| In case of pancreatic cancer's combined therapy, the chemo-therapeutic effectwas not satisfactory all the time. We know that MDR (multidrug resistance) and mdr1gene (multidrug resistance gene 1) were the main cause of clinical chemotherapyfailure. In order to alter the multidrug resistance of pancreatic cancer, we tried todetect the alteration of mdr1 gene methylation in pancreatic cancer and to control thetranscription of methylation by using action of the MBD1(methyl-CpG bindingdomain protein 1)Experimental study: The upper stream in the main transcription domain of mdr15'-flanking sequence had promoter activity. It was rich of CAAT-box, C-C-box andlack of TATA-box. The -110GC-box and -50GC-box among them were methylatedbinding sites and were bind of transcriptional repressor and enhanser respectively. Wedetected mdr1 and its transcriptional domain binding site methylated expression levelfrom pancreatic cancer tissues by the techniques of FQ-PCR (Real-time fluorescentquantitative PCR)and MSP (methylation—specific PCR). The results showed thatexpressions of mdr1 and P-gp were closely correlated, the methylated expressions of-110GC-box and -50GC-box were correlated significantly also. So we thought thatDNA's methylation was one kind of important mechanism, to influence the geneticexpression. We also supposed that altering the status of DNA's methylation could bethe one of important method to regulate the tumor's multidrug resistance.By using the technique of cDNA microarray to analyze the pancreatic cancergenetic expression spectra, we found that the abnormal transcription controlmechanism between numerous methylation related gene took very important role inthe genetic expression difference. Among them, the expression of MBD1 wasobviously up-regulated in pancreatic cancer. As a important transcriptional controlfactor, MBD1 mediated methylated transcription control in pancreatic cancer might bethe related reason that made the expression and transcription of multidrug resistancegene up/down, and changed the pancreatic cancer's multidrug resistance.BxPC-3 was cultured from pancreatic cancer in situ, and the expression of mdr1 and MBD1 were higher. So BxPC-3 was chosen as a further research platform. ByRNA interference (RNAi) technology, the siRNA was designed and synthesied whichaimed at the MBD1 gene. MBD1 siRNAs eukaryotic expression vector RotroSuper-MBD 1 was constructed and had been successfully integrated into the plasmid.MBD1 siRNAs vector was transfected into BxPC-3 pancreatic cancer cell byliposome and had regulated the level of MBD1 gene expression down ward.Techniques of quantitative immunohistochemical analysis, FQ-PCR, MSP and MTTcolormetric assay was used to detect the difference of mdr1/P-gp protein level, geneticexpression level, genetic methylation level and tumour cell mukiplication activitybefore and after transcription control. It was demonstrated that MBD 1 siRNAs couldsignificantly suppress the expression of MBD 1 mRNA in BxPC-3, which induced thedown-regulation of mdr1 mRNA and P-gp and up-regulation of -110GC-box and-50GC-box's methylation. Among those changes, the MDR (multidrug resistance) ofBxPC-3 were also downgraded and drug sensitivity were upgraded.Clinical study: Utilizing methods including quantitative immunohistochemicalanalysis, FQ-PCR and MSP to detect the protein, genetic expression and methylationlevel in pancreatic carcinoma tissue sample and peripheral blood sample accordingly.After investigation, it was proved that the revariation of expression and methylation inmultidrug resistance gene were dependable concordant between tumor tissue sampleand blood sample of pancreatic carcinoma patients. We could predict the effect andchange of multidrug resistance in pancreatic cancer especially after chemotherapythrough the detection of expression and methylation of multidrug resistance genemerely from peripheral blood sample.Conclusion: Our experimental result indicated that the expressions of mdr1 andP-gp were closely correlated, the methylated expressions of -110GC-box and-50GC-box were correlated significantly also. So the methylation of mdr1 played avery important role in the process of pancreatic cancer's multidrug resistance. Theexpression of mdr1 and MBD1 in BxPC-3 pancreatic cancer cell were high. Theexpression of MBD1 was down-regulated by transfecting MBD1 siRNAs vector intoBxPC-3 mediated with liposome. The level of mdr1/P-gp protein, genetic expression,genetic methylation and tumour cell multiplication activity had been altered, and eventually also changed pancreatic cancer's multidrug resistance indirectly. Thevariation of expression and methylation in multidrug resistance gene were dependableconcordant between tumor tissue sample and blood sample of pancreatic carcinomapatients. We could predict the effect and change of multidrug resistance in pancreaticcancer especially after chemotherapy through the detection of expression andmethylation of multidrug resistance gene merely from peripheral blood sample. |
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