| Lung cancer is one of the leading causes of cancer death around the world. According to the newest statistical analysis, lung cancer has the highest cancer mortality among all cancers in males worldwide and the high mortality burden among females in developing countries. Besides, in the year 2008, both the morbidity and mortality of lung cancer ranked first in all the cancer diseases in China. The morbidity of lung cancer in our country was 57 per 100,000 and the mortality was 46 per 100,000. The death toll caused by lung cancer accounted for 22.7% of the total death caused by cancer diseases. As a result, it is urgent and meaningful to investigate the molecular mechanism of lung cancer formatting and further to develop effective therapeutic methods for lung cancer therapy.Lung cancer could be divided into two types including small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). SCLC accounts for 20% of lung cancer and the rest 80% of that is NSCLC. SCLC has the ability of fast proliferating, early metastasis and high invasion. NSCLC mainly include lung squamous cell carcinoma, lung adenocarcinoma and pulmonary large cell carcinoma. Compared with SCLC, the proliferating rate of NSCLC cells is lower and the time of cancer cells metastasis is not as early as SCLC. Although NSCLC is less malignant than SCLC, the five year survival rate of NSCLC is still low partially because when NSCLC was detected, the cancer cells had already spread to other places of the bodies in about 75% NSCLC patients. Therefore, it is urgent to develop the effective treatments for preventing non-small cell lung cancer and promoting the survival rate.The human NOB1 (Nin1/RPN12-binding Protein 1. NOB1) located on human chromosome 16q22.1 and includes nine exons and eight introns. The length of NOB 1 cDNA sequence is 1749bp and contains an open reading frame of 1239bp long. NOB1 protein is consisted of 412 amino acids and the molecular weight is 46KDa. The N terminal of NOB 1 protein has a PIN (PilT aminoterminus, PIN) domain and the C terminal has a ZNRD1 (zinc ribbon domain containing 1, ZNRD1) domain. PIN domain consisted of about 100 amino acids is essential for NOB1 to process 20S pre-rRNA to the mature 18S rRNA.As a ribosome assembly factor, NOB1 is essential for processing of the 20S pre-rRNA to the mature 18S rRNA because PIN domain is a part of it. NOB1 also serves as a part of a pre-40S ribosomal particle that is transported from nucleus to the cytoplasm and consequently cleaved site D at the 3’end of mature 18S rRNA. More importantly. NOB1 is essential for 26S proteasome assembling.26S proteasome is an ATP (adenosine triphosphoric acid, ATP) dependent protein degradation complex and is the core component of UPP (ubiquity-proteasome pathway, UPP). As the most important protein degradation mechanism, UPP is mainly consisted of ubiquitin, E1 (ubiquitin-activatingenzyme, E1), E2 (ubiquitin-conjugating enzymes. E2), E3 (ubiquitin-proteinligases, E3),26S proteasome and deubiquitinatingenzymes. UPP has the ability to specifically degraded proteins including cell cycle regulating proteins in mammalian cells. As a part of UPP, NOB1 may serve as an important regulator of cell cycle. Therefore, it is valuable to investigate the function of NOB 1 in cancer cells for effective therapeutic methods development.In the current study, we first detected that the expression of NOB 1 in the tumor tissues of NSCLC patients were specifically high expressing. Based on the knowledge that NOB1 may serve as a regulator of cell cycle, we further studied the function of NOB1 in lung cancer cells. Our studies mainly include three parts. In the first part, we investigated the expression of NOB 1 in the NSCLC tumor tissues. In the second part, we mainly studied the effect on cancer cells proliferation of NOB1. In the third part, we investigated the molecular mechanism of NOB1 regulating cancer cells proliferation.First, NOB1 is highly expressed in non-small cell lung cancer.Previous studies indicated that the expression of NOB 1 is involved in several carcinomas. To investigate the function of NOB 1 in non-small cell lung cancer, we evaluated the expression in 29 non-small cell lung cancer specimens using immunohistochemical staining. The twenty-nine non-small cell lung cancer specimens were collected for immunohistochemistry (15 male,14 female; 16 specimens from patients younger than 60 years old,13 from patients older than 60 years old). Ten normal lung specimens were used as control. All the above tissues samples were provided by the Department of Thoracic Surgery in First Hospital of shanghai, and patients were informed consent form conforming to the guidelines of the Declaration of Helsinki.Of the 29 non-small cell lung cancer samples,4 (13.7%) were hadro-paositive,21 (72.4%) were positive and 2 (6.9%) were weak positive, which was significantly higher than the normal lung tissue samples (none of them show hadro-positive,2 of 10 (20.0%) are positive and 8 (80.0%) are negative). These results suggest that NOB1 is highly expressed in the non-small cell lung cancer. The high expression level of NOB1 in non-small cell lung cancer suggests it might be involved in the pathogenesis of non-small cell lung cancers. Second, NOB1 regulate non-small cell lung cancer cell proliferation. For the NOB1 is highly expressed in non-small cell lung cancer, we next evaluated the effect on cancer cells proliferation of NOB1. RNAi technology has been proved to be an efficient, specific and stable method to silence the target genes. Taking advantage of the prevalence and availability of RNAi technology in cancer therapy, as well as the relatively high and stable transfection rate of viral vector, a lentivirus shRNA system was used to knock down NOB1 expression in non-small cell lung cancer cells. We first cloned NOB1 shRNA targets into the recombinant lentivirus plasmid, which was utilized to infect two established non-small cell lung cancer cell lines A549 and H1299. The non-silencing sequences were also inserted into the vector as a control. The GFP florescence imaging was used to indicate the lentivirus infection as GFP was transfected to the cancer cells together with NOB1 shRNA. In our study, more than 80% cells were infected by lentivirus as assessed by GFP fluorescence, indicating the successful transfection in both A549 and H1299 cells. NOB1 knockdown efficiency was determined by real-time PCR and western blot. Lentivirus-mediated RNAi could markedly decreased endogenous NOB1 mRNA expression, by 95.4% reduction in A549 cells and 40.7% reduction in H1299 cells.The protein level of NOB 1 was also significantly reduced in both cells after lentivirus infection. Hence, lentivirus infection was confirmed to be effective to inhibit the expression of NOB 1 in non-small cell lung cancer cells.The effect of silencing NOB1 on non-small cell lung cancer cell proliferation was examined by MTT assay. Our data indicated that there was no significantly difference in cell viability between Lv-shCon infected and uninfected cells, suggesting no cytotoxic effect of lentiviral system on both cells. Whereas, the proliferation rates of NOB 1 silenced A549 cells and H1299 cells were significantly reduced as compared with control groups from day 3. On day 5. the cell viability was decreased by 74.9% in A549 cells and 27.5% in H1299 cells after lentivirus infection, respectively. The inhibition rate on A549 cells was higher than H1299 cells, consistent with the suppression of NOB 1 expression by Lv-shNOB1. These results revealed the important functional role of NOB1 in the proliferation of non-small cell lung cancer cells, and its inhibitory effect was dependent on specific cell lines.Additionally, we utilized cell colony formation assay to evaluate the proliferation of cancer cells. The colony formation assay by Giemsa staining was performed to evaluate the effect of NOB1 depletion on the colony forming capability of A549 cells. Three groups of A549 cells (Con. Lv-shCon and Lv-shNOB1) were cultured for 9 days. The number of colonies in Lv-shNOBl infected A549 cells was markedly reduced by 96.8% as compared with control groups observed under light microscope). The results indicated that knockdown of NOB1 could also inhibit the colony formation of non-small cell lung cancer cells, representing its oncogenicity in vitro.Third, NOB1 suppression leads to G0/G1 cell cycle arrest.To find out the underlying mechanisms of NOB1 silencing induced cell growth inhibition, we analyzed the cell cycle distribution of A549 cells after lentivirus infection. Our data indicated that more cells were accumulated in G0/G1 phase of cell cycle (69.32 ± 0.45%) after Lv-shNOB1 infection, compared with the Con group (57.05 ± 0.37%) and Lv-shCon group (59.21 ± 0.86%). The percentage of cells in the S phase and G2/M phase was dramatically decreased after lentivirus infection. These results suggested that knockdown of NOB 1 suppressed the growth of non-small cell lung cancer cells possibly via inducing cell cycle arrest.Furthermore, the expression alterations of cell cycle markers were detected in A549 cells, including Cyclin D1, CDK4, CDKN2A, and CDKN2B. Western blotting showed that depletion of NOB 1 resulted in a significant decrease in Cyclin D1 and CDK4 expression, and no significant change was observed in CDKN2A and CDKN2B expression. These results suggested that knockdown of NOB 1 in non-small cell lung cancer cells blocked cell cycle progression via downregulation of Cyclin D1 and CDK4.NOB1 facilitates the maturation of 20S proteasome, and then regulates the biogenesis of 26S proteasome which contributes to protein degradation by ubiquitin-proteasome system (UPS) in universal biological processes including cell cycle progression in eukaryotes. Thus, inhibitory effect on the proliferation of the non-small cell lung cancer cells induced by NOB1 repression may be attributed to the influences on degradation of cell cycle protein and some complicated aspects in the cell cycle progression. The subsequent studies would elucidate the mechanism how NOB1 regulates cell cycle of non-small cell lung cancer cells and its underlying relationship with the proteasome-mediated degradation. |
PDF Full Text Request