The Research Of DNA And Histone Epigenetic Differences Between Non-small Cell Lung Cancer Cell Lines And Their Cisplatin Resistant Subclones

Objective: To explore the epigenetic differences of DNA and histone between Non-small cell lung cancer cell lines and their cisplatin resistant clones.Methods: We selected wild type non-small cell lung cancer cell lines A549 and H838, and established the cisplatin-resistant cell strains A549/DDP and H838/DDP by combining gradually increasing concentration of cisplatin with large dosage impact. We the extracted DNA, RNA and protein et al at the days of 1st, 6 th, 11 th, 16 th and 21 st after the induction with normal medium, and did the detections as following: 1. Methylation-Specific-PCR(MSP) was used to detect the methylated status of DNMT1 and DNMT3 b in their promoter regions.2. Im munoprecipitation-chrom atin(Ch IP) was utilized to explore the relationship between DNMT1, DNMT3 b and H3K4Me2, H3K9Me2 in A549/DDP and H838/DDP cells with their progenitor cells. 3. Infinium was carried out to validate the genomic methylation statuses, according to which we can adjust the check-point time. 4. Real-time fluorescence quantitative PCR(RT-PCR) was applied for mRNA expression of DNMT1, DNMT3 b and MDR1.5. Flow cytometry(FCM) was implemented to check up the cell cycle andapoptosis. 6. Western blotting was put into use to analysis the protein expression of DNMT1, DNMT3 b and MDR1 genes at the different check-points. 7.Cispatin resistance and cell growth activity were measured by CCK-8 in the cell lines. Results: 1. With 30 weeks cisplatin induction, two strains of NSCLC can proliferate stably in the medium at the target cisplatin concentration-2.0 u g/mL,which suggested that the cisplatin-resistant cell strains- A549/DDP and H838/DDP were established successfully. 2. As the time went on, the expression of the DNMT1 and DNMT3 b in cisplatin-resistant cell strains- A549/DDP and H838/DDP were depressed at different check-points in the culture medium without cisplatin, presenting the trend of decreasing to increasing, whose expression of mRNA, protein, histone and genomic methylation statuses was consistent with the trend above. Further more, it can lead to cell cycle arrest, and inhibition of cell proliferation. Conclusions: 1. The cisplatin resistant subclones in NSCLC can not only reverse the methylated status of DNMT1 and DNMT3 b but also illustrate that the nature of the epigenetic could be reversed, providing the basis for long-term clinical epigenetic therapy. 2. On the account of the close relationship among the DNMT1, DNMT3 b with depressed expression of their m RNA, protein, histone and genomic methylation statuses and the blocking in the cell cycle, this research suggested that the repressive role of DNMT1 and DNMT3 b is coming to play mainly by curbing their formation. 3. DNMT1 and DNMT3 b gene may be important factors in the occurrence and development of NSCLC and their cisplatin resistant subclones, with which providing a new way of epigenetic molecular targeted therapy for NSCLC in the future.