Effects Of YB-1 Expression In Breast Cancer On Cell Growth, Invasion And Drug Resistance

Background: Breast cancer is one of the most common tumors that threaten women’s health.The incidence of Europe and the United States is high,Asian incidence is low but increased year by year.Although there are many treatment methods,breast cancer patients still face the dilemma of drug resistance after multi-line treatment.YB-1 is a transcription and translation factor,It is considered as the oncogene of a variety of solid tumors,which is involved in the processes of mRNA cutting,transcription,translation,cell proliferation,apoptosis and drug resistance by binding to RNA,It is related to the prognosis and drug resistance.The purpose of this study was to observe the effect of YB-1 expression level on the growth,proliferation,migration and invasion of breast cancer cells;To understand the effect of YB-1 expression level on the chemotherapy effect of adriamycin treated breast cancer resistant cells;To analyze the correlation between YB-1 and breast cancer drug resistance protein(BCRP)in drug resistance mechanism.Through the research and discussion on the above issues,the effect of yb-1 on breast cancer and breast cancer resistant cells was further clarified.Part one: The relationship between the expression level of YB-1 and the clinicopathological parameters in breast cancer patients.Objective: To observe the expression level of yb-1 in breast cancer tissues and adjacent tissues,and to analyze the relationship between the expression level of YB-1and the clinicopathological parameters in breast cancer.Methods: The cancer tissues of 48 patients with stage II-III invasive ductal carcinoma and the paracancer tissues of 20 patients were collected as control,and immunohistochemical staining was performed.The percentage of positive cells was 0to 4 scores,assigning negative a score of 0,≤ 10% positive cells a score of 1,11–50%positive cells a score of 2,51–75% positive cells a score of 3,and ≥ 75% positivecells a score of 4.The expression levels of YB-1 in cancer tissues and paracancer tissues were compared,and the data of 48 patients were statistically followed up.The relationship between YB-1 expression level and various clinicopathological parameters of breast cancer was studied by independent sample t test and one-way ANOVA statistical method.Results: Compared with breast cancer tissues,the expression level of YB-1 in paracancer tissues was significantly decreased,The expression of YB-1 in paracancer tissues of 17 cases was 0-1,accounting for 85%,and no nuclear staining was observed(median score: 2.52 vs 1.10,F=12.048,sig=0.001).The numbers of breast cancer patients with regional lymph node positive were 27 cases(accounted for65.2%),negative were 21 cases(accounted for 43.8%),according to the immunohistochemical staining,found that the YB-1 expression level in patients with regional lymph node positive was significantly higher than those with the regional lymph node negative,difference was statistically significant(P = 0.017).There were11 patients whose age ≤35 years old,among whom 7 patients had YB-1 expression level score of 3-4 scores.The patients with high YB-1 expression tended to be younger,but there was no statistical difference.There was no statistical difference in age,mass size,histological grade,molecular typing and postoperative stage.Conclusion: Patients with high expression of YB-1 were prone to regional lymph node metastasis and show younger trend.Part two: Effects of YB-1 expression on the growth,proliferation,migration and invasion in breast cancer cells.Objective: To investigate the effect of YB-1 expression on the growth,proliferation,migration and invasion in breast cancer cells.Methods: 1.The effect of adriamycin,cisplatin and 5-Fu at different concentrations on the proliferation of MCF-7 /ADR cells was detected by MTT method,and IC50 was calculated to verify the drug-resistant cell line MCF-7 /ADR.2.The lentiviral transfection technique was used to construct YB-1 overexpr-ession vector and knock down vector to be transfected into MCF-7 /ADR cells,and the expression level of YB-1 protein was detected by Western blot after stable transformation.To clarify the transfection effect,MCF-7 /ADR was set as the control group,MCF-7 /ADR+ YB-1 overexpression group,MCF-7 /ADR+ YB-1 knock down group,and MCF-7 /ADR+ empty group as the experimental group.3.The growth of cells in each group was observed by electron microscope;Flow cytometry was used to detect cyclins.The cell proliferation of each group at 24 h,48h and 72 h was detected by CCK-8 method,and the proliferation curve was drawn.Transwell invasion assay was used to detect the migration and invasion ability of cells in each group.4.Fluorescence quantitative RT-PCR and Western blot were used to detect the expression levels of YB-1 and corresponding mRNA in cells of each experimental group.5.Growth in good shape of breast cancer cells MCF-7 and resistant MCF-7cell/ADR were selected,subcutaneous tissue inoculation in nude mice,give ADM(4mg/kg),nude mice were divided into four groups,respectively is the MCF-7group,the MCF-7 + ADR group,the MCF-7 / ADR group and MCF-7 / ADR +ADR group,feeding 14 days after transplanted tumor tissue,measure the tumor weight and volume,observe the change of the growth curve,by using flow cytometry to detect the cell cycle proteins,The expression level of YB-1 protein was detected by qRT-PCR and Western blot.Results: 1.Nude mouse experiment: The tumor weight and volume of MCF-7/ADR transplantation were greater than that of MCF-7 transplantation with or without adriamycin treatment.The expressions of YB-1,BCRP,Cyclin E,Cyclin D and P53 in MCF-7 /ADR were all higher than those in MCF-7 cells,but the expression of GAPDH was not different in the four groups.2.Cell experiment: The expression level of YB-1mRNA in MCF-7 /ADR-resistant cell lines was significantly increased;In the CCK-8 experiment,the proliferation capacity of MCF-7 /ADR+ YB-1 overexpressed cells was the strongest in each group at 24 h,48h and 72 h,while that of MCF-7 /ADR+ YB-1 was the weakest.MCF-7 /ADR with YB-1 overexpression was enhanced in migration andinvasion,while the ability of YB-1 knock down with cell migration and invasion was weakened.The difference between the two groups was statistically significant(P <0.05).Cell cycle detection: MCF-7 /ADR+ YB-1 knock down cells accounted for65.67% in G1 phase,the number of cells in S phase and G2 phase was relatively small,while the proportion of MCF-7 /ADR and MCF-7 /ADR+ YB-1 overexpressed cells decreased in G1 phase and increased in S phase,and about 90% of them remained in G1/S phase.Conclusion: YB-1 overexpression promotes breast cancer cells proliferation,migration and invasion.Part three: Effect of YB-1 expression level on chemotherapy sensitivity of Adriamycin in drug-resistant breast cancer cells.Objective: To investigate the effect of YB-1 expression level on chemotherapy sensitivity of Adriamycin in drug-resistant breast cancer cells.Methods: Three group cells were given by Adriamycin,like MCF-7 /ADR+EV,MCF-7 /ADR+ YB-1 overexpression and MCF-7 /ADR+ YB-1 knock down.CCK-8kit was used to detect the proliferation ability of them.Transwell detected the migration and invasion ability of cells in each group.Immunofluorescence assay was used to detect the changes of cells in each group before and after Adriamycin treatment.Results: The proliferation capacity of YB-1 overexpressed MCF-7 /ADR cells was slightly reduced,but the difference was not statistically significant after Adriamycin treatment.There was no significant decreased in the migration and invasion ability of drug-resistant breast cancer cells with YB-1 overexpression,and the difference was not statistically significant(P > 0.05).The migration and invasion ability of MCF-7/ADR+YB-1 knock down were significantly decreased,and the difference was statistically significant(P < 0.05).In immunofluorescence assay,the expression level of YB-1 in MCF-7 /ADR+ YB-1 overexpressed cells was not significantly decreased after Adriamycin treatment,while the expression level ofYB-1 in MCF-7 /ADR+ YB-1 knock down cells was significantly decreased.Conclusion: YB-1 overexpression reduces the chemotherapy sensitivity of M CF-7 /ADR to Adriamycin in drug-resistant breast cancer cells.Part four: The relationship between BCRP and YB-1Objective: To investigate the correlation between BCRP and YB-1 in chemotherapy resisitance in breast cancer.Methods: The expression level of BCRP in MCF-7 /ADR was detected,and the correlation between BCRP and YB-1 was detected by immunoprecipitation.Results: 1.The expression level of BCRP in MCF-7 /ADR increased with or without Adriamycin treatment.2.The expressions level of YB-1 and BCRP were significantly enhanced in MCF-7 / ADR cells with YB-1 overexpression,while the expressions level of YB-1and BCRP were significantly decreased in MCF-7 /ADR cells with YB-1 knock down.The expressions level of YB-1 and BCRP were in the middle of both in no-load cells.The same was true for yb-1 and BCRP expression levels in Adriamycin treated cells.Conclusion: The expression level of BCRP was increased in MCF-7 /ADR,and the expressions level of YB-1 and BCRP were synchronized in YB-1overexpression,knock down and no-load cell groups.Therefore,there is a certain correlation between the expression of YB-1 and BCRP,but the mechanism is not clear.