cell lung cancer (NSCLC). We examined TRIM31 expression in 116 NSCLC tissues and 20 corresponding normal lung tissues by immumohistochemistry. We found TRIM31 downregulation in 47 out of 116 (40.5 %) cancer samples, which correlated with tumor status (p=0.0132), advanced p-TNM stage (p=0.001), and nodal metastasis (p=0.0382). TRIM31 expression was lower in lung cancer cell lines than normal bronchial cell line HBE. Transfection of TRIM31 plasmid was performed in H157 and H1299 cells. TRIM31 overexpression inhibited cell growth rate and colony formation ability in both cell lines. In addition, expression of cell cycle regulator cyclin D1 and cyclin E were decreased after TRIM31 transfection. In conclusion, TRIM31 might serve as a tumor suppressor in non-small cell lung cancer.
Keywords TRIM31 . Immunohistochemistry . NSCLC . Cyclin D1 . Cyclin E
direct role in tumor progression is an important task for developing a novel therapeutic strategy [4–6]. TRIM31 was a member of RBCC protein, which was composed of RING finger, B-box and coiled-coil domains. TRIM31 expression was found to be upregulated in patients with chronic gastritis and stomach cancer [7]. Transfection of TRIM31 suppresses colony formation while its knockdown enhances cancer cell growth, making it a negative regulator of cell proliferation [7]. TRIM31 was reported to interact with p52 (Shc) and its overexpression suppressed c-Src induced anchorage-independent growth [8]. However, the expression pattern of TRIM31 protein and its clinical significance in human NSCLC have not been well characterized to date. The biological roles of TRIM31 in NSCLC have not been explored. In order to address these questions, we examined TRIM31 expression in NSCLC by immunohistochemistry and analyzed its clinical significance. Also, the potential molecular mechanism for and biological significance of TRIM31 were investigated in vitro.
Materials and methods Introduction
Patients and specimens
Lung cancer is one of the leading causes of all cancer-related deaths worldwide [1]. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. The development and progression of NSCLC is a complex process. Tumor proliferation and invasion is the main cause of malignant lung cancer progression [2, 3]. Understanding the molecular basis of its development and identification of tumor suppressor playing a
This study was conducted with the approval of the local institutional review board at the Shengjing Hospital. One hundred sixteen cases of NSCLC samples and 20 tumor adjacent normal lung tissues were obtained from the Shengjing Hospital of China Medical University during the period of 2008 to 2012. The histological diagnosis and grade of differentiation of the tumors were defined by evaluation of the hematoxylin and eosin-stained tissue sections, according to the World Health Organization guidelines of classification (2004). All 116 specimens were reevaluated with respect to their histological subtypes, differentiation status, and tumor
H. Li : Y. Zhang : Y. Zhang : X. Bai : Y. Peng : P. He (*) Department of Geriatrics, Shengjing Hospital of China Medical University, 36 Sanhao Road, Shenyang 110004, China e-mail: [email protected]
Tumor Biol.
stages. The p-TNM staging system of the International Union Against Cancer (seventh edition) was used to classify specimens. Immunohistochemistry Surgically excised tumor specimens were fixed with 10 % neutral formalin and embedded in paraffin, and 4-μm-thick sections were prepared. Immunostaining was performed using the avidin–biotin–peroxidase complex method (Ultrasensitive™, MaiXin, Fuzhou, China). The sections were deparaffinized in xylene, rehydrated with graded alcohol, and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min in an autoclave. Hydrogen peroxide (0.3 %) was applied to block endogenous peroxide activity, and the sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with TRIM31 antibody (1:150, Prteintech, USA). Antibody was diluted in PBS and antibody incubation was performed at 37 °C in humidity box for 2 h. Biotinylated goat anti-rabbit serum IgG was used as a secondary antibody. After washing, the sections were incubated with streptavidin–biotin conjugated with horseradish peroxidase, and the peroxidase reaction was developed with 3,3′diaminobenzidine tetrahydrochloride. Counterstaining with hematoxylin was performed, and the sections were dehydrated in ethanol before mounting. Two independent investigators examined all tumor slides randomly. Five views were examined per slide, and 100 cells were observed per view at ×400 magnification. Immunostaining of TRIM31 was scored following a semiquantitative scale by evaluating in representative tumor areas, the intensity and percentage of cells. Cytoplasmic staining of the tumor cells was considered as positive immunostaining. The intensity of TRIM31 cytoplasmic staining was also scored as 0 (no staining), 1 (weak), and 2 (marked). Percentage scores were assigned as 1(1–25 %), 2 (26–50 %), 3 (51–75 %), and 4 (76–100 %). The scores of each tumor sample were multiplied to give a final score of 0 to 8, and the total expression of TRIM31 was determined as either negative or low expression (−): score
