The Study Of Histone Demethylase KDM2B Promotes Proliferation Of Triple Negative Breast Cancer

BackgroundBreast cancer originated in the mammary epithelium,is a malignant tumor due to abnormal proliferation and differentiation of cells in the mammary duct and lobular cells.Breast cancer occurs mostly in the female population and is the most common malignancy in women.Breast cancer remains the leading cause of cancer deaths in women,despite the advances in diagnostic techniques and the more elaborate treatment modalities.Breast cancer is a heterogeneous group of tumors that can be subdivided into four molecular subtypes:luminal A,luminal B,HER-2 overexpression and Basal-like,with a worse prognosis of Basal-like subtypes.The Basal-like type is also known as triple negative breast cancer(TNBC)due to a lack of estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor 2(HER2).TNBC accounts for 15%-20%of all types of breast cancer,because of its high metastasis and recurrence rate,there is still no effective treatment.The occurrence and development of breast cancer is a complex process involving many factors.Triple negative breast cancer is a special subtype in breast cancer.The study of molecular markers and functional molecules involved in this process can deepen our understanding of triple negative breast Cancer awareness,and then for triple negative breast cancer patients seeking a new treatment.Epigenetic changes are important aspects of tumorigenesis.Histone demethylase KDM2B,a demethylase of the JMJc family,can methylate H3K36me2 and H3K4me3.It can regulate the Self-renewal ability and involvement in the development and progression of tumors.As a transcriptional regulator,its function and mechanism of action in different types of cells are not the same.This study mainly explores the relationship between KDM2B and triple negative breast cancer and its role in triple negative breast cancer cell proliferation,and then explore its mechanism of regulation,provide a new therapeutic target for the treatment of triple negative breast cancer.Methods1.The expression of KDM2B in breast cancer and its relationship with the survival rate of breast cancer were analyzed in Breast Cancer Integrative Platform database.The KDM2B knocked down triple negative breast cancer cell lines SUM159PT,MDA-MB-231 and KDM2B over-expressed triple negative breast cancer cell line HS578t were constructed as follow-up experimental cell lines.2.The effects of KDM2B on the proliferation of triple negative breast cancer cells were examined by CCK-8 assay and by the plate cloning assay.The effects of KDM2B on the cell cycle progression were detected by cell cycle assay.3.Real-time PCR was used to screen the proliferation related genes that could be regulated by KDM2B and the related proteins were detected by Western blotting in triple negative breast cancer cells.4.Chromatin immunoprecipitation to explore the relationship between KDM2B and related gene promoter in triple negative breast cancer cells and its mechanism of transcription.Dual luciferase reporter gene assay further determined the direct effect of KDM2B and related gene promoters.5.Rescue test by proliferation and cell cycle experiments further confirmed that the related genes were essential on KDM2B regulated triple negative breast cancer cell proliferation.6.The experimental data were validated independently for at least three independent replicates,and the significance was analyzed using the SPSS t-test.P values<0.05 were considered significant.Flow results were processed using FlowJo and histograms and line graphs were generated using GraphPad Prism version 5.0.Results1.KDM2B expression is significantly associated with poor prognosis in triple negative breast cancer patients.KDM2B is highly expressed in breast cancer,and its expression is higher in triple-negative breast cancer than in non-triple-negative breast cancer.The high level of KDM2B is associated with poor prognosis of triple negative breast cancer.Thus,KDM2B is an oncogenic factor that plays an important role in the development of triple negative breast cancer.Therefore,we want to explore the effect of KDM2B on the function of triple negative breast cancer cells.The expression of KDM2B in different breast cancer cell lines was detected by Western blot.There were significant differences in the expression of KDM2B in different breast cancer cells.We knocked down KDM2B highly expressed triple negative breast cancer cell HS578t and overexpressed triple negative breast cancer cells SUM159PT or MDA-MB-231.2.KDM2B promoted cell proliferation in triple negative breast cancer cells.The results of CCK-8 assay and plate clone assay showed that KDM2B overexpression significantly promoted the proliferation of triple negative breast cancer cells.In order to explore the ways in which KDM2B affects the proliferation of triple negative breast cancer cells,over-expression KDM2B in HS578t reduced the proportion of cells in G0/G1 phase and increased cells in S phase and G2/M phase Ratio,indicating that KDM2B promotes the cell cycle progression by promoting the transition from GO/G1 phase to S phase and G2/M phase in triple negative breast cancer cells.3.KDM2B promoted triple negative breast cancer cell proliferation by repressing p151NK4B,p16INK4A and p57 KIP2 transcription.Screening of proliferation-related genes affected by KDM2B by real-time fluorescence quantitative PCR found that overexpression of KDM2B could reduce the transcriptional level of cyclin-dependent kinase inhibitors p15INK4B,p16INK4A and p57KIP2.After knockdown of KDM2B expression,p15INK4B,p16INK4A and p57KIP2 Transcriptional level increased.In addition,we detected the protein expression level of p15I5K4B、P16INK4A和 P57KIP2 by Western blot and found that the protein level was consistent with the change of transcription level,indicating that KDM2B affects the cell cycle progression by down-regulating the transcription ofp15INK4B,p16INK4A and p57KIP2.4.KDM2B repressed p15INK4B,p16IN4A and p57KIP2 transcription through binding promoters and reducing H3K4me3 and H3K36me2 level.Chromatin immunoprecipitation experiments showed that KDM2B inhibits gene transcription by binding to specific promoter regions of p15INKB,p16INK4A and p57KIP2,and reducing the methylation level of H3K4me3 and H3K36me2 in the promoter region of these genes.To further prove the regulatory mechanism of KDM2B on p15INK4B,p16INK4A and p57KIP2 promoters,the p15INK4B,p16INK4A and p57KIP2 promoters were inserted into the dual luciferase reporter gene,and it was found that over-expression of KDM2B significantly inhibited the transcription of the luciferase reporter gene Activity,indicating that KDM2B inhibits transcription of p15INK4B,p16INK4A and p57KIP2 by direct binding to the promoter region of these genes.5.p15INK4B,p16INK4A and p57KIP2 is essential for KDM2B induced cell proliferation in triple negative breast cancer cells.In the recovery experiment,p15INK4B,p16INK4A and p57KIP2 were silenced in KDM2B knockdown triple negative breast cancer cell,and cell proliferation experiments showed that the proliferation of triple negative breast cancer cells was enhanced after the silencing of p15INK4B,p16INK4A and p57KIP2 expression.In addition,cell cycle experiments showed that silencing p15INK4B,p16INK4A cand p57KIP2 decreased the cell proportion in GO/G1 phase and caused an increase in the proportion of G2/M phase cells,indicating that silencing p15IN4B,p16INK4A and p57KIP2 could restore the effect of KDM2B on the cell cycle of triple negative breast cancer cells,promote the proliferation of triple negative breast cancer cells.It is further demonstrated that the effect of KDM2B on the proliferation of triple negative breast cancer cells is achieved by regulating the transcription of p15INK4B,p16INK4A and p57KIP2.ConclusionsAs an oncogene,histone demethylase KDM2B plays an important role in the proliferation of triple negative breast cancer cells.It inhibits the transcription of p15INK4B,p16INK4A and p57KIP2 by down-regulating the levels of H3K4me3 and H3K36me2 and further promoting the proliferation of triple negative breast cancer cells.It may become Potential target for the treatment of triple negative breast cancer.Thus,our study uncovered a novel regulatory mechanism for triple negative breast cancer proliferation that deepens our understanding of triple negative breast cancer and offers a new treatment for triple negative breast cancer patients.