ONCOLOGY REPORTS 31: 1271-1276, 2014
Downregulation of NOB1 suppresses the proliferation and tumor growth of non-small cell lung cancer in vitro and in vivo YANG LI1, CHENGYUAN MA2, MING QIAN3, ZHONGMEI WEN1, HONGYU JING1 and DONGHUA QIAN1 Departments of 1Respiration, and 2Neurosurgery, The First Hospital of Jilin University, Changchun, Jilin 130021; Prosthodontics Department of the Stomatological Hospital, Jilin University, Changchun, Jilin 130021, P.R. China
3
Received December 1, 2013; Accepted January 8, 2014 DOI: 10.3892/or.2014.2991 Abstract. Non-small cell lung cancer (NSCLC) is a lethal disease due to the absence of effective diagnostic biomarkers and therapeutic targets. Therefore, novel molecular targets are critically needed to formulate new approaches for this devastating disease. In the present study, using quantitive real-time PCR and immunohistochemistry. we initially found that expression of the ribosome assembly factor NIN/ RPN12 binding protein (NOB1) was elevated in the majority of NSCLC tissues when compared to that in the normal lung tissue counterparts������������������������������������� , and its expression level was correlated with key pathological characteristics including tumor differentiation, stage and metastasis. Then, the recombinant lentiviral shRNA expression vector carrying NOB1 was constructed and infected into the human NSCLC A549 cell line. Cell proliferation, cell apoptosis, cell cycle distribution and colony formation ability in A549 cells were assessed following downregulation of NOB1 by siRNA. In addition, tumor growth ability in nude mice was evaluated to define the function of NOB1 in cell transformation and tumorigenesis. It was found that downregulation of NOB1 expression using the RNA silencing approach in A549 tumor cells significantly suppressed the proliferation and colony formation ability, and induced tumor apoptosis in vitro. Tumor growth was also suppressed in vivo. These data suggest that NOB1 is an important regulator of the tumorigenic properties of human NSCLC and may be used as a new promising diagnostic biomarker and a potential anticancer therapeutic target for NSCLC. Introduction Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, with less than 15% of patients surviving beyond 5 years due to the difficulty of early
Correspondence to: Professor Donghua Qian, Department of
Respiration, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China E-mail: [email protected]
Key words: non-small cell lung cancer, NOB1, RNA silencing, tumorigenesis
diagnosis and the absence of effective treatment methods (1,2). Cytotoxic chemotherapy remains the therapeutic foundation for treatment in both the adjuvant and metastatic settings (3-5). Although much effort has been made in the advancement of chemotherapeutic regimens in NSCLC, these therapies are toxic and are almost never curative in the case of metastatic disease. Thus, it is crucial to develop novel molecular diagnostic markers and therapeutic targets for the treatment of NSCLC. The NIN1/RPN12 binding protein 1 homologue (NOB1) is a subunit of the 26 S proteasome and is composed of nine exons and eight introns, and is located on chromosome 16q22.1 (6). NOB1 protein, an evolutionarily conserved protein, comprises a PilT N terminus (PIN) domain and a C terminal zinc ribbon domain (7,8) and is expressed mainly in the liver, lung and spleen (6). The PIN domain was postulated as the enzymatic domain of Nob1 since cells expressing the mutant PIN failed to cleave the 20S pre-rRNA, strengthening the notion that NOB1 is the long-sought D-site endonuclease (9,10). It has been found that genetic depletion of Nob1 strongly suppresses the processing of the 20S pre-rRNA to the mature 18S rRNA, producing markedly high levels of the 20S pre-RNA with novel degradation intermediates (11). These studies showed that NOB1 plays a crucial role in protease function and RNA metabolism. Recently, increased NOB1 expression has been reported in breast and ovarian cancer, and hepatocellular carcinoma (12-14). Lu et al (13) found that NOB1 is an important regulator of the tumorigenic properties of human hepatocellular carcinoma and may be used as a candidate therapeutic target in human hepatocellular carcinoma. Lin et al (14) found that downregulation of NOB1 expression by siRNA suppresses cell proliferation and survival and may be used as a therapeutic marker in ovarian cancer. Huang et al (12) showed that NOB1 plays an essential role in breast cancer cell proliferation, and its gene expression may be a therapeutic target. These results suggest that NOB1 may be involved in the progression of various types of tumors, although little information concerning the expression of NOB1 and its role in other tumors is available. To the best of our knowledge, the correlation between the expression of the NOB1 gene and the pathological characteristics of NSCLC has not been determined, and whether NOB1 affects tumor cell proliferation or tumor growth in NSCLC remains unclear. The aims of
LI et al: NOB1 siRNA SUPPRESSES NSCLC GROWTH
1272
Table I. Correlation of NOB1 overexpression with clinicopathological features of the NSCLC cases. NOB1 expression ------------------------------------------Clinical factors Positive Negative
P-value
Age (years)
Downregulation of NOB1 suppresses the proliferation and tumor growth of non-small cell lung cancer in vitro and in vivo YANG LI1, CHENGYUAN MA2, MING QIAN3, ZHONGMEI WEN1, HONGYU JING1 and DONGHUA QIAN1 Departments of 1Respiration, and 2Neurosurgery, The First Hospital of Jilin University, Changchun, Jilin 130021; Prosthodontics Department of the Stomatological Hospital, Jilin University, Changchun, Jilin 130021, P.R. China
3
Received December 1, 2013; Accepted January 8, 2014 DOI: 10.3892/or.2014.2991 Abstract. Non-small cell lung cancer (NSCLC) is a lethal disease due to the absence of effective diagnostic biomarkers and therapeutic targets. Therefore, novel molecular targets are critically needed to formulate new approaches for this devastating disease. In the present study, using quantitive real-time PCR and immunohistochemistry. we initially found that expression of the ribosome assembly factor NIN/ RPN12 binding protein (NOB1) was elevated in the majority of NSCLC tissues when compared to that in the normal lung tissue counterparts������������������������������������� , and its expression level was correlated with key pathological characteristics including tumor differentiation, stage and metastasis. Then, the recombinant lentiviral shRNA expression vector carrying NOB1 was constructed and infected into the human NSCLC A549 cell line. Cell proliferation, cell apoptosis, cell cycle distribution and colony formation ability in A549 cells were assessed following downregulation of NOB1 by siRNA. In addition, tumor growth ability in nude mice was evaluated to define the function of NOB1 in cell transformation and tumorigenesis. It was found that downregulation of NOB1 expression using the RNA silencing approach in A549 tumor cells significantly suppressed the proliferation and colony formation ability, and induced tumor apoptosis in vitro. Tumor growth was also suppressed in vivo. These data suggest that NOB1 is an important regulator of the tumorigenic properties of human NSCLC and may be used as a new promising diagnostic biomarker and a potential anticancer therapeutic target for NSCLC. Introduction Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, with less than 15% of patients surviving beyond 5 years due to the difficulty of early
Correspondence to: Professor Donghua Qian, Department of
Respiration, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China E-mail: [email protected]
Key words: non-small cell lung cancer, NOB1, RNA silencing, tumorigenesis
diagnosis and the absence of effective treatment methods (1,2). Cytotoxic chemotherapy remains the therapeutic foundation for treatment in both the adjuvant and metastatic settings (3-5). Although much effort has been made in the advancement of chemotherapeutic regimens in NSCLC, these therapies are toxic and are almost never curative in the case of metastatic disease. Thus, it is crucial to develop novel molecular diagnostic markers and therapeutic targets for the treatment of NSCLC. The NIN1/RPN12 binding protein 1 homologue (NOB1) is a subunit of the 26 S proteasome and is composed of nine exons and eight introns, and is located on chromosome 16q22.1 (6). NOB1 protein, an evolutionarily conserved protein, comprises a PilT N terminus (PIN) domain and a C terminal zinc ribbon domain (7,8) and is expressed mainly in the liver, lung and spleen (6). The PIN domain was postulated as the enzymatic domain of Nob1 since cells expressing the mutant PIN failed to cleave the 20S pre-rRNA, strengthening the notion that NOB1 is the long-sought D-site endonuclease (9,10). It has been found that genetic depletion of Nob1 strongly suppresses the processing of the 20S pre-rRNA to the mature 18S rRNA, producing markedly high levels of the 20S pre-RNA with novel degradation intermediates (11). These studies showed that NOB1 plays a crucial role in protease function and RNA metabolism. Recently, increased NOB1 expression has been reported in breast and ovarian cancer, and hepatocellular carcinoma (12-14). Lu et al (13) found that NOB1 is an important regulator of the tumorigenic properties of human hepatocellular carcinoma and may be used as a candidate therapeutic target in human hepatocellular carcinoma. Lin et al (14) found that downregulation of NOB1 expression by siRNA suppresses cell proliferation and survival and may be used as a therapeutic marker in ovarian cancer. Huang et al (12) showed that NOB1 plays an essential role in breast cancer cell proliferation, and its gene expression may be a therapeutic target. These results suggest that NOB1 may be involved in the progression of various types of tumors, although little information concerning the expression of NOB1 and its role in other tumors is available. To the best of our knowledge, the correlation between the expression of the NOB1 gene and the pathological characteristics of NSCLC has not been determined, and whether NOB1 affects tumor cell proliferation or tumor growth in NSCLC remains unclear. The aims of
LI et al: NOB1 siRNA SUPPRESSES NSCLC GROWTH
1272
Table I. Correlation of NOB1 overexpression with clinicopathological features of the NSCLC cases. NOB1 expression ------------------------------------------Clinical factors Positive Negative
P-value
Age (years)
