Effect Of Methyltransferase PRMT5 On Stabilizing USP15 To Promote The Occurrence And Development Of Breast Cancer

ObjectiveTo explore the mechanism by which protein arginine methyltransferase 5(PRMT5)up-regulates the expression of ubiquitin-specific protease 15(USP15)through methylation modification,regulates breast cancer cell proliferation,epithelial to mesenchymal transition(EMT)and other biological processes,and promotes the occurrence and development of breast cancer.MethodsUsing public databases such as The Cancer Genome Atlas(TCGA)database,Gene Expression Profiling Interactive Analysis(GEPIA2)online database,and Kaplan-Meier Plotter survival curve analysis,to explore the expression difference of PRMT5 in tumor tissue and normal tissue and its relationship with clinical prognosis of breast cancer patients.The expression difference of PRMT5 in normal breast cell line and different types of breast cancer cell lines was detected.Using immunoaffinity purification and silver staining combined with mass spectrometry analysis,the substrate protein USP15 combined with PRMT5 was obtained.Design PRMT5 truncated mutants,in vitro protein affinity purified PRMT5 truncated mutants,and in vitro transcribed and translated USP15 for GST pulldown experiment,and found the site where the substrate protein USP15 directly bound to PRMT5.PRMT5 was overexpressed or knocked down in MCF-7 cell by lentiviral stabilizing technology,and the expression of related proteins in cell was detected by quantitative real-time PCR(qRT-PCR)and Western blotting technology.In methylation Co-immunoprecipitation(Co-IP)experiment and protein half-life(Cycloheximide,CHX)experiment were used to verify the methylation modification of the substrate protein USP15 by PRMT5.USP15 was knocked down or overexpressed by small interfering RNA(siRNA)or plasmid transfection technology,and the expression of related protein F-box and WD repeat domain containing 7(FBXW7)in cells was detected by qRT-PCR and Western blotting technology.The effects of FBXW7 on the proliferation and invasion of breast cancer cells were detected by growth curve analysis,Colony formation,Transwell assay,and Western blotting assay.After overexpressing USP15 in MCF-7 cell,Cell counting kit-8(CCK8),Colony formation,Wound healing and Transwell experiments were used to detect the effect of USP15 on the proliferation and invasion of breast cancer cells.After the PRMT5 arginine methylase activity was inhibited by PRMT5 small molecule inhibitor EPZ015666(GSK3235025),Western blotting was used to detect the expression changes of related proteins in the cells.CCK8 and Transwell assays were used to detect the effects on the proliferation and invasion of breast cancer cells.ResultsBy analyzing the breast cancer data in the public database and detecting the expression of PRMT5 in breast cancer cell lines,it was found that PRMT5 is highly expressed in breast cancer tissues and breast cancer cell lines.Immunoaffinity purification and silver staining combined with mass spectrometry and verified by Co-IP experiment showed that PRMT5 and USP15 were mutually bound,and GST pulldown experiment confirmed that the C-terminus of PRMT5 directly bound to USP15.In breast cancer cell MCF-7,overexpression of PRMT5 significantly increased the protein expression level of USP15,but did not affect the mRNA level of USP15,and knocking down PRMT5 had the opposite result.Through methylation Co-IP experiment,PRMT5 enzyme activity mutation and protein half-life experiment found that PRMT5 up-regulated the protein expression level of USP15 in a manner dependent on methyltransferase activity.In MCF-7 cell,overexpression of USP15 can deubiquitinate histone H2BK120ub,thereby reducing the mRNA and protein levels of FBXW7.Through cell function experiments and detection of EMT-related markers,it was found that FBXW7 inhibited the proliferation and invasive ability of breast cancer cells,while USP15 promoted the proliferation and invasion of breast cancer.The small molecule inhibitor EPZ015666 reduced the protein level of USP15 by inhibiting the methyltransferase activity of PRMT5,thereby weakening the proliferation and invasion ability of breast cancer cells.ConclusionIn this study,we found that the expression of PRMT5 was increased in breast cancer tissues and cell lines.PRMT5 interacted with USP15 in breast cancer cells and upregulated USP15 protein levels in a methyltransferase activity-dependent manner.The increase of USP15 protein level can catalyze the deubiquitination modification of histone H2BK120ub,inhibit the transcriptional activation of FBXW7 gene,enhance the proliferation and invasion ability of breast cancer cells,and then promote the occurrence and development of breast cancer.The above findings of this study suggested that PRMT5 is a potential diagnostic and therapeutic target for breast cancer.