| Breast cancer has a variety of different pathological entities and belongs to malignant diseases with a variety of clinical manifestations.Estrogen receptor(ER)is an important cell surface marker for breast cancer,which promoted the occurrence and development of breast cancer and regulated the sensitivity of cells to drugs by activating both the Src-Ras-PI3K-Akt and MAPK/ERK downstream pathways.With the development of biochip and high-throughput sequencing technology,many gene SNP sites associated with ER phenotype have been identified,which promoted the occurrence and development of breast cancer and regulated the sensitivity of cells.However,the methylation studies involved in regulating the expression of these genes are still in the qualitative stage,whether the methylation pattern of these genes promote breast cancer development and regulation of drug sensitivity or not has not yet been clearly defined.Therefore,a combination of TCGA platform and pyrosequencing here was used to analyze the methylation level of genes associated with ER phenotype in different breast cancer cells and 160 cases of clinical samples to determine the relationship between these genes and the occurrence,stage,classification and drug resistance of breast cancer.Furthermore,the CpG loci in different pathological characteristics and resistance characteristics were screened.The transcriptome data of 547 breast cancer patients were download from the TCGA database.Then ESR1,FOXA1,GATA3,EGFR and GSTP1 genes which have a significant expression difference between ER+ and ER-breast cancer were screened based on TTEST and SAM analysis.And the pyrosequencing method of 75 C.pG sites methylation was optimized respectively.The methylation and expression characteristics of these genes were further analyzed respectively in different breast cancer cell lines and resistant cell lines,the same cell lines with different drugs treatment and 160 breast cancer cases with different pathological features according to non parametric Mann-Whitney(or Kruskall-Wallis)test.The results show that ESR1 and GATA3 gene methylation was positively correlated with the malignant lesions of breast cancer,tamoxifen and fulvestrant resistance and negatively correlated with the ER+phenotype and Luminal subtype.Moreover,GSTP1 gene methylation was positively correlated with malignant lesions of breast cancer,lymph node metastasis,TNM staging,ER+ phenotype,Luminal subtype and high-recurrence risk and negatively associated with the resistance of epirubicin and paclitaxel.These results suggest that ESRI,GATA3 and GSTP1 methylation are involved in the regulation of breast cancer development and drug sensitivity.For in-depth analysis of the mechanism of these gene methylation patterns mediating multidrug-resistance of breast cancer cells,a paclitaxel-resistant breast cancer cell line MCF-7PTXR was constructed,where the PTX resistance ratio is up to 315 times.And in cell line MCF-7PTXR the GATA3 gene is hypermethylation,while EGFR and GSTP1 genes are hypomethylation.Furthermore,the cell line MCF-7PTXR is cross-resistance of epirubicin,cisplatin and fulvestrant.Plasmid pGL3-GATA3-MP and pGL3-GSTP1-MP were transformed respectively into cells.The results show that GATA3 and GSTP1 gene promoter hypermethylation inhibit downstream genes transcription,while overexpression plasmid pGL3-UP-GATA3 can upregulate GATA3 and ER alpha expression of MDA-MB-231 and MCF-7PTXR cells,downregulate the expression of GSTP1,reverse its resistance to tamoxifen and fulvestrant,and increase its sensitivity to epirubicin and paclitaxel.The results proved that long-term administration of paclitaxel can confer MCF7 cell abnormal hypermethylation of ESR1 and GATA3 genes,thereby inhibiting the expression of ER alpha which resulted in the decrease of DNMT1 transmethylase activit and reduced the methylation of GSTP1,thereby inducing the overexpression of GSTP1 protein which resulted in the increase of drug-metabolizing enzyme activity of MCF7 cells,leading to the multidrug resistance of MCF7 cells.In addition,to some extent the drug resistance can be reversed by the demethylation of 5-aza-dC,or the pGL3-UP-GATA3.Spearman’s correlation analysis confirmed that the methylation levels of ESR1 541,ESR1555,ESR1 589,GATA3-1091,GATA3-1033,GATA3-1013,GSTP1 23 and GSTP1 38 sites were significantly related with breast cancer development,lymph node metastasis,TNM staging,molecular typing and drug resistance.The experimental results show that these CpG loci involved in core methylation structure of hypervariable region,regulating the development and resistance of breast cancer by specific methylation model,which provides a novel target for molecular diagnosis and new drug development of breast cancer. |
