MUS81 Inhibits Breast Cancer Progression Through NPM1-Mediated CDKN2A Stabilization And Expression

ObjectiveMethyl methanesulfonate(MMS)and ultraviolet(UV)sensitive gene 81(MUS81),a family member of xeroderma pigmentosum group F-complementing protein(XPF),is a structural specific endonuclease.MUS81 plays an important role in the recognition and repair of DNA damage and the maintenance of chromosome stability.There have been studies using mouse models to prove that the MUS81 gene has strong tumor suppressor function,but no studies have reported whether MUS81 has a similar effect in breast cancer.Therefore,this study aims to clarify the expression of MUS81 gene and its coded product in breast cancer tissues and cells,explore the biological function of Mus81 in breast cancer,and study whether the gene is involved in the process of proliferation,invasion and migration of breast cancer and its possible mechanism,so as to provide theoretical basis for the treatment of breast cancer.MethodsThe expression of MUS81 in breast cancer and its influence on the prognosis of breast cancer were predicted through bioinformatics.The expression of MUS81 m RNA and protein in breast cancer tissues and cell lines was verified by real-time fluorescent quantitative PCR(RT-q PCR)and Western Blot.Human breast cancer cell lines were transfected with MUS81 overexpression lentivirus(MUS81)or MUS81 interference lentivirus(MUS81 RNAi).CCK8 and plate clone formation assays were used to detect the proliferation of transfected cells;Transwell and scratch assays were used to detect the invasion and migration of transfected cells.Breast cancer cells stably overexpressing MUS81 were inoculated subcutaneously under the armpit of nude mice to establish subcutaneous tumor model of nude mice and evaluate the effect of MUS81 on the proliferation of breast cancer.The expression of MUS81 and Ki-67 in subcutaneous tumors were detected by immunohistochemistry.The interaction proteins of MUS81 were predicted by mass spectrometry and Bio GRID website.The nucleophosmin(NPM1)binding to MUS81 was identified by Co-IP.After transfection of MUS81 or MUS81 RNAi into breast cancer cells,the localization and expression of NPM1 were detected by immunofluorescence and Western Blot.The bioinformatics website and Co-IP were used to screen out the protein CDKN2 A that interacts with NPM1,and the co-localization of NPM1 and CDKN2 A in breast cancer cells was detected by immunofluorescence.The expression of CDKN2 A and the co-localization of NPM1 and CDKN2 A in breast cancer cells were detected by immunofluorescence.Overexpression or knockdown of MUS81 in breast cancer cells,Co-IP and Western Blot experiments to detect the interaction of NPM1 and CDKN2 A in cells.CCK8 assay,plate clone formation assay and Transwell assay were used to detect the proliferation and invasion of breast cancer cells transfected with MUS81(Vector)or MUS81RNAi(RNAi NC)and co-transfected with si-NPM1(si-NC)or NPM1(Vector).The expression of NPM1 protein in breast cancer cells was detected by western blot,and the website was used to predict the effect of NPM1 on the prognosis of breast cancer.After overexpression or knockdown of NPM1,CCK8 assay and plate clone formation assay detected cell proliferation ability;Transwell and scratch assays detected cell invasion and migration ability.ResultsMUS81 is low expressed in breast cancer tissues and cells,and its low expression is related to poor prognosis of breast cancer.In vitro experiments show that MUS81 can inhibit breast cancer cell proliferation,invasion and migration;in vivo experiments show that overexpression of MUS81 can inhibit breast cancer proliferation.Mechanistically,Mus81 binds to NPM1 to promote its physical interaction with CDKN2 A,while NPM1 stabilizes CDKN2 A by inhibiting proteasome mediated degradation,thus promoting the expression of CDKN2 A.Knockdown or overexpression of NPM1 can rescue the changes in breast cancer cell proliferation and invasion ability induced by changes in MUS81 expression.NPM1 is low expressed in breast cancer cells,and its low expression is associated with poor prognosis of breast cancer.Cell experiments show that NPM1 can inhibit the proliferation,invasion and migration of breast cancer cells.ConclusionMUS81 is low expressed in breast cancer,and it plays an important biological function in breast cancer.MUS81 inhibits the proliferation and metastasis of breast cancer by promoting the stabilization and expression of CDKN2 A mediated by NPM1,thereby affecting the progression of breast cancer.