| Canine mammary gland tumor,a common tumor disease in female dogs,is a good model for human mammary gland tumor research.In the diagnosis and treatment of canine mammary gland tumors,the role of cytokines has been paid more and more attention.Transforming growth factorβ1(TGF-β1),a multifunctional cytokine,plays a variety of roles in tumors.It may play a role in inhibiting cancer by promoting apoptosis and cell cycle arrest before or at the early stage of cancer.It may also play a role in promoting cancer by promoting cell invasion and migration,tumor angiogenesis,and immune escape at the late stage of cancer.It is one of the hotspots of human oncology research.However,there are few studies on canine mammary gland tumors.Similar to the role of TGF-β1,autophagy can inhibit cell carcinogenesis by eliminating aging and mutating organelles before tumor occurrence,but it can also provide nutrients for cells and promote the development of cancer after cell carcinogenesis.Therefore,good use of autophagy plays an important role in the treatment of tumors.However,the effect of TGF-β1 on autophagy on canine mammary gland tumors is not clear.In this study,the m RNA and protein expressions of TGF-β1 in canine mammary gland tumor tissue were detected first.In the subsequent cell experiment,canine mammary gland tumor cell line,CHMm,were treated with different concentrations of TGF-β1,and the CCK-8 method and plate clone formation experiment were used to detect the effect of TGF-β1 on cell proliferation activity.Through the MDC staining test,the effect of TGF-β1 on the autophagy of CHMm cells was observed.Then,the scratch healing test and Transwell chamber test were used to determine the effects of TGF-β1 and inhibiting TGF-β1-induced autophagy on the invasion and migration of CHMm cells.Finally,the effects of TGF-β1 on the expression levels of autophagy-related proteins,epithelial-to-mesenchymal transition(EMT)-related proteins,TGF/SMAD pathway,and MEK/ERK pathway were analyzed by immunofluorescence assay and Western blot.The results showed that the expression levels of TGF-β1 m RNA and protein in malignant canine mammary gland tumors were significantly higher than those in benign mammary gland tumors and normal adjacent tissues.In vitro experiments showed that TGF-β1 could promote the proliferation and colony formation of CHMm cells.MDC staining showed that the fluorescence intensity of cells in the TGF-β1 treatment group was significantly enhanced,while the fluorescence intensity was weakened when the cells were treated with Baf A1 and TGF-β1.The scratch healing test and Transwell chamber test showed that TGF-β1 could significantly promote the invasion and migration ability of CHMm cells,while the invasion and migration ability decreased significantly after the addition of autophagy inhibitor.In immunofluorescence test and Western blot test,TGF-β1 can promote the expression of autophagy-related proteins and EMT-related proteins,promote the phosphorylation of SMAD2 and SMAD3,activate TGF/SMAD pathway,promote the phosphorylation of MEK1/2 and ERK1/2,and activate MEK/ERK pathway.Meanwhile,autophagy inhibitor Baf A1 can alleviate the promoting effect of TGF-β1.To verify the effect of autophagy on the above pathways,CHMm cells were treated with autophagy promoter RAPA and autophagy inhibitor Baf A1,respectively.The results showed that the promotion or inhibition of autophagy could not change the expression of autocrine TGF-β1,but could promote the phosphorylation of SMAD2 and SMAD3,and also activate the MEK/ERK pathway.In conclusion,TGF-β1 could activate TGF/SMAD pathway and MEK/ERK pathway to induce autophagy in canine mammary gland cancer CHMm cells.Autophagy induced by TGF-β1 in canine mammary gland tumor CHMm could promote cell proliferation,invasion,migration,and EMT. |
