| BackgroundBreast cancer(BCa)has surpassed lung cancer to become the most common cancer in the world,which severely impairs the survival outcomes of patients and quality of life.However,tumor recurrent or drug resistance often lead to a failure to successful therapy,so it is of great research value and clinical interests to discover novel molecular targets to improve therapeutic efficacy in breast cancer.Our group used proteomics,bioinformatics and large-scale data analysis to find the correlation between NR2F6(Nuclear receptor subfamily 2,group F,member 6)expression and cisplatin sensitivity and resistance in some tent.Then we conducted primary experiments to confirm earlier findings in breast cancer cell lines,which provided a new research idea for NR2F6 in tumor drug sensitivity and resistance.However,studies on whether NR2F6 could induce cisplatin resistance as a therapy target in breast cancer are still limited.Therefore,in this study,we aimed to explore whether NR2F6 has an impact on the proliferation,apoptosis,invasion and migration of breast cancer and whether NR2F6 mediates cisplatin sensitivity in breast cancer and its possible mechanism.Method1.Real-time PCR(qPCR)assay was used to validate the expression of NR2F6 mRNA levels in five human breast cancer cell lines(MCF-7,UACC-812,SK-BR-3,BT-474,MDA-MB-231)and one human normal breast epithelial cell line(MCF-10A).We detected NR2F6 expressions at the protein level by Western blot.In order to further explore the biological role of NR2F6,MDA-MB-231-NR2F6 cell line(231NR2F6),which stably overexpressed NR2F6 was established by lentivirus vector and MCF-7-shNR2F6(MCF7-shNR2F6)cell line using lentivirus vectors,which stably represses NR2F6 expression were successfully established.We used western blot to verify NR2F6 expression at the protein level in these cells.We used CCK8 assay and colony formation assay to detect MDA-MB-231-NR2F6 cell proliferation ability,Transwell assay and wound healing assay to detect MDA-MB-231-NR2F6 cell invasion and migration ability,then we could verify whether NR2F6 had an effect on MDA-MB-231-NR2F6 cell proliferation,invasion and migration ability.2.Drug-resistant breast cancer cell line MCF7/DDP and its parent cell MCF7 were used to validate the function of NR2F6 when exposed to DDP,We used western blot to verify NR2F6 expression at the protein levels in these cells.CCK8 method was used to compare proliferation in MCF7/DDP,MDA-MB-231-NR2F6,MCF7shNR2F6 and their control cells when exposed to escalating concentrations of DDP,and dose-response curves were constructed to indicate the half maximal inhibitory concentration(IC50)of each cell.A series of phenotypic experiments including CCK8 assay,wound healing assay and Transwell assay were conducted to verify whether NR2F6 affected breast cancer cell proliferation,invasion and migration ability under the treatment of cisplatin.3.Study on the mechanism of cisplatin resistance to NR2F6.RNA-seq was used to screen drug resistance gene BCL2A1,Real-time PCR(qPCR)and Western blot were used to verify drug resistance gene BCL2A1,and NR2F6 RNA interference plasmid was transfected to observe whether NR2F6 caused changes in drug resistance of breast cancer.Result1.The TIMER database and ULCAN database results suggested that the expression levels of NR2F6 were significantly higher in breast cancer tissues than that in normal tissues adjacent to carcinoma.Kaplan Meier Plotter database results suggested that patients with high NR2F6 expression had poorer prognosis(P<0.05).The results of CCK8 assay,colony formation assay,wound healing assay and transwell assay showed that overexpression of NR2F6 significantly inhibited MDAMB-231 cell proliferation,invasion and migration ability(P<0.05).2.DDP could stimulate the expression of NR2F6 in MDA-MB-231 cells,and NR2F6 expression was observed to be increased in a time-dependent manner.Pearson analysis results showed that the IC50 values were positively correlated with the expression of NR2F6.The IC50 values for DDP in MCF-7/DDP and parental MCF-7 cell line were(11.98±0.35)μmol/L and(6.65±0.93)μmol/L,respectively(P<0.05).The results of western blot showed that NR2F6 expression were lower in MCF7/DDP cells than that in MCF-7 cell(P<0.0001).The IC50 values for DDP in MDAMB-231-NR2F6 and MDA-MB-231-NC cell lines were(12.02±0.65)μmol/L and(13±0.44)μmol/L,respectively.The IC50 values for DDP in MCF-7-shNR2F6 and MCF-7-control cell lines were(23.3 1±0.92)μmol/L and(17.49±0.7)μmol/L,respectively.3.The results of wound healing assay and transwell assay showed that overexpression of NR2F6 significantly inhibited MDA-MB-231 cell invasion and migration ability(P<0.05).Overexpression of NR2F6 significantly increased the protein levels of cleaved caspase 3 and cleaved caspase 7,induced the protein level of P21 waf,p-H3 and p-weel when exposed to DDP.4.Anti-apoptosis protein BCL2A1 was screened out by proteomic and RNAseq assays.The IC50 values for DDP in shNR2F6-siNR2F6 and shNR2F6-siNC cell lines were(9.98±0.65)μmol/L and(12.07±0.70)μmol/L,respectively(P<0.0001).Conclusion1.Overexpression of NR2F6 inhibited MDA-MB-231 cell proliferation,invasion and migration ability.And when cells were exposed to cisplatin,overexpression of NR2F6 inhibited MDA-MB-231 cell proliferation,invasion and migration ability.2.NR2F6 maintains the sensitivity of cisplatin therapy in breast cancer by BCL2A1. |
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