| Objectives:Protein arginine methyltransferases play an important role in various biological processes.CARM1 is one of this family,and it has been reported that it promotes the development and metastasis of ERα-positive breast cancer.However,few articles reported the role of CARM1 in triple-negative breast cancer with higher malignancy and poor prognosis.The aim of our research is to explore the molecular mechanism and biological function of CARM1 and HIF1A interaction in triple-negative breast cancer and to study the effect on the regulatory mechanism and expression level of the common downstream target genes.Therefore,new ideas for prevention and treatment for triple-negative breast cancer could be provided.The purpose could be divided specifically into the following points:1.To explore the expression of PRMT family and the effect of each molecule on triple-negative breast cancer.2.To find the effects of CARM1 on the proliferation,invasion,epithelialmesenchymal transition and stemness of triple-negative breast cancer.3.To find the downstream target genes regulated by CARM1 transcription.4.To investigate CARM1 interacting proteins and specific mechanisms of CARM1 and HIF1A transcription regulation in the proliferation,invasion and stemness of triple-negative breast cancer cells.5.To explore the expression of CARM1 in various types of tumors and the relationships between CARM1,HIF1A and CDK4.Method:1.Through the bioinformatic analysis of the PRMT family on GEO and TCGA databases to determine the high expression molecules in triple-negative breast cancer.To study the effect of PRMT family on triple-negative breast cancer through EdU and wound healing experiments,and then determine the expression of CARM1 in breast cancer through immunohistochemical analysis.2.In triple-negative breast cancer cell lines,growth curve,colony formation,transwell,sphere formation experiments,detection of epithelial-mesenchymal transition and stemness markers were carried out to explore the effects of CARM1 on proliferation,invasion,epithelial-mesenchymal transition and stemness.3.The whole genome analysis of CARM1 were carried out by ChIP-seq and the downstream target genes of CARM1 were found.qChIP experiments were performed to verify the results.RNA-seq on CARM1 were performed to find CARM1 target genes,and subsequent RT-PCR were to verify the expression of these genes in mRNA levels.4.Silver staining and mass spectrometry were carried out to find the CARM1 interacting protein and the results were verified by western blotting,immunoprecipitation,and GST pull-down experiments.Meanwhile,the HRE reporter were used to explore the impact of CARM1 on the hypoxic pathway.qChIP and ChIP/Re-ChIP experiments were performed to determine the occupancy of CARM1 and HIF1A on the promoters of downstream target genes under normoxia and hypoxia,and the expression of target genes were detected by western blotting and RT-PCR.In triple-negative breast cancer cell lines,growth curve,transwell and sphere formation experiments were used to explore whether C ARM 1 carcinogenesis effect depends on transcriptional regulation or enzyme activity.The effects of CARM1,HIF1A and CDK4 on the proliferation and invasion of triplenegative breast cancer were further explored through transwell and colony formation experiments.5.Immunohistochemical staining on multi-tumor tissue microarray specimens was performed to explore the expression level of CARM1.The expression of CARM1,HIF1A and CDK4 in the TCGA database were analyzed to explore the relationships among the three.Results:1.Compared with other PRMT family members,CARM1 is highly expressed and promotes the proliferation and invasion of triple-negative breast cancer cells.At the same time,the expression of CARM1 in breast cancer increases with the progression of pathological grade.2.CARM1 promotes the proliferation,invasion,epithelial-mesenchymal transition and stemness of triple-negative breast cancer cells.3.CARM1 occupies the promoters of multiple target genes,which are mainly enriched in metabolism,cell cycle,hypoxia pathway and Wnt pathway,etc.Besides,CARM1 has a certain relationship with lncRNAs.4.CARM1 interacts with HIF1A directly and they play an epigenetic regulatory role as a whole.CARM1’s role of proliferation,invasion and stemness in triple-negative breast cancer depends on its transcriptional regulation and enzyme activity.CARM1 and HIF1A jointly participate in the process of transcriptional regulation as a whole,transactivate the expression of the downstream target gene CDK4,and promote the proliferation and invasion of triple-negative breast cancer.5.Immunohistochemical analysis of various tumor tissues suggests that CARM1 is highly expressed in a variety of cancer tissues,and published clinical data sets show that CARM1,HIF1A and CDK4 are positively correlated in multiple cancer tissues.Conclusion:Our research found that CARM1 directly interacts with HIF1A and they act as an organic whole.By transactivating the expression of CCND1,CDK4,CTNNB1,HIF1A,MALAT1 and SIX1,they regulate the proliferation,invasion,epithelial-mesenchymal transition and sternness of breast cancer cells,and promote the occurrence,development and metastasis of triple-negative breast cancer.Our research shows that CARM1 is an important target and potential biomarker in triple-negative breast cancer,which provides new ideas for the prevention and treatment of triple-negative breast cancer. |
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