Preliminary Study Of DNA Methylation Mechanism Effect The Development Of Nasopharyngeal Carcinoma Taxol Resistance

Chapter One Combined analysis of DNA methylated microarry and gene expression microarry to screen candidate genes in Taxol resistant nasopharyngeal carcinoma cellsObject:To study the relationship between DNA methylation and nasopharyngeal carcinoma (NPC) Taxol resistance. Compared the differential methylation and expression profiling in parental and Taxol resistant nasopharyngeal carcinoma cells by the Combined analysis of DNA methylated microarry and gene expression microarry.Method:Detect the genomic DNA methylation in nasopharyngeal carcinoma by HPLC assay and analyze the relationship between DNA methylation and Taxol resistance.NimbleGen HG18DNA methylated microarry integrated with Affymetrix HG-U133Plus2.0gene expression microarry to screen the differential candidate genes in Taxol resistant nasopharyngeal carcinoma cells. The screened genes were classified by GO and KEGG signal pathway database with DAVID online software. The candidate genes were furtherly validated by MS-PCR and Real-time PCR.Result:Taxol resistant cell lines CNE-1/Taxol, HNE-2/Taxol and5-8F/Taxol were globally hypermethylated, furthermore, the methylation level raise with the increase of drug resistance. Regression analysis indicated that DNA methylation level was positive related with drug resistant.117commonly methylation differential genes were screened out in three Taxol resistant cells,83(70.94%)genes were hypermethylated and34(29.06%) genes were hypomethylated.Moreover,in the Taxol resistant cells327genes were detected to be differentially expressed by gene expression microarry.129(39.45%)genes were over-expressed, and198(60.55%) genes were down-expressed. Integrated the results of that two microarray,48genes were traped. Among these genes,13were hypermethylated and down-expressed,14were hypomethylated and over-expressed. Six screened candidate genes (CHFR,PEG10,ABCC5, CDKN1C. DLC1and ERBB2) were validated to be consistent with the results of the microarrays by MS-PCR and Real-time PCR.Conclusion:DNA methylation was identified to be closely related with Taxol resistance. Aberrant gene methylation associated gene expression variation played an crucial role in chemo-agent drug resistance development. Combined analysis of DNA methylation microarray and expression microarray was a good tool to study the mechanism of the cancer drug resistance development to identify effective and specific gene markers. The screened genes were proved to be involved in the formation of drug resistance by functional classification. CHFR,PEG10,ABCC5,CDKN1C,DLC1and ERBB2were identified to be potential biomarkers for drug resistant cancer. Chapter Two Reversal of the Taxol-resistant phenotype by5-aza-dC in nasopharyngeal carcinomaObject:To compare the difference of cell growth inhibition between Taxol-resistance NPC cells and its parental cells treated by5-aza-dC, and observed the reversal effection of5-aza-dC to the Taxol-resistance phenotype in NPC.Method:The cell growth inhibitory effect of5-aza-dC and Taxol were detected by colony formation assay. Hochest fluorescence staining was applied to detected cell apoptosis. Apoptotic death and cell cycle were measured by flow cytometry.Result:Both parental NPC cells and Taxol-resistant NPC cells were inhibited by different concentration of5-aza-dC,the IC50value of parental cells CNE-1,HNE-2and5-8F were9.7±0.2uM,10.5±0.6uM,11.2±0.5uM, respectively. IC50value for CNE-1/Taxol,HNE-2/Taxol and5-8F/Taxol were4.9±0.3uM,5.3±0.2uM,6.1±0.3uM. It refers that Taxol-resistant showed significantly higher sensitivity to5-aza-dC than parental cells (p<0.01).When the cells were pretreated with low-dose5-aza-dC(5uM), the growth inhibition rate of taxol in parental cells remained unchanged, however, a significantly increase was observed in Taxol-resistant cells(p<0.01), its IC50value was changed to4.87±0.29nM,5.35±0.24nM,5.79±0.34nM, and RI value were3.43±0.11,3.86±0.13,4.53±0.17for CNE-1/Taxol, HNE-2/Taxol and5-8F/Taxol, respectively. Result showed that significantly increased sensitivity to Taxol was detected in drug-resistant cells (p<0.01), oppositely, no obvious change was found in parental cells (P<0.05).Conclusion:Both Taxol-resistant NPC cells and drug-sensitive cells were inhibited by5-aza-dC, and Taxol-resistant cells showed an more significant sensitivity. Pretreated with5-aza-dC can partially reverse the Taxol-resistant phenotype in NPC cells.Chapter Three Preliminary study of the mechanism involved in the reverse of NPC Taxol resistance by5-aza-dCObject:Explored the possible effect of candidate genes (CHFR, PEG10,ABCC5,GSTP1,DLC1and ERBB2) in reverse of Taxol resistance by5-aza-dC.Method:Both Taxol resistat and sensitive cells were pretreated by different doses of5-aza-dC. MS-PCR was used for gene methylation detection. The mRNA expression of genes was determined by Real time-PCR, protein expression was measured by western blot.Result:Hypermethylated genes CHFR, PEG10and DLC1were demethylated by5-aza-dC,and no change was detected in ABCC5, GSTP1and ERBB2methylation status after5-aza-dC treatment. The mRNA and protein express were inactivated by both low and high dose of5-aza-dC in down-regulated CHFR,PEG10,and DLC1gene (p<0.01). However, there was no significant difference between5uM and20uM group (P>0.05)Conclusion:Aberrant CHFR,PEG10,DLC1methylation play important roles in NPC Taxol resistance development.5-aza-dC can reverse the Taxol resistance by inactivating the silenced CHFR, PEG10and DLC1genes.