| Objective: ZBTB4 plays an important role in tumorigenesis,proliferation and differentiation.The changes in the expression of ZBTB4 in tumor cells indicate that ZBTB4 can act as a tumor suppressor or an oncogene.However,it is still unclear about the role and specific mechanism of ZBTB4 in tamoxifen-resistant breast cancer cells.Here,we will use in vivo and in vitro experiments to study the expression of ZBTB4 in breast cancer drug-resistant cells and its effect on the malignant progression of breast cancer,explore the effect of ZBTB4 on the metabolic reprogrammingof resistant cells,and further explore its potential specific mechanisms.Method:1.The expression and clinical significance of ZBTB4 in breast cancer cells: The expression of ZBTB4 in breast cancer tissues and normal breast tissues was searched through the TCGA database,and then the expression of ZBTB4 in breast tissues was verified by Western blottingand chromatin immunoprecipitation experiments.Protein expression levels in cancer and normal breast.In addition,the expression of ZBTB4 in paraffin-embedded tumor tissues from breast cancer patients was detected,and the patients were divided into low or high ZBTB4 expression groups based on ROC analysis.Kaplan-Meier analyzed the difference in overall survival rate between the two groups of patients.2.The effect of ZBTB4 on the malignant progression and metabolism of breast cancer cells: construct breast cancer cells stably overexpressing ZBTB4,and verify the overexpression by Western blotting experiments.Through CCK-8 experiments and scratch experiments,explore the effects of ZBTB4 on breast cancer cell proliferation.And the effect of migration ability;then specifically knock down the ZBTB4 gene in breast cancer cells overexpressing ZBTB4,and explore whether the knockdown of ZBTB4 can reverse the effect of this gene on the proliferation and migration of breast cancer cells.Through in vivo experiments,breast cancer cells with stable overexpression and low ZBTB4 expression were injected into the second pair of fat pads in nude mice to construct xenograft tumor models to explore the effect of ZBTB4 on tumor growth in vivo.The contents of glutamate,glutamine,glucose and lactic acid in breast cancer cells overexpressing and underexpressing ZBTB4 were detected according to the instructions of the kit to verify the effect of ZBTB4 on the metabolic transformation of breast cancer cells.3.ZBTB4 affects the sensitivity of tamoxifen-resistant breast cancer cells: a tamoxifen-resistant stable breast cancer cell line was obtained by continuously increasing the concentration of tamoxifen for a period of 6 months,and retained Parental cells for subsequent comparison experiments.The expression of ZBTB4 was detected and compared by Western blotting.Then,the drug-resistant and parental cells of breast cancer cells stably expressing ZBTB4 were constructed by transfection,and the protein expression levels of the two were detected.Then CCK-8 experiment and nude mouse experiment were used to detect the changes in the sensitivity of drug-resistant cells after tamoxifen treatment.4.The mechanism of ZBTB4 regulating breast cancer cell proliferation,metabolism and drug resistance: Western blotting and immunoprecipitation were used to detect the expression level of c-Myc in breast cancer cells and drug-resistant cells,and breast cancer cells overexpressing ZBTB4 and The expression level of c-Myc in drug-resistant cells was tested to detect the effect of up-regulation of ZBTB4 on the expression of c-Myc.Co-immunoprecipitation was used to identify whether ZBTB4 interacts with c-Myc in vivo and in vitro.Plasmid transfection was used to construct breast cancer cells and drug-resistant cells stably overexpressing c-Myc,and the expression level was verified by Western blotting.Then,in vivo and in vitro experiments were conducted to confirm whether the overexpression of c-Myc could reverse the proliferation and proliferation of breast cancer cells by ZBTB4.The role of metabolism and drug-resistant cell sensitivity.Result:1.By querying the TCGA database,we found that compared with normal breast tissues,ZBTB4 is under-expressed in breast cancer tissues,and the results of western blot and chromatin immunohistochemistry are consistent with the former.In addition,the examination of paraffin-embedded breast cancer tissue sections from 97 breast cancer patients found that compared with the high-expressing ZBTB4 group,the overall survival rate of the low-expressing ZBTB4 group was worse.2.CCK-8 and scratch experiments showed that overexpression of ZBTB4 significantly inhibited the proliferation and healing ability of breast cancer cells in vitro.Nude mouse experiments showed that overexpression of ZBTB4 inhibited the proliferation of breast cancer cells in vivo.On the contrary,knockdown of ZBTB4 expression relieves this inhibitory effect.These results indicate that ZBTB4 inhibits the progression of breast cancer cells.In addition,by separately detecting the uptake of glucose and glutamine and the content of lactic acid and glutamate,it was found that breast cancer cells with low ZBTB4 expression showed higher glutamine metabolism and glutamine metabolism than breast cancer cells overexpressing ZBTB4 glycolysis rate.3.Western blot experiments and CCK-8 experiments showed that compared with normal breast cancer cells,ZBTB4 was significantly lower expressed in acquired drug-resistant cells,while overexpression of ZBTB4 significantly increased the sensitivity of drug-resistant cells to tamoxifen,And measured the tumor volume and weight in nude mice in in vivo experiments,showing the same results as in vitro experiments.4.Compared with parental breast cancer cells,c-Myc is highly expressed in breast cancer tamoxifen-resistant cells,and ZBTB4 interacts with c-Myc in vivo and in vitro,and up-regulatingthe expression of ZBTB4 in breast cancer resistant cells can be significant Inhibition of c-Myc expression in tamoxifen-resistant cells,and up-regulation of c-Myc expression reversed the inhibitory effect of ZBTB4 on the proliferation and metabolism of breast cancer cells and the sensitivity of breast cancer resistant cells to tamoxifen.Conclusion: ZBTB4 is down-regulated in human breast cancer cells,and it plays an important role in breast cancer cell proliferation,metabolism and chemotherapy resistance.It plays a regulatory role mainly by mediating the c-Myc transcription pathway. |
PDF Full Text Request