Analysis The Relationship Between The Aberrant Methylation Of BRCA1 Promoter And Expressed Region And The Expression Of BRCA1 In Triple Negative Breast Cancer

Breast cacer is the highest incidence of female cancer in China.It's a serious threat to women's health.In the clinic,for TNBC expresses negatively to progesterone receptor(PR),estrogen receptor(ER)and human epidermal receptor 2(Her2),it neutralizes hormonal or Herceptin therapy and lacks targeted therapies.The treatment of TNBC is currently one of the hottest and the most difficult points in the treatment of breast cancer.BRCA1 is anti-oncogene.The loss of BRCA1 can lead to the development of breast cancer.Some studies have confirmed that the mutation of BRCA1 gene can inhibit the expression of BRCA1 gene,which leads to the occurrence and propagation of breast cancer.But in sporadic breast cancer,the sequence of BRCA1 gene was complete,but the expression of mRNA and protein were decreased significantly.These results suggest that BRCA1 gene is involved in BRCA1 gene expression by other mechanisms.At present,it is thought that aberrant methylation of genes plays an important role in the malignant transformation of normal cells and in the process of malignant tumor invasion.It was found that the methylation rate of BRCA1 gene was higher in Triple negative breast cancer(TNBC).Objective: Explore the relationship between the aberrant methylation of BRCA1 promoter and the first expressed region and the expression of BRCA1 in triple negative breast cancer.Provide new ideas for triple negative breast cancer treatment.Methods:1 The protein expression of BRCA1 in breast cancer tissues with different immunophenotype were detected by immunohistochemistry assay.2 The mRNA expression of BRCA1 in breast cancer cell lines withdifferent immunophenotype were detected by RT-qPCR.3 Effect of medicine on expression of BRCA1 mRNA in breast cancer cell lines with different immunophenotype detected by RT-qPCR.4 The protein expression of BRCA1 in breast cancer cell lines with different immunophenotype were detected by immunocytochemistry assay.5 The methylation rate of the BRCA1 promoter and the first expressed region in breast cancer cell lines with different immunophenotype were detected by bisulfite sequencing PCR(BSP).Results:1 The expression levels of BRCA1 protein in breast cancer tissues with different immunophenotype.Immunohistochemistry assay demonstrated that: the expression rate of BRCA1 protein in triple negative breast cancer tissues was 57.9%(11/19);the expression rate of BRCA1 protein in Luminal breast cancer tissues was 85.5%(59/69);the expression rate of BRCA1 protein in HER2+ breast cancer tissues was 76.9%(10/13).The expression rate of BRCA1 protein in triple negative breast cancer tissues was lower than Luminal breast cancer(P<0.05).The expression rate of BRCA1 protein in triple negative breast cancer tissues was lower than HER2+ breast cancer(P>0.05).the expression rate of BRCA1 protein in HER2+ breast cancer tissues was lower than Luminal breast cancer(P>0.05).2 Effect of immunophenotype on the overall survival of patients with BC.Our results of the overall survival demonstrated that: Immunophenotype has poor effect on the overall survival of patients with BC.Compared with the Luminal group,overall survival of the TNBC group and the HER2+ group were shorter(P<0.05).There was no statistically significant difference in overall survival between the TNBC group and the HER2+ group(P>0.05).3 Effect of BRCA1 expression on the overall survival of patients with BC.The result of the overall survival demonstrated that: Compared with patients with positive BRCA1 protein expression,the overall survival wasshorter in patients with negative BRCA1 protein expression,and there was no statistical difference(P>0.05).4 The expression levels of BRCA1 mRNA in breast cancer cell lines with different immunophenotype.The expression levels of BRCA1 mRNA in MDA-MB-231?BT-549?MCF-7 and MDA-MB-453 werevdetected by RT-qPCR.The expression levels of BRCA1 mRNA was expressed in 2-??CTvalue.The results showed: The expression levels of BRCA1 mRNA in MDA-MB-231 was 1.00�0.08,BT549 was 1.11�0.14,MCF-7 was 2.42�0.21,MDA-MB-453 was 5.89�0.65.The expression levels of BRCA1 mRNA in MDA-MB-231 and BT549 had no statistical difference;The expression levels of BRCA1 mRNA in MDA-MB-231 and BT549 were lower than MCF-7 and MDA-MB-453;MCF-7 was lower than MDA-MB-453,there were statistically significant(P<0.001).5 Effect of 5-Aza-DC on expression of BRCA1 mRNA in breast cancer cell lines with different immunophenotype.Effect of 5-Aza-DC on expression of BRCA1 mRNA in breast cancer cell lines(MDA-MB-231,BT-549,MCF-7,MDA-MB-453)detected by RT-qPCR.The results showed:MDA-MB-231: The expression levels of BRCA1 mRNA in 1?mol/L5-Aza-DC treatment group was(1.94�0.24).The expression levels of BRCA1 mRNA in 2?mol/L 5-Aza-DC treatment group was(2.35�0.66).There were higher than control group(1.00�0.08)and statistically significant(P<0.001).BT-549: The expression levels of BRCA1 mRNA in 1?mol/L 5-Aza-DC treatment group was(1.22�0.08).The expression levels of BRCA1 mRNA in control group was(1.01�0.13).They had no statistical difference(P>0.05).The expression levels of BRCA1 mRNA in 2?mol/L 5-Aza-DC treatment group was(2.69�0.34).It was higher than control group and statistically significant(P<0.01).MCF-7: The expression levels of BRCA1 mRNA in 1?mol/L 5-Aza-DC treatment group was(1.15�0.19).The expression levels of BRCA1 mRNA incontrol group was(1.00�0.09).They had no statistical difference(P>0.05).The expression levels of BRCA1 mRNA in 2?mol/L 5-Aza-DC treatment group was(1.42�0.18).It was higher than control group and statistically significant(P<0.05).MDA-MB-453: The expression levels of BRCA1 mRNA in 1?mol/L5-Aza-DC treatment group was(3.18�0.70).The expression levels of BRCA1 mRNA in 2?mol/L 5-Aza-DC treatment group was(5.97�0.87).There were higher than control group(1.00�0.11)and statistically significant(P<0.01).6 The expression levels of BRCA1 protein in breast cancer cells with different immunophenotype.The expression of BRCA1 protein in breast cancer cell lines detected by immunocytochemistry assay.The positive signals were yellow and located in the cytoplasm.Immunocytochemistry assay demonstrated that the expression level of BRCA1 protein in MDA-MB-231 and BT-549 of triple negative breast cancer cells were lower than MCF-7 and MDA-MB-453 and the expression level of BRCA1 protein in MCF-7 were lower than MDA-MB-453.It consistent with the results of BRCA1 mRNA expression.7 Total methylation rate of BRCA1 promoter in breast cancer cell lines with different immunophenotype.The methylation rate of BRCA1 promoter(-908~-714)can be detected by BSP.The results showed:BRCA1 promoter methylation rate of MDA-MB-231,BT-549,MCF-7,MDA-MB-453 were respectively(26/180)14.44%,(7/180)3.89%,(4/180)2.22%,(2/180)1.11%.The correlation between BRCA1 promoter methylation rate and the expression levels of BRCA1 was negative.8 Total methylation rate of BRCA1 the first expressed region in breast cancer cell lines with different immunophenotype.The methylation rate of BRCA1 the first expressed region(-70~ 165)can be detected by BSP.The results showed:BRCA1 the first expressed region methylation rate of MDA-MB-231,BT-549,MCF-7,MDA-MB-453 were respectively(6/182)3.30%,(1/182)0.55%,(13/182)7.14%,(0/180)0.00%.There was not correlation between BRCA1 expressed region methylation rate and the expression levels of BRCA1.Conclusion:1 The expression levels of BRCA1 in triple negative breast cancer was lower,and the correlation between it and the methylation rate of BRCA1promoter(-908~-714)was positive.2 There was not correlation between the expression levels of BRCA1 and the methylation rate of BRCA1 the first expressed region(-70~165).