The Analysis Of NDRG1 Gene Aberrant Methylation In Breast Cancer And The Effect Of Transcription Inhibition Regulated By BRCA1 Gene Promoter CpG Specific-site Methylation

Part 1 The analysis of NDRG1 gene aberrant methylation in breast cancerObjective:To explore the correlation between NDRG1 promoter methylation and NDRG1 gene expression and clinicopathological significance in human breast cancer.Method:NDRG1 mRNA expression was detected using semi-quantitative RT-PCR method in human breast cancer cell lines(T47D and SK-BR-3 and MCF7).NDRG1 protein expression was detected using western blot method in human breast cancer cell lines T47D,SK-BR-3 and MCF7.NDRG1 gene promoter methylation in distribution was tested using BSP cloning sequencing method in breast cancer cell lines(T47D and SK-BR-3 and MCF7).NDRG1 gene promoter region methylation status was detected using Methylation specific PCR(MSP)method in T47D,SK-BR-3 and MCF7 cell lines.NDRG1 gene mRNA and protein expression were detected after a demethylation drug 5-Aza-dC(AZA)treatment in T47D cell lines using semi-quantitative RT-PCR and western blot methods.NDRG1 gene mRNA expression was detected using semi-quantitative RT-PCR method in breast cancer tissues.NDRG1 gene promoter region methylation status was detected using MSP method in breast cancer tissues.Results:The results of RT-PCR and western blot methods showd that NDRG1 expressed in breast cancer cell lines(MCF7 and SK-BR-3),while NDRG1 expression was not detected in T47D cell line.BSP cloning sequencing results showed that the methylation percentage for NDRG1 gene in breast cancer cell line T47D was 66%,in SK-BR-3 and MCF7 cell lines were 9%and 15%,respectively.NDRG1 gene in its expression and non-expression cell lines,the different methylation sites were CpGs 13-17,20-23 and 35-40(the first CpG was considered as 1 in the region from-379 to +86).The primer of methylation specific PCR(MSP)based on the different methylation site CpGs 13-17,35-40 was designed.The results of MSP assay showed that NDRG1 gene was methylated in T47D cells and unmethylaed in SK-BR-3 and MCF7 cell lines.With increasing of AZA drug concentration in T47D cells,NDRG1 gene expression at mRNA and protein expression levels were gradually increased.The presence of NDRG1 mRNA was detected in 45 out of 87 breast tumors or in 10 out of 87 normal tissues.Therefore,loss of NDRG1 expression was significantly more frequent in tumors than in the corresponding adjacent tissues(P = 0.000).We used MSP analysis to study the methylation status of NDRG1 CpG islands.NDRG1 promoter methylation was detected in 32 of 87 tumors and in 13 corresponding adjacent tissues;in 121 of 389(31.1%).Therefore,NDRG1 promoter methylation was observed more frequently in breast cancer tissues than in the corresponding adjacent tissues(P = 0.000).NDRG1 in 27 out of 32 methylated breast cancer tissues was not expressed,and 18 out of 55 cases with unmethylation was NDRG1 lost,the difference was statistically significant(P = 0.000).NDRG1 gene expression in 9 cases with methylation for adjacent breast cancer tissues was all not detected,in 78 cases with unmethylation was only one case detected,the difference was statistically significant(P =0.000).Clinico-pathological correlation analysis showed that NDRG1 promoter methylation was associated with histological grade in Ⅱ[ORs:1.89(1.20 3.20)95%CIs],Ⅲ stage[ORs:2.96(1.19 7.39)95%CIs]and metastasis[ORs:1.94 95%CIs,(1.10-1.10)],TNM stageⅢ/Ⅳ[ORs:2.82(1.41 5.62)95%CIs,]and lymphoid invasion(OR:2.22(95%CI,1.194.11)].There was no relationship between NDRG1 gene methylation and menopausal status,tumor size,p53 mutation,Her2 status and Ki67 proliferation.Conclusion:NDRG1 gene expression was reduced in breast cancer and regulated by the abnormal promoter region methylation.NDRG1 gene promoter methylation was associated with histological grade,metastasis,Tumor Node Metastasis(TNM)and lymph invasion.Part 2 The investigation of transcription inhibition regulated by BRCA1 gene promoter CpG specific-site methylationObjective:The aim of this study was to examine the important CpG sites of BRCA1 gene novel methylation for transcriptional regulation.Method:Five different fragments around the transcription start site(from-247 to +86)of BRCA1 were cloned and examined putative promoter activities before and after M.SssI treatment by luciferase reporter assay system.Results:The luciferase activity of fragment F422 and F281 after transfection in MCF7 cell line is significantly higher than of other segments.Meanwhile,the activity of fragment F204 were also detected,was significantly higher compared with the activity of F178 and blank control Basic fragments.The luciferase activity of F178 and F200 fragment was not detected significantly different.Moreover,there was no significant difference for the luciferase activity between F178 fragment and blank control Basic.After M.SssI treatment,compared with non-methylated F281 fragment,the luciferase activity of methylated F281 fragments was reduced by 60%,and was significantly different.Compared with non-methylated F422 fragment,the luciferase activity of methylated F422 fragments was reduced by 58%,and was significantly different.Compared with non-methylated F204 fragment,the luciferase activity of methylated F204 fragments was reduced by 20%,and was significantly different.However,the activity of other fragments was not found to be significantly reduced.Conclusion:BRCA1 gene promoter region methylation methylaion-specific site for inhibitory transcription was in-37～-19 position.