The Expression Of FANCF Related Genes In Breast Cancer Tissues And The Effects Of FANCF On Cisplatin-resistant Breast Cancer Cells

Objectives:The mRNA expression profiles of FANCF related genes(FANCA,FANCF,FANCD2,FANCI,BRCA1/FANCS and BRCA2/FANCD1)in different subtypes of breast cancer in non-chemotherapy group and neoadjuvant chemotherapy group were examined in order to find which of these genes are closely related to breast cancer.The expression of FANCF in cisplatin-resistant breast carcinoma was further investigated.In addition,the differential expression of FANCF was verified in an induced MDA-MB-231 cisplatin-resistant cell model before and after drug resistance,and the role and mechanism of FANCF in cisplatin-resistant breast cancer were investigated by RNAi knockdown technology.Methods:Ninety-three breast cancer tissues from invasive ductal carcinoma and normal breast tissues were collected during surgical resection.Among them,20 cases each of triple-negative breast cancer,Luminal breast cancer,and HER2 over-expression breast cancer underwent surgical operation.The cases of these three types of breast cancer who underwent neoadjuvant chemotherapy were 11,13 and 9 cases,respectively.The mRNA expression levels of the FANCF related genes FANCA?FANCD2?FANCF?FANCI?BRCA1 and BRCA2 in breast samples were detected by means of real-time quantitative PCR(RT-PCR).Tissues from twenty-seven patients of relapsed breast cancer who were treated by chemotherapy based on cisplatin were collected,and the expression of FANCF was examined by using immunohistochemistry in cisplatin-sensitive and cisplatin-resistant relapsed tissues embeded in paraffin.Cisplatin-resistant MDA-MB-231 cell line was established by means of long-term cisplatin-exposed cultures.The sensitivity of the cells to cisplatin was determined by the CCK8 assay.The cell cycle distribution was examined by flow cytometry after exposure to cisplatin.The inRNA and protein expression of FANCF gene was examined using qRT-PCR and western blotting,respectively.The expression of FANCF in the cells was reduced by RNAi interference technology and the effect of the RNAi was verified.The sensitivity of the 'two cells to cisplatin was examined by CCK8 assay,and the apoptosis of the cells was analyzed by flow cytometry.Results:1.The mRNA expression levels of FANCA?FANCF.FANCD2?FANCI?BRCA1 and BRCA2 were detected in both cancer tissues and normal breast tissues.In non-chemotherapy group,the mRNA expression of FANCF gene in breast cancer tissues of the three subtypes was lower than that in normal breast tissues(P<0.01).The expression of BRCA1,BRCA2,FANCD2 and FANCI was significant higher in HER2 over-expression breast cancer tissues than normal breast tissues and also higher when compared with those in the other two subtypes cancer tissues.The expression of FANCD2 and FANCI in cancer tissues was higher than that of normal breast tissues in TNBC.The expression level of FANCA in Luminal breast cancer tissues is higher than that in HER2 over-expression type and triple-negative type breast cancer tissues(P<0.05).The expression levels of the other five genes except FANCF were found to be low in neoadjuvant chemotherapeutic triple-negative breast cancer tissues.In neoadjuvant chemotherapy group,the expression levels of BRCA1 and FANCI of Luminal and HER2 subtype in breast cancer tissue are higher than those in normal breast tissue.The expression levels of BRCA1?BRCA2?FANCA and FANCI in Luminal breast cancer tissues were found to be higher than those in HER2 over-expression type and triple-negative type breast cancer tissues(P<0.05).The expression of FANCF was higher in neochemotherapy group than that in non-chemotherapy group(P<0.05)and the difference between the TNBC group was the most significant.The mRNA expression levels of BRCA1?BRCA2?FANCD2.FANCI gene in breast cancer tissues of TNBC and HER2 subtypes who underwent neochemotherapy were lower than those in untreated group(P<0.05).The results of gene correlation analysis showed that the expression of FANCF was negatively correlated with the other five gene expression levels,and the expression level of FANCA.FANCD2?FANCI?BRCA2 and BRCA1 was positively correlated with each other.2.Twenty-seven samples of relapsed breast cancer were examined by immunohistochemical method and the result showed that the expression of FANCF was higher in cisplatin-resistant tissue than that in cisplatin-sensitive breast cancer tissue(P<0.05).3.MDA-MB-231 cell had a 13.5-fold resistance to cisplatin after being induced.chemoresistance by means of longterm exposure to cisplatin.Cell cycle analysis indicated that the cisplatin treatment significantly induced GO/Gl arrest and the number of cells in phase S and G2/M decreased after exposure to cisplatin.The mRNA and protein expression levels of FANCF were significantly higher in cisplatin-resistant cells(P<0.05).4?SiRNA-mediated interference of FANCF not only increased sensitivity of both MDA-MB-231 and MDA-MB-231/Pt to cisplatin but also able to upregulate apoptosis of both cells in response to cisplatin.Conclusions:1.The mRNA expression level of FANCF gene in breast cancer tissues in non-chemotherapy group was lower than that in corresponding normal breast tissue and the mRNA expression level of FANCF in neoadjuvant chemotherapy group were upregulated,suggesting that the use of chemotherapeutic drugs might play a role in the expression of FANCF.The mRNA expression levels of FANCF between the non-chemotherapy group and the neochemotherapy group were different,and the difference between the TNBC group of genes was the most significant.The mRNA expression levels of FANCF related genes in three different subtypes of breast cancer tissues differ significantly.To a certain extent,neoadjuvant chemotherapy can affect the expression of BRCA1,BRCA2,FANCD2 and FANCI genes.2.The finding that the expression level of FANCF was significantly increased in cisplatin-resistant relapsed cancer tissue indicated that FANCF might play a role in cisplatin-resistance of breast cancer.3.MDA-MB-231 cells were induced to become cisplatin-resistant in vitro by means of incremental increase of cisplatin concentrations and the cisplatin-resistant cell line MDA-MB-231/Pt was successfully established.The expression levels of FANCF mRNA and protein in MDA-MB-231/Pt cells were significantly higher than those in MDA-MB-231 cells which suggested that the upregulation of FANCF expression in TNBC cells might be related to cisplatin-resistance.4.When FANCF gene was knockdown by FANCF-siRNA,the sensitivity to cisplatin and the induction of apoptosis of MDA-MB-231/Pt and MDA-MB-231 cells were sigrnificantly increased,which suggested that FANCF gene might play an important role in cisplatin-resistance of TNBC cell MDA-MB-231 and indicated that FANCF might be a novel target to develop treatment for breast cancers.