Detection The Methylation Status Of CDH1and RUNX3Promoter Region CpG Islands In IPF

BackgroudIdiopathic pulmonary fibrosis(IPF)is a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause, without effective therapy. Its pathological character is the fibroblastic foci which comprises of aggregates of mesenchymal cells and extra cellular matrix(ECM). More and more evidences from clinic and basic research show that IPF is a diseae with similarities and links to cancer. Further research on IPF from an oncologist’s view will help us get a more comprehensive understanding of IPF. Epigenetic alterations, espeically DNA methylation, participate in the pathogenesis of cancer. DNA methylation research in IPF is gradually conducted. Myofibroblasts are key effector cells in pulmonary fibrosis and one of their cellular origins is considered to be pulmonary epithelial cell which go under epithelial mesenchymal transition(EMT). The loss of CDH1and RUNX3promotes EMT process. CDH1and RUNX3are thought to be function as tumor suppressor genes and are inactivated or downregulated by promoter hypermethylation in a variety of tumors. However, the promoter hypermethylation of CDH1and RUNX3in IPF have not been reported.ObjectiveTo detect the methylation status of CDH1and RUNX3promoter region CpG islands in IPF and analyse the relationship between genes’methylation frequency and clinic parameters.MethodsIn the present study, we examined the methylation status of CDH1and RUNX3promoter region CpG islands in10cell lines,16cases of IPF,17cases of lung cancer and17cases of matching non-neoplastic tissue by methylation-specific PCR. CDH1and RUNX3gene expression were examined in10cell lines by real-time quantitative PCR. The protein expression of CDH1(E-cadherin) was examined in10cell lines by Western Blot and in17cases of lung cancer,17cases of matching non-neoplastic tissues and16cases of IPF tissues which have CDH1promoter hypermethylation by immunohistochemistry. We also reviewed the clinic data of IPF patients and analysed the correlations between the two genes’methylation frequency and clinic parameters. We also analysed the correlations between E-cadherin expression and CDH1promoter methylation status in17cases of lung cancer.Results1. The expression of CDH1mRNA were lower in those cell lines which were promoter hypermethylation, including293T, BGC823, RKO,MGC803, Hela, MKN45than in those cell lines which were promoter unmethylaion, including MKN74, MCF7, HCT116,AGS.2. Those cell lines which were completely methylated, including RKO, Hela, MGC803, were not detected in expression of CDH1protein level(E-cadherin).3. The expression of RUNX3mRNA were lower in those cell lines which were promoter hypermethylation, including RKO,293T, MCF7, MKN74, AGS, MGC803, Hela than in those cell lines which were promoter unmethylaion, including BGC823, HCT116,MKN45.4. Aberrant hypermethylation of CDH1was detected in11of16(68.75%) IPF tissues,7of17(41.18%) lung cancer tissues. Whereas none of the matched normal lung tissues presented hypermethylation of CDH1. Statistical analyses of the correlation between CDH1promoter hypermethylation and clinic parameters demonstrated that there was no significant difference between methylated and unmethylated patients with regard to age, gender, smoke history and duration of disease.5. Aberrant CDH1protein(E-cadherin) expression was detected in9/17(52.94%) lung cancer tissues. In CDH1methylated lung cacer patients, aberrant E-cadherin expression(71.4%)is higher than normal expression(28.6%). Alveolar epithelial cells covering fibroblastic foci showed negative or slight positive immunoreactivity to E-cadherin in two cases of IPF tissues which are CDH1promoter hypermethylation.6. Aberrant hypermethylation of RUNX3was detected in7of16(43.75%) IPF tissues,9of17(52.94%) lung cancer tissues and3of17(17.65%) matched normal lung tissues. Statistical analyses of the correlation between RUNX3promoter hypermethylation and clinic parameters demonstrated that there was no significant difference between methylated and unmethylated patients with regard to age, gender, smoke history and duration of disease.Conclusions1. The cell lines experiments show that the transcription of CDH1and RUNX3may be downregulated by promoter hypermethylation.2. Promoter hypermethylation of CDH1and RUNX3are observed in IPF, similar to lung cancer.The gene CDH1methylation incidence shows significantly higher when it compares to matching normal lung tissues. Tumor suppressor genes’promoter hypermethylation in IPF tissues is one of the reasons that IPF patients are susceptible to tumor.3. Alveolar epithelial cells covering fibroblastic foci showed negative or slight positive immunoreactivity to E-cadherin in IPF tissues may be related with CDH1promoter hypermethylation. The loss of E-cadherin due to CDH1methylation may take part in EMT process of IPF.