The Effect And Mechanism Of H3K27me3 On The Stemness Maintenance Of Breast Cancer Stem Cell

Background and objectiveBreast cancer is the top of incidence rate cancer among Chinese females.With the advancement of early diagnosis techniques and standardized comprehensive treatment strategies,the treatment of breast cancer has made great progress.However,the recurrence and metastasis of breast cancer remains a major clinical problem.Tumor heterogeneity is considered to be an important factor in breast cancer recurrence and metastasis.Cancer stem cell theory has been considered to be one of the important causes of tumor heterogeneity.Intriguingly,previous studies shows that H3K27me3 plays an important role in the stemness maintenance of both prostate cancer and ovarian cancer stem cells,nevertheless,its role in BCSCs is still scarce.Based on the above hypotheses and research results,we conjectured whether H3K27me3 also plays an important role in the stemness maintenance of BCSCs.Subsequently,we focused on the role of H3K27me3 in the stemness maintenance of BCSCs,to explore the difference in the global protein levels of H3K27me3 among different molecular subtypes of breast cancer cell lines and BCSCs enriched mammosphere,and the mechanism of epigenetic modification in maintaining the stemness of BCSCs.What's more,owing to improve the survival rate and life quality of the recurrent and metastatic breast cancer,looking for a promising agent targeting BCSCs is emergency.Methods1.BCSCs enriched culture and identification?1?Using serum-free medium to induce BCSCs enriched mammosphere in MDA-MB-231 and MCF7 cell lines.?2?Western blotting and RT-QPCR were used to detect the expression of BCSCs stemness markers in MDA-MB-231 and MCF7 breast cancer mammospheres from translational and transcriptional levels.2.comparing the different Protein level of H3K27me3 in breast cancer adherent cells and their suspension microspheresThe protein levels of H3K27me3 in MCF7 and MDA-MB-231 adherent cells and their suspension microspheres were detected by Western blotting.3.The role of H3K27me3 in the stemness maintenance of BCSCs?1?The effect of H3K27me3 inhibitor GSKJ4 on the proliferation of breast cancer adherent cells.The cell viability of MDA-MB-231 and MCF7 adherent cells treated with gradient concentration of GSKJ4 was detected by CCK8 cytotoxicity assay,and the optimal concentration was screened.?2?The effect of H3K27me3 on self-renewal ability of BCSCs..MCF7,MDA-MB-231 adherent cells were treated with 6uM and 20uM GSKJ4for 72 hours,respectively,and the control group was set up.Subsequently,the two group of cell lines were plated in 6-well plate for colony formation experiments.After14 days,Comparing the difference of the number of clones formed between the control group and the experimental group of MCF7 and MDA-MB-231 cell lines.MCF7 and MDA-MB-231 breast cancer mammospheres enriched cultured for 5days were treated with 6uM and 20uM GSKJ4 for 72 hours,respectively,and the control group was set.Then,the difference in the number of mammospheres formed between the experimental group and the control group was statistically compared.?3?The effect of H3K27me3 on the proportion of BCSCs subsets in breast cancer adherent cells.6uM and 20uM GSKJ4 were treated with MCF7 and MDA-MB-231 adherent cells for 72 hours,respectively,and the control group was set up.Subsequently,the MCF7 and MDA-MB-231 cell lines in the experimental group and the control were detected by flow cytometry.The proportion of CD44⁺CD24^-BCSCs in breast cancer cell lines.6uM and 20uM GSKJ4 were treated with MCF7 and MDA-MB-231 adherent cells for 72 hours,and the control was set up.Subsequently,the MCF7 and MDA-MB-231 cell lines in the experimental group and the control were detected by flow cytometry.The difference in the proportion of ALDH1⁺BCSCs subsets in breast cancer cell lines.?4?The effect of H3K27me3 on tumoneogenicity of nude miceMDA-MB-231 adherent cells were treated with 20uM GSKJ4 for 72 hours,and the control was set up.Subsequently,the tumor formation models of nude mice were constructed by using these two groups of cells,and the volume and weight tumor formed of the two groups were supervised regularly.4.Mechanism of stemness maintenance of H3K27me3 in BCSCs?1?The protein and mRNA levels of JMJD3 and UTX in MCF7 and MDA-MB-231 adherent cells and suspension cells were detected by Western blotting and RT-QPCR,respectively.?2?Western blotting and RT-QPCR were used to detect the protein and mRNA expression levels of Nanog,Sox2,Oct4,JMJD3 and UTX in GSKJ4 treated and non-treated MCF7 and MDA-MB-231 adherent cells,respectively.?3?The function of JMJD3 and UTX was silenced by siRNA transient transfection technique,siNC was used as the control,and the protein and mRNA expression levels of JMJD3,UTX,Nanog,Sox2,Oct4 and the protein level of H3K27me3 in the two groups were detected.Result:1.Compared with breast cancer adherent cells,the protein level of H3K27me3decreased in suspension microspheres.2.GSKJ4 can effectively inhibit the proliferation of breast cancer cells?P<0.05?,and the IC₅₀ of MDA-MB-231 is significantly higher than that of MCF7?P<0.05?.3.GSKJ4 can reduce the colony formation rate?P<0.05?,the proportion of stem-like cell subsets in breast cancer adherent cells?P<0.05?of MCF7 and MDA-MB-231 adherent cells and the tumorigenic ability of nude mice of MDA-MB-231 adherent cells?weight P<0.05,volume P<0.01?.4.Compared with adherent cells of breast cancer,the protein?P<0.05?and mRNA?P<0.05?expression level of UTX and JMJD3 increased significantly in breast cancer suspension microspheres.5.GSKJ4 can reduce the protein and mRNA expression levels of JMJD3?P<0.05?,UTX?P<0.01?,and increase the protein level of H3K27me3 expression in MCF7 and MDA-MB-231 adherent cells.6.After silencing the function of JMJD3 and UTX,the protein level of H3K27me3 increased,and the protein and mRNA expression levels of JMJD3,UTX,Nanog,Sox2,Oct4 decreased significantly in MCF7 and MDA-MB-231 adherent cells?P<0.05?.Conclusion:1.To enhence the protein level of H3K27me3 can effectively inhibit the self-renewal ability and stemness maintenance of BCSCs.2.GSKJ4,H3K27me3 demethylation inhibitor,affects the stemness maintenance of BCSCs by inhibiting the activity of JMJD3 and UTX demethylase.3.GSKJ4 is expected to become a targeted promising biologic agent for BCSCs.