| Lung cancer, with high morbidity and mortality, is one of the most common malignant cancers worldwide. Cigarette smoke is the principal risk factor of estimated 90% of all lung cancers. OLC1 (Overexpressed in lung cancer 1) gene using suppression subtractive hybridization (SSH) was identified and named in this laboratory previously. OLC1 is considered as an oncogene in lung cancer, which notablely overexpressed in the early stages (atypical hyperplasia and carcinoma in situ) of lung squamous cell carcinoma (SCC); and up-regulated expression of OLC1 was more common in the patients with a cigarette smoking history. In addition, OLC1 protein level was elevated when treated with cigarette smoke condensate (CSC) in human lung cancer cell lines, and human immortalized bronchial epithelia cells as well. These results indicate that the OLC1 is related with carcinogenesis and cigarette smoking. The aim of present study is to explore the role(s) of OLC1 in cigarette smoke-associated lung cancer.In the first part of study, we examined the OLC1 protein in the tumor tissues of non-small cell lung cancer (NSCLC) patients, with immunohistochemical (IHC) analysis. IHC staining was applied on formalin-fixed paraffin-embedded sections derived from 172 cases of NSCLC, including 77 squamous cell carcinomas (SCCs) and 95 adnocarcinomas (ADCs). The results showed that the level of OLC1 protein was significantly higher in the tumor tissues compared with that in adjacent normal tissues. Moreover, the levels of the OLC1 were associated with smoking history in SCCs.In the second part of study, human lung cancer cell lines A549 and H1299 were used for in vitro investigations. Treatment of three carcinogens in cigarette smoke, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),3-methylcholanthrene (MCA) and benzo[a]pyrene (B[a]P) did not affect OLC1 protein level in the lung cancer cells by Western blot analysis. When the cells were treated with cigarette smoke condensate (CSC), the results indicated that genes up-regulated were related to cellular defend mechanisms, including metabolic process, cellular response to oxidative stress, cellular detoxification, etc. Genes up-regulated after CSC treatment were significantly enriched in alveolar primary septum development and epithelial cell differentiation; while down-regulated genes were involved in cell cycle process. In the lung cancer cells stably overexpressed exogenous OLCl, extensive biological processes were altered, such as morphogenesis, cell adhesion, neuron projection, circulatory system process, cellular respiration, regulation of transporter activity, etc. Furthermore, the mRNA profiles derived from CSC-treated stably transfected OLCl lung cancer cells revealed that the up-regulation of cell adhesion and down-regulation of transporter activity were enhanced. Using the data sets of immune and inflammation from GEO database, the immune and inflammation associated differentially expressed genes were screened in the A549 cell mRNA profiles. Furthermore, combined with the data set derived from a model of multi-stages carcinogenesis of squamous cell carcinomas generated in this laboratory,8 specific candidate genes in A549/OLC1 cells were selected.In the third part of study, OLC1 genetic engineering mice were prepared for cigarette smoke exposure experiments, including OLC1 general expression transgenic mouse (C57BL/6J/OLC1) and OLC1 general knock-down transgenic mouse (C57BL/6J/OLC1si)-The mice were sacrificed for further mRNA expression profile analysis after three-month or seven-month exposure, respectively. The mRNA expression profiling data showed that OLC1 played an important role in nervous system and organ development in the mice. Additionally, the GSEA result revealed that aberrant expression of OLC1 was related to sensory perception and response to stimulus in C57BL/6J/OLC1 mice, while immune system in C57BL/6J/OLCl si mice. Genes involved in immune system arose among short-term cigarette smoke exposure mice. C57BL/6J/OLC1 mice were more sensitive in response to hypoxia. The data of C57BL/6J/OLC1si mice implied that there is a connection among smoke, OLC1 and olfactory transduction. Cell adhesion pathway was enriched in both OLC1 genetically engineered mice while conducting KEGG pathway enrichment analysis, which implied OLCl may be involved in cell migration. The up-regulated genes in wild type mice were still associated with defend mechanisms under long-term cigarette smoke exposure, like humoral response, inflammatory response, response to external stimulus, etc. while the down-regulated genes involved in organ development. In contrast, the up-regulated genes were associated with organ development in C57BL/6J/OLC1 mice, like respiratory system development. The development related genes overexpression de novo is a critical signal of carcinogenesis. The genes involved in proteoglycan biosynthetic process, protein kinase B signaling, cell cycle indicated the defend mechanisms toward cigarette smoke exposure were suppressed, which did not appear in C57BL/6J/0LC/si mice. Forty-three differentially expressed genes associated with immune and inflammation were screened out from the mRNA expression profile of the smoke exposed OLC1 genetic engineering mice by the similar methods applied in the A549 cell. And followed with enrichment by KEGG pathway analysis,13 candidate genes were selected finally.In conclusion, OLC1 protein level was increased in the tumor tissues of NSCLC patients. Genes up-regulated after CSC treatment were significantly enriched in alveolar primary septum development and epithelial cell differentiation in A549 cells, while down-regulated genes were involved in cell cycle process in H1299 cells. Comparing to control group, the stably transfected OLC1 lung cancer cells showed that the up-regulation of cell adhesion and down-regulation of transporter activity were enhanced when treated with CSC. Eight candidate genes associated with immune, inflammation and carcinogenesis in the A549/OLC1 cells were found. OLC1 played an important role in nervous system and organ development in mice. After short-term cigarette smoke exposure, cell adhesion pathway was enriched in both OLC1 genetically engineered mice. The genes associated with organ development were up-regulated and the genes involved in defend mechanisms toward cigarette smoke exposure were down-regulated in the C57BL/6J/OLC1 mice under long-term cigarette smoke exposure. Thirteen candidate genes associated with immune, inflammation and carcinogenesis were obtained in the smoke exposed OLC1 genetic engineering mice for further studies. |
