The Research On The Relationship Between EGFR Promoter Region Methylation And Secondary Resistance Which May Be Induced By Gefitinib

Aims:This research is to explore whether the gefitinib secondary resistance can be induced by gefitinib.Explore whether EGFR promotor methylation correlate with the gefitinib-resistance in PC9/GR cell lines.To find a new therapeutic target to overcome the gefitinib secondary resistance in lung adenocarcinoma.Method:In vitro cultivation of lung adenocarcinoma PC9cell lines, apply gefitinib on lung adenocarcinoma PC9cell lines, and improve drug concentration.MTT for test of gefitinib resistance index in PC9cell and PC9/GR cell. Bisulfite sequencing PCR(BSP) and real-time PCR for detection of EGFR promoter methylation status and mRNA expression. In vitro cultivation of lung adenocarcinoma PC9cell lines, apply1μM5-Aza-dc on lung adenocarcinoma PC9/GR cell lines for72h.MTT method for test of gefitinib resistance index in PC9/GR cell.Results:After improving the gefitinib concentration,MTT results shows IC50of PC9cell lines increase from (0.01±0.002)μM to (3.95±0.23)μM(P<0.05).BSP results show abnormal methylation sites compared the degree of methylation change:PC9:59%; PC9/GR:74% (P<0.05).RT-PCR results showed in PC9/GR cell lines, EGFR mRNA expression quantity increased (P<0.05). After applying5-Aza-dc on PC9cell lines, IC50of PC9/GR decrease from (3.87±0.034μM) to (2.55±0.14μM)Conclusion:1.The PC9cell line which is induced by improving gefitinib concentration will be resistant to gefitinib, and the gefitinib-resistant cell line PC9/GR could be built.2.EGFR gene promoter methylation may be one of the mechanisms for the secondary resistance to gefitinib.