The Role And Mechanism Of LAP3 In IFN-γ Induced Malignant Transformation Of Mammary Epithelial Cells

Background and Objective:Breast cancer is one of the most common malignant tumors in women all over the world,and the cause of cancer has always been a hot and difficult research topic.Inflammation is closely related to cancer,but the mechanism of inflammation-induced cancer still lacks in-depth understanding.The previous research results of our research group found that long-term low-level IFN-γ could promote the malignant transformation of Bovine mammary epithelial cells（BMECs）by inducing arginine depletion,and arginine supplementation could antagonize the malignant transformation of BMECs.Furthermore,it was found by transcriptomics and metabonomics that 3(Leucine aminopeptidase 3（LAP3）was related to arginine depletion induced by IFN-γ.LAP3 is highly expressed in various cancer tissues,which is related to tumor migration,invasion and poor prognosis of patients.However,the relationship between LAP3 and arginine metabolism and its function in malignant transformation of healthy mammary epithelial cells have not been reported.Therefore,this study assumes that LAP3 is related to IFN-γ-induced malignant transformation of BMECs,and reveals the role and mechanism of LAP3 in IFN-γ-induced malignant transformation of mammary epithelial cells from the perspective of cell proliferation and arginine-related enzyme metabolism.Methods:In this study,firstly,the malignant transformation of cow mammary epithelial cells induced by IFN-γ and normal cow mammary epithelial cells MAC-T were selected as research objects,and the effects of LAP3 on the malignant transformation of BMECs were observed by cell proliferation（CCK-8 experiment）,cell migration experiment and flow detection cell cycle experiment.The regulation mechanism of LAP3 on cell proliferation-related proteins and arginine metabolism-related enzymes induced by IFN-γ was verified by q PCR and Western blot experiments.ELISA and immunohistochemistry were used to identify key proteins in clinical samples related to human breast cancer.Results:（1）LAP3 shows high expression in long-term IFN-γ-stimulated BMECs as compared with primary BMECs.LAP3 can participate in the malignant transformation of BMECs.The specific manifestation is that down-regulating the expression of LAP3 in malignant transformed BMECs can inhibit the proliferation of cells,slow down the migration speed of cells,and reduce the proliferation in the S phase of the cell cycle,thus affecting the process of transformation of malignant transformed BMECs from G1 phase to Sphase.（2）Compared with normal MAC-T cells not treated with IFN-γ,LAP3 plays a key role in IFN-mediated malignant transformation cells.When stimulated by IFN-γ,LAP3 has a greater effect on the enzymes related to arginine metabolism.LAP3 3 inhibits ASS1 expression in malignant transformed cells,which in turn limits arginine synthesis.LAP3 3participates in malignant transformation of cells by regulating HDAC2 protein and thus affecting the cell cycle.（3）Further verification by clinical research: The plasma LAP3 content of human breast cancer patients is higher than that of healthy people undergoing physical examination,and the content of ASS1 is lower than that of healthy people undergoing physical examination.In breast cancer tissues,LAP3 shows high expression relative to paracancerous tissues,and ASS1 shows low expression.Conclusion:LAP3 can promote the malignant transformation of BMECs mediated by IFN-γ.The regulation of arginine metabolism-related enzymes leads to arginine depletion in cells and its influence on cell cycle-related proteins is involved in the mechanism of malignant transformation of BMECs.From the perspective of cell function,this study reveals the regulation process of malignant transformation of normal cells induced by chronic inflammation,and provides new ideas and targets for the research of inflammation-related tumor diseases.