| Backgroud and Objective:Lung cancer is the most common cause of cancer-related mortality in China, as well as in the United States and Europe. Effective early diagnosis and targeted therapies to reduce incidence and mortality would benefit from a better understanding of the key molecular changes from normal cells to precancerous lesions to malignant tumor cells. However, the molecular events responsible for lung cancer initiation and development are still largely unknown.The occurrence of lung cancer is a multiphase and multistep process. Take lung squamous cell carcinoma for example, it will experience several stages from bronchial epithelial to malignant lesions, including hyperplasia, squamous metaplasia, dysplasia (mild, moderate, and severe), carcinoma in situ (CIS) and infiltrating carcinoma. Previous studies have shown that there is an accumulation of many genetic changes that occur during the process of lung tumorigenesis, including DNA mutations, deletions, amplification, rearrangements, chromosome aberrations, and so on. In recent years, DNA methylation as another important mechanism for the regulation of gene expression has been shown to play an important role in the lung carcinogenesis, but the relevant research is still in the initial stage.Methylation of DNA, which occurs at the cytosine residues of cytosine- phospho-guanine (CpG) dinucleotides by an enzymatic reaction that produces 5-methycytosine (5-mC), is an extensively characterized mechanism for epigenetic gene regulation. Neoplastic cells may simultaneously harbor widespread genomic hypomethylation and regional areas of hypermethylation. The prevalence of global hypomethylation in many types of human cancers suggests that such hypomethylation plays an important role in tumorigenesis. Global DNA hypomethylation may induce neoplastic transformation, genomic instability, and abnormal chromosomal structures. Alterations in normal DNA methylation patterns are frequently observed in cancer cells, and hypermethylation in the promoter regions of tumor suppressor genes is associated with an epigenetically mediated gene silencing. This is a common feature of human carcinomas, and is a key to the tumorigenic process, contributing to all of the typical hallmarks of a cancer cell. With regard to abnormal DNA methylation in lung cancer, many literatures have found that tumor-related genes hypermethylation occurred in the tumor tissues. These genes are involved in a number of intracellular signal pathways: cell cycle regulation, cell apoptosis, DNA repair, cell adhesion and so on. Thus, epigenetic mechanisms representative of DNA methylation and genetic mechanisms are of equal importance during the process of lung cancer initiation, development, and progression. However, research about DNA methylation in precancerous tissues of lung cancer is relatively small and the underlying mechanisms are unclear.Previous studies have suggested that there are many similarities in not only the morphological process, but also the level of molecular biology, alterations in 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN)-induced lung squamous cell carcinoma and human lung cancer. Therefore, the animal model may be particularly informative and helpful in gaining a better understanding of the epigenetic alterations that occur during human lung carcinogenesis.Based on these considerations, the aim of this study was to determine the role of DNA methylation alterations in the progression of MCA and DEN-induced rat lung carcinogenesis from normal bronchial epithelium to cancer. We focused on the changes in DNA methylation associated with the progression of lung carcinogenesis, including the global methylation status, the presence of differentially methylated DNA fragments, the alterations in methylation of tumor-related genes and their regulation of protein expression, DNA methyltransferases changes and their expressions association with tumor-related genes methylation, and demethylation experiments.Materials and Methods:1. Wistar rats of both sexes (6 weeks old, 200±20g) randomly assigned to two groups. The 80 animals assigned to Group 1 were treated with a single dose of 10mg MCA and 10μl DEN in iodized oil by left intra-bronchial instillation. The 10 animals assigned to Group 2 were instilled with 0.1 ml iodized oil without any carcinogen into the left lung as a control. Rats were killed by exsanguination under anesthesia on days 15, 35, 55, 65 and 75 after instillation. The left lungs were split into two halves. One was rapidly frozen in liquid nitrogen and kept at -80℃. The other was fixed by immersion in 4% formaldehyde in phosphate buffer for 24 hours, embedded in paraffin, sectioned to 4μm and 10μm thickness, and routinely processed for hematoxylin and eosin staining. Laser capture microdissection (LCM) was employed to obtain near-pure normal, precancerous and malignant cells for methylation analysis.2. The status of genomic methylation during rat lung carcinogenesis was detected by immunohistochemistry with anti-5-methycytosine (5-mC) antibody, and the mean optical density (OD) and integrated optical density (IOD) were measured by image analysis system. We also used methylation-sensitive arbitrarily-primed PCR (MS-AP-PCR) to screen differentially methylated DNA fragments in 11 tumor and 11 precancerous tissues, and their paired normal bronchial epithelial tissues isolated using LCM. Differentially methylated fragments products were separated cloned into vector and sequenced. Sequence data were used to determine genomic information, including homology to characterized or novel genes and cytogenetic map positions, employing the NCBI's BLAST program.3. Methylation-specific PCR (MSP) and sequencing were used to detect the methylation status of tumor-related genes, including p15, p16, p27, p57, RASSF1A, TSLC1, TIMP-3, E-cadherin, N-cadherin, DAPK1, FHIT and SOCS-3 during MCA and DEN-induced rat lung carcinogenesis. Immunohistochemistry was used to examine the expressions of p16, p27, p57, RASSF1A, TSLC1, TIMP-3, N-cadherin, DAPK1, FHIT and SOCS-3 proteins during rat lung carcinogenesis. Normal tissues, precancerous and tumor tissues were examined by Western blot for the expressions of these proteins.4. Immunohistochemistry was used to examine the expressions of DNA methyltransferases (DNMTs) 1, 3a and 3b proteins during MCA and DEN-induced rat lung carcinogenesis. Correlation analyses were applied to study the relationship between DNMTs expressions and tumor-related genes methylation. After purification through a series of pathologic, morphological and immunologic identifications, primary rat lung squamous cell carcinoma cells with methylated p16, p57 and TSLC1 were cultured and exposed to different concentrations (2μM, 5μM, 10μM) of 5-aza-2′-deoxycytidine (5-aza-dC). DNA and RNA were harvested on day 3 after initial treatment. The methylation status and mRNA expression of p16, p57 and TSLC1 were detected by MSP and reverse transcription-polymerase chain reaction (RT-PCR).5. Statistical analyses were performed with the SPSS 13.0 software. Chi-square analyses were applied to study the correlation between expression and CpG methylation, the differences in methylation, and differences in expression between normal, precancerous, and tumor tissues. Multiple comparisons in 5-mC immunostaining intensity were evaluated using one-way ANOVA and significant differences between two groups were analyzed by Tukey's test. The level of significance was set at P < 0.05.Results:1. Rats treated with carcinogens had no obviously pathological lesions in any of the main organs, except the treated lung. No pathological changes were found in any of the control groups. We obtained different morphological tissues representative of hyperplasia, squamous metaplasia, dysplasia, CIS and infiltrating carcinoma in the multistep process of rat lung tumorigenesis by selecting different durations of time after instillation of the carcinogens to sacrifice the animals. All of the lung cancers induced by MCA and DEN were diagnosed as squamous cell carcinoma by a professional pathologist. In all, 20 samples of normal bronchial epithelium (including 10 samples from chemically-treated and 10 samples from untreated rats), 25 samples of hyperplasia, 27 samples of squamous metaplasia, 37 samples of dysplasia, 30 samples of CIS and 25 samples of infiltrating carcinoma were included.2. Anti-5-mC antibody was expressed in the nucleus of bronchial epithelial cells. Staining levels gradually decreased from normal with nucleus brown to tumor with nucleus yellow during the process of carcinogenesis. Nuclear staining in basal cells was deeper than that in luminal cells of normal and precancerous stages. The OD and IOD of combined 5-mC scores of different types of tissues decreased gradually during the progression of carcinogenesis (P < 0.01). The degree of global methylation was, in general, higher in basal cells compared to luminal cells of normal and precancerous tissues (P < 0.01).3. We chose 11 tumor and 11 precancerous tissues, and their paired normal bronchial epithelial tissues, for screening differentially methylated DNA fragments by MS-AP-PCR following LCM. Results showed that abnormal methylation fragments can be found in majority of samples. In the range of 200bp-700bp, we identified a total of eight differentially methylated DNA fragments, including seven hypomethylated and one hypermethylated DNA fragment in both precancerous tissues and tumor tissues. DNA sequence analysis of these fragments revealed that seven of the eight DNA fragments had significant homology matches (99-100% homology) to sequences in the GENBANK database after a BLAST search. These sequences were found to reside on rat chromosomes 1, 3, 9, 12, 13, 20 and X, indicating putative target genes in these regions. The methylation status of hypermethylated fragment CpG islands, which confirmed the MS-AP-PCR results.4. p15 and E-cadherin were unmethylated in normal, precancerous and tumor tissues. Other tumor-related genes methylation gradually increased during the process of carcinogenesis. The prevalence of DNA methylation of at least one gene and the average number of methylated genes were significantly increase from normal to precancerous and tumor tissues. The frequency of each tumor-related gene methylation in the stage of normal, hyperplasia, squamous metaplasia, dysplasia, CIS and infiltrating carcinoma was as follows: p16: 0%, 8.00%, 22.22%, 40.54%, 50.00% and 56.00%; p27: 0%, 0%, 0%, 0%, 3.33% and 8.00%; p57: 0%, 0%, 11.11%, 18.92%, 26.67% and 36.00%; RASSF1A: 0%, 0%, 3.70%, 8.11%, 6.67% and 16.00%; TSLC1: 0%, 0%, 18.52%, 24.32%, 40.00% and 48.00%; TIMP-3: 10.00%, 12.00%, 18.52%, 24.32%, 30.00% and 60.00%; N-cadherin: 0%, 0%, 7.41%, 18.92%, 26.67% and 36.00%; DAPK1: 0%, 0%, 7.41%, 10.81%, 26.67% and 36.00%; FHIT: 0%, 0%, 3.70%, 16.22%, 43.33% and 48.00%; SOCS-3: 0%, 0%, 7.41%, 16.22%, 26.67% and 48.00%. On the contrary, protein expression of tumor-related genes decreased during the process of carcinogenesis except the increase of N-cadherin expression. The rates of positive protein expression in the stage of normal, hyperplasia, squamous metaplasia, dysplasia, CIS and infiltrating carcinoma were as follows: p16: 90.00%, 84.00%, 70.37%, 59.46%, 40.00% and 32.00%; p27: 95.00%, 88.00%, 74.07%, 67.57%, 60.00% and 44.00%; p57: 95.00%, 92.00%, 81.48%, 75.68%, 63.33% and 56.00%; RASSF1A: 100.00%, 92.00%, 70.37%, 64.86%, 53.33% and 48.00%; TSLC1: 95.00%, 88.00%, 55.56%, 48.65%, 43.33% and 40.00%; TIMP-3: 100.00%, 96.00%, 77.78%, 70.27%, 53.33% and 24.00%; N-cadherin: 0%, 0%, 14.81%, 21.62%, 33.33% and 44.00%; DAPK1: 100.00%, 100.00%, 88.89%, 81.08%, 70.00% and 56.00%; FHIT: 100.00%, 96.00%, 81.48%, 62.16%, 43.33% and 36.00%; SOCS-3: 100.00%, 100.00%, 88.89%, 86.49%, 73.33% and 56.00%. Results of Western blot showed that samples with unmethylation and positive by immunohistochemistry showed strong protein expression, while samples with methylation and negative by immunohistochemistry showed weak or no expression. The correlation between methylation of p16, p57, TSLC1, TIMP-3, DAPK1, FHIT, SOCS-3 and their protein expressions was significant.5. The rates of positive DNMTs protein expression in the stage of normal, hyperplasia, squamous metaplasia, dysplasia, CIS and infiltrating carcinoma were as follows: DNMT1: 0%, 16.00%, 29.63%, 40.54%, 46.67% and 64.00%; DNMT3a: 0%, 0%, 18.52%, 37.84%, 40.00% and 68.00%; DNMT3b: 0%, 0%, 0%, 5.41%, 10.00% and 12.00%. The expression of DNMT1 was significant positively correlated with DNMT3a and DNMT3b expression (P < 0.05). The expression of DNMT3a was not correlated with DNMT3b expression (P > 0.05). Correlation analyses between DNMT1, DNMT3a, DNMT3b expression and 10 tumor-related genes with hypermethylation showed that the expression of DNMT1 and DNMT3a were significantly positively correlated with p16, p57 and TSLC1 gene methylation, respectively (P < 0.01). In addition, RASSF1A, TIMP-3, N-cadherin gene methylation was significantly positively correlated with the expression of DNMT3a (P < 0.01). The average numbers of methylated genes were significantly higher in samples with positive expression of DNMT1 and DNMT3a (2.43±1.85 and 2.65±1.82) than those with negative expression of DNMT1 and DNMT3a (1.35±1.98 and 1.34±1.95), respectively. After the primary tumor cell lines were treated with 5-aza-dC, the mRNA expression of p16, p57 and TSLC1 significantly increased compared to the untreated cells.Conclusions:1. Using bronchial instillation, two kinds of carcinogens MCA and DEN successfully established animal model for lung squamous cell carcinoma model in Wistar rats.2. Global hypomethylation not only occurred in tumors, but also in precancerous tissues. The decrease in the degree of genomic methylation and coexist of hypermethylation and hypomethylation may play an important role during carcinogenesis of lung squamous cell carcinoma in rats. Screened differential methylated fragments may locate in sensitive regions that are altered during MCA and DEN-induced lung carcinogenesis. In the future, these sequences could be explored as potential biomarkers of carcinogen exposure. Identification of Hyper-1 suggests that it might be new gene and the aberrant hypermethylation may be the main mechanism of gene inactivation.3. Dynamic changes in hyperthylation of cell cycle regulation-related genes p16, p27, p57, RASSF1A, cell adhesion-related gene TSLC1, TIMP-3, N-cadherin, cell apoptosis-related genes DAPK1, FHIT, and SOCS-3 gene, accounting for their defective expression, are an early and frequent event in tumorigenesis and play an important role during the progression of MCA/DEN-induced multistep rat lung carcinogenesis.4. DNMT1 and DNMT3a overexpression associated with accumulation of DNA methylation of multiple tumor-related genes are involved in multistage carcinogenesis from normal to early precancerous stages to malignant progression.5. DNA methylation is an important mechanism for tumor-related genes inactivation and CpG island hypermethylation leading to gene silencing is a reversible process.To sum up, the present investigation explored the underlying epigenetic mechanisms of chemically induced rat lung carcinogenesis and enriched the traditional"cancer genetics theory". The experimental model used in present study may be particularly informative and helpful in gaining a better understanding of the epigenetic alterations that occur during genotoxic carcinogen-induced lung carcinogenesis. Established experimental system has laid a foundation for further comprehensive assessment of environmental factors and DNA methylation interactions associated with the occurrence of lung cancer. It also plays a key role in traditional evaluation system for carcinogenic compounds based on the"mutation detection". At the same time, this study provide information for further study on how related genes on the signal pathway involved in cancer through epigenetic/genetic interactions. The experimental model can be used for screening new demethylation agents for lung cancer treatment and evaluating the interaction between DNA methylation and environmental factors in the mechanism of lung cancer in China.
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