| OBJECTIVE: Chronic Obstructive Pulmonary Disease(COPD)is an incurable and progressive lung disease characterized by airway(chronic bronchitis),small airways and alveoli(emphysema).Exposure to cigarette smoke is a major risk factor for COPD.Chronic bronchitis is associated with chronic inflammatory cell infiltration and excessive airway mucus secretion.For smokers and COPD patients,the excess mucus secretion is an important cause of symptoms such as cough and dyspnea.The mucus is produced by goblet cells in the airway,and long-term smoke exposure can result in an increase in the number of goblet cells and an increase in mucus secretion.There are several signaling pathways involved in the promotion of goblet cell differentiation and mucus production in epithelial cells.A study of smokers in the Groningen Asthma and COPD Research Center showed that smokers without lung disease and smokers with COPD accompanied by cough and excessive mucus secretion had multiple genes highly expressed in the epithelial cells.One of the most important genes is SPDEF,which is a master factor in this entire regulatory network.There are multiple studies shown that SPDEF can promote the differentiation of goblet cells and mucus production.The SPDEF expression can be regulated,"on" or "off",by the epigenetic mechanism.The overall aim of this thesis was to 1)examine epigenetic mechanisms related to goblet cell differentiation and COPD,and 2)establish epigenetic editing as a tool to reduce mucus production.Methods: This project is divided into 2 parts.Part I:(1)Primary bronchial epithelial cells were isolated from the airway of COPD patients and non-COPD control individuals,and differentiated to goblet cells and ciliated cells by culture at the air-liquid interface(ALI),where cytokine interleukin(IL)-13 was used to promote the goblet cell differentiation.(2)Realtime PCR was used to monitor the expression of genes related to mucus secretion: transcription factors SPDEF and FOXA2,mucin MUC5 AC,and potential chaperone for mucin packaging AGR2.(3)Pyrosequencing was performed to detect DNA methylation of Cp G sites in the promoter region of SPDEF and FOXA2 during the goblet celldifferentiation.(4)The expression level and methylation level of SPDEF and FOXA2 were analyzed and compared in the ALI cultures derived from COPD individuals and controls in the absence of IL-13,to investigate the relationship between the DNA methylation of SPDEF and FOXA2 and mucus hypersecretion in COPD.Part II:(1)Gene expression of SPDEF and MUC5 AC and the DNA methylation of SPDEF gene were analyzed and compared in different human lung epithelial cell lines.The cell line with high expression of SPDEF and MUC5 AC would be selected as a model system for epigenetic editing.(2)Zinc fingers and CRISPR/d Cas platforms were engineered to target repressors(KRAB,DNA methyltransferases,histone methyltransferases)to the SPDEF promoter.(3)Zinc fingers and CRISPR/d Cas platforms were transduced to the A549 cells,and cells were harvested to analyze the expression of SPDEF and downstream mucus-related genes MUC5 AC and AGR2.(4)Expression level of multiple irrelevant genes were analyzed to investigate the specificity of SPDEF targeted epigenetic editing.(5)Transduced A549 cells were cultured for two weeks with regular passage,and the expression of SPDEF was analyzed to investigate the stability of the obtained SPDEF silencing.Result: Part I:(1)During the goblet cell differentiation(IL-13 stimulated ALI-cultures derived from non-COPD controls),the m RNA expression of genes related to mucus production,such as MUC5 AC,AGR2 and SPDEF,were upregulated,and the gene associated with inhibition of mucus production FOXA2 was downregulated.(2)There was a positive correlation between the increase of SPDEF gene expression and the change of DNA methylation in the promoter region of SPDEF during differentiation.(3)ALI-cultures derived from COPD cases had higher expression levels of SPDEF and MUC5 AC,and lower DNA methylation levels of SPDEF.Part II:(1)A549 cells line was selected as model system because of its high expression of SPDEF and MUC5 AC.(2)In A549 cells,all SPDEF targeting inhibitory fusion proteins were able to inhibit the m RNA and protein expression of SPDEF effectively,which were also accompanied by repression of mucus related genes MUC5 AC and AGR2,but no expression changes of other irrelevant genes.(3)For the histone methyltransferase G9 A,and not its mutant or other effectors,theobtained silencing was mitotically stable.Conclusion: Part I: The DNA methylation level and expression level of SPDEF gene were changed in bronchial epithelial cells derived from COPD patients,suggesting that SPDEF gene may play an important role in the pathogenesis of COPD and SPDEF gene may be a good target for future epigenetic editing and treatment of COPD.Part II: Targeted increase the DNA/ histone methylation of SPDEF gene could inhibit the expression of SPDEF and mucus related genes,which is the first step in achieving the goal of COPD treatment by targeted epigenetic editing,and which also opens up broad prospects for regulating the expression of any target gene by targeted epigenetic editing. |
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