Study On The Mechanism Of Doxorubicin Resistance And Abnormal Glucose Metabolism In Breast Cancer Stem Cells

PART ONE:MiR-124 Reversed the Doxorubicin Resistance of Breast Cancer Stem Cells Through STAT3/HIF-1 Signaling PathwaysObjective: To investigate the reversal of adriamycin resistance of breast cancer stem cells by miR-124 through STAT3 / HIF-1 signaling pathway.Methods: Using gpl571 based gse24460 microarray analysis,gse24460 was divided into two parental controls and two drug-resistant MCF7 cells,and the differential expression genes(DEG)between the two groups were analyzed.MCF7 cells were collected and hydrolyzed into single cell suspension.The synthetic microrna-124 analog and non-specific miRNA control were developed by genepharma(Shanghai,China).Pcdna3.1-stat3 plasmid was constructed and transfected with STAT3 siRNA and control siRNA.Cell viability was measured by MTT.Stat33'utr containing the putative miR-124 binding site was analyzed by targetscan(www.targetscan.ORG),and the miRNA site was inserted into the downstream of the fluorescent enzyme gene(Promega,USA)and transfected to determine the fluorescent enzyme activity.The expression of STAT3,ALDH1,Sox2,Oct4 and HIF-1 ? were detected by Western blot.Real time quantitative reverse transcription PCR(QRT PCR)was used to analyze the expression of STAT3 and miR-124.Flow cytometry was used to detect apoptosis;after collecting cells and digesting cell suspension,cells were fixed with 75% ethanol at 4 ? for 4 hours,then incubated with RNA enzyme containing iodide(PI,40%,sigma Aldrich),and cell cycle was detected with FACS Calibur.Transwell method was used to analyze the cell invasion ability to verify the drug resistance.Results: The expression of STAT3 in mcf7-r cells was higher than that in MCF7 cells,and affected the adriamycin resistance of BCSC.MiR-124 reversed the adriamycin resistance of breast cancer stem cells by targeting STAT3 to control HIF-1signaling pathway.Conclusion: The recovery of miR-124 and the inhibition of STAT3 in the treatment of breast cancer are helpful to overcome drug resistance.PART TWO:Estradiol influences doxorubicin resistance and glycolysis in breast cancer stem cells through the Shh-Gli pathwayObjective: The purpose of this study was to investigate the mechanism by which estradiol affects doxorubicin resistance and glycolysis in breast cancer stem cells through the Shh-Gli pathway.Methods: Human breast cancer MCF-7 cells were purchased from the American Type Culture Collection and then incubated with a medium containing 10% fetal bovine serum penicillin-streptomycin solution at a temperature of 37 ? and a carbon dioxide content of 5%.The medium was changed every 2 days.The drug resistance of the cell line MCF-7 / ADR(M / A)was maintained by adding 1.0 mg / l doxorubicin.Two weeks before the experiment,MCF-7 / ADR(M / A)cells were cultured in a medium without adriamycin.The experiment was divided into 3 groups: MCF-7 /ADR group(MCF-7 / ADR cell line,untreated);Gli mimic group(MCF-7 / ADR cell line + Gli mimic plasmid transfection,Gli overexpression);Sh-Gli transfection group(MCF-7 / ADR cell line + Sh-Gli plasmid transfection,Gli low expression).Cell survival rate was measured by MTT;total RNA was extracted from cells using TRIzol reagent,and Shh and Gli mRNA levels were detected by RT-PCR;48 hours after transfection,each group of cells was digested and cell migration was detected by Transwell;Akt was analyzed by Western blot,GLUT1 and HK-2 protein content;HIF-1? and LDHA levels of breast cancer stem cells were detected by ELISA.Cellular lactic acid accumulation was quantified using two different techniques,nuclear magnetic resonance and gas chromatography-mass spectrometry.Results: Compared with the control group,the cell proliferation rate was increased at 3 hours,6 hours,12 hours,and 24 hours in the estradiol treated group,and 3 hours in the estradiol + doxorubicin group compared with the estradiol treated group Cell proliferation rate decreased at 6 hours,12 hours,and 24 hours(P<0.05).Compared with the control group(0.83 ± 0.12,0.74 ± 0.23),the levels of Shh(5.74 ±0.86)and Gli(6.63 ± 0.45 mRNA)increased in the estradiol-treated group,and the estradiol + increased Compared with MCF-7 / ADR group,Shli(3.13 ± 0.15)and Gli(4.28 ± 0.16)mRNA levels were decreased(P<0.05)in the solubizin group.Compared with the MCF-7 / ADR group,Gli expression was increased in Gli mimic group,cell proliferation and cell migration Increased(P<0.05).Gli expression decreased,cell proliferation and cell migration decreased in the Sh-Gli transfection group(P<0.05).Compared with MCF-7 / ADR group,p-AKT,GLUT1 and HK2 expression in the Gli mimic group Increased(P<0.05).The expression of p-AKT,GLUT1,and HK2 decreased in the Sh-Gli transfection group(P<0.05).Compared with the MCF-7 / ADR group,the levels of HIF-1?,LDHA and lactic acid in the Gli mimic group Increased(P<0.05).The levels of HIF-1?,LDHA,and lactic acid decreased in the Sh-Gli transfection group(P<0.05).Conclusion: The aerobic glycolytic phenotype of MCF7 / ADR cells increased,and it passed Shh-Gli Signaling regulation of p-AKT,GLUT1,HK2,LDHA,and HIF-1? may be attributed to changes in glycolytic metabolism in doxorubicinresistant cells.