| As a common disease in humans and animals,breast tumors are mostly malignant clinically,and their morbidity and mortality increase year by year,seriously threatening the life and health of humans and animals.Mastitis is an important disease that seriously restricts the dairy breeding and dairy industries worldwide.Epithelial-mesenchymal transformation(EMT)is the important cause of breast tumor cell migration,invasion,and breast epithelial cell fibrosis.However,the exact nmechanism of action of EMT in breast cancer and breast fibrosis is unclear.The first section was designed to investigate the molecular details of TGF-β1/Smad3 and IL-6/JAK1/STAT3 pathways-mediated epithelialmesenchymal transformation in mouse breast tumor cells,to analyze the role and molecular mechanism of 6-bromo-indigo-3’-oxime(6BIO)in intervening in epithelial-mesenchymal transformation of breast tumor cells,so as to provide a new strategy for the treatment of breast cancer in the clinic.The second part of the study was designed to preliminarily explore the mechanism of lipopolysaccharide(LPS)promoting TGF-β1 induced epithelial interstitial transformation in bovine mammary epithelial cells,and provide a new direction for the clinical development of drugs for the prevention and control of mastitis.q PCR,transwell,colony forming assay,immunofluorescence,wound healing assay,siRNA interference techniques,overexpression vector construction,western blot,flow cytometry and other experimental methods were used for verification.The specific results of the study are as follows:1.Study on the mechanism of TGF-β1 induced epithelial-mesenchymal transformation in mouse breast tumor cellsStimulation of mouse breast tumor cells with 10 ng/m L TGF-β1 for 60 h,the m RNA expression of of epithelial cell marker E-cadherin reduced significantly(p < 0.001),and the m RNA expressions of mesenchymal cell markers MMP9,Fibronectin,α-SMA and Snail1(p < 0.001)increased prominently.Morphologically,TGF-β1 promotes the transition of4T1 cells from paving stone-like to slender morphology in a dose-dependent manner.At the protein level,the expression of E-cadherin(p < 0.001)decreased notably,and the expression of MMP9(p < 0.001)and P-Smad3(p < 0.05)increased significantly.Compared with the unhandled blank control group,the percentage migration of 4T1 cells in the TGF-β1 group with wound healing assay increased significantly(p < 0.001),the number of invaded(p < 0.01)and migrated(p < 0.05)in the TGF-β1 group increased significantly in the Transwell assay.Compared with the blank control group,the DNA replication activity of 4T1 cells in the TGF-β1 group was remarkably raised(p < 0.01),and the number of cell clones was significantly increased(p < 0.001).In addition,TGF-β1 can prominently promote the expression of apoptotic protein PARP and Bcl-2(p < 0.001),inhibit the expression of apoptotic protein Caspase 3(p < 0.01),and reduce the apoptosis rate(p <0.001).2.Study on the mechanism of IL-6 induced epithelial-mesenchymal transformation in mouse breast tumor cellsAt the m RNA and protein levels,TGF-β1 can promote IL-6 secretion,but IL-6 cannot upregulate TGF-β1 expression,and IL-6 is presumed to be located downstream of TGF-β1.Stimulation of mouse breast tumor cells with 25 ng/m L IL-6 for 60 h,the m RNA expression of of E-cadherin reduced significantly(p < 0.001),and the m RNA expression of Fibronectin(p < 0.05),Snail1(p < 0.001),Zeb2(p < 0.001)and Twist1(p < 0.001)increased strikingly.With regard to cell morphology,IL-6 promotes the transformation of 4T1 cells from paving stone-like to slender morphology.At the protein level,the expression of E-cadherin decreased significantly(p < 0.01),and the expression of MMP9,Vimentin,α-SMA and PSTAT3 increased notably(p < 0.01).Compared with the untreated blank control group,the percentage migration of 4T1 cells in the IL-6 group of wound healing assay increased notably(p < 0.001),the number of invaded(p < 0.001)and migrated(p < 0.01)cells in the IL-6 group increased significantly in the Transwell assay.Compared with the blank control group,the DNA replication activity of 4T1 cells in the IL-6 group was conspicuously enhanced(P < 0.01),and the number of cell clones was remarkably increased(p < 0.05).Moreover,IL-6 can significantly promote the expression of apoptotic protein PARP(p <0.01),inhibit the expression of apoptotic protein Caspase 3(p < 0.01),and reduce the apoptosis rate(p < 0.01).3.Study on the mechanism of 6BIO inhibition of TGF-β1 induced epithelial mesenchymal transformation in 4T1 cellsWe found that 25-1000 n M 6BIO did not affect the viability of 4T1 cells through CCK-8 detection.Under the action of 5 n M 6BIO,the m RNA expression of E-cadherin significantly increased(p < 0.05),MMP9(p < 0.01),Fibronectin(p <0.05),N-cadherin(p< 0.01),Snail1(p < 0.05),α-SMA(p < 0.01)and IL-6(p < 0.001)m RNA expression decreased notably.In terms of cell morphology,6BIO can inhibit the transition of TGF-β1-induced 4T1 cells from paving stone-like to slender morphology in a dose-dependently manner.At the protein level,the expression of E-cadherin(p < 0.01)raised remarkably,and the expression of Vimentin(p < 0.01),IL-6(p < 0.001)and P-Smad3(p < 0.001)decreased prominently after the action of 6BIO.Compared with the TGF-β1 group,the migration percentage of 4T1 cells in the 6BIO and TGF-β1 co-treated group of wound healing assay decreased significantly(p < 0.001),the number of invaded(p < 0.001)and migrated(p <0.001)cells in the 6BIO and TGF-β1 co-treated group increased significantly in the Transwell assay.Compared with the blank control group,the DNA replication activity of4T1 cells in the 6BIO-treated group was observably decreased(p < 0.05),and the number of cell clones was remarkably reduced(p < 0.05).Furthermore,6BIO significantly raised the apoptosis rate of 4T1 cells(p < 0.01).4.Study on the mechanism of IL-6-mediated TGF-β1/Smad pathway regulating epithelial-mesenchymal transitionSmall interfering RNA(SiRNA)was used to knockdown the IL-6 gene in 4T1 cells.Under the action of Si IL-6,the m RNA expression of E-cadherin increased significantly(p< 0.05),and the m RNA expression of Fibronectin and MMP9 decreased prominently(p <0.01).At the protein level,the expression of E-cadherin increased significantly,and the expression of IL-6(p < 0.001),P-STAT3(p < 0.001),Snail1(p < 0.001),Vimentin(p <0.01)and P-Smad3(p < 0.01)decreased notably after Si IL-6 treatment.Compared with the TGF-β1 group,the migration percentage of 4T1 cells in the Si IL-6 and TGF-β1 cotreated group of wound healing assay decreased remarkably(p < 0.001),the number of invaded(p < 0.001)and migrated(p < 0.001)cells in the Si IL-6 and TGF-β1 co-treated groups of Transwell assay decreased prominently.Compared with the blank control group,the DNA replication activity of 4T1 cells in the Si IL-6 group was remarkly reduced(p <0.01),and the number of cell clones was significantly decreased(p < 0.001).Overexpression IL-6 gene in 4T1 cells,and the m RNA expression of Fibronectin(p <0.01),MMP9(p < 0.001),and Snail1(p < 0.01)increased notably.At the protein level,the expression of IL-6(p < 0.001),P-STAT3(p < 0.001),Fibronectin(p < 0.05),Vimentin(p< 0.001)and P-Smad3(p < 0.001)increased prominently.Compared with the TGF-β1group,the migration percentage of 4T1 cells in the co-treated group of OE IL-6 and TGF-β1 in wound healing assay increased significantly(p < 0.001).The number of invaded(p <0.001)and migrated cells(p < 0.001)in the OE IL-6 and TGF-β1 co-treated group of the Transwell assay reduced prominently.Compared with the blank control group,the DNA replication activity of 4T1 cells in the Si IL-6 group was significantly inhibited(p < 0.001),and the number of cell clones was significantly promoted(p < 0.001).5.Study on the mechanism of 6BIO-targeted IL-6 inhibition of TGF-β1-induced epithelial interstitial transformationAt the protein level,TGF-β1 and OE IL-6 alone and in combination can inhibit the expression of E-caherin,promote the expression of Vimentin and P-Smad3.Moreover,TGF-β1 cowork with OE IL-6 has a more significant effect on the induction of epithelialmesenchymal transformation.6BIO and Si IL-6 promoted the expression of E-cadherin,inhibited Vimentin and P-Smad3 expression,6BIO cowork with Si IL-6 has a more remarkable effect on the inhibition of epithelial-mesenchymal transformation.Compared with TGF-β1 and 6BIO co-treated groups,E-cadherin expression(p < 0.001),Vimentin(p< 0.001)and P-Smad3(p < 0.05)expressions in TGF-β1+6BIO+Si IL-6 group were prominently decreased,E-cadherin expression in TGF-β1+6BIO+OE IL-6 group inhibited significantly(p < 0.05),Vimentin(p < 0.001)and P-Smad3(p < 0.001)expressions in TGF-β1+6BIO+OE IL-6 group were significantly increased.6.Research mechanism of lipopolysaccharide promoting TGF-β1-induced epithelial-mesenchymal transformation in BMECsStimulation of bovine mammary epithelial cells by 15 ng/m L TGF-β1 for 48 h,the m RNA expression of E-cadherin decreased significantly(p < 0.05),and the m RNA expression of N-cadherin,MMP9 and Zeb1 promoted notably(p<0.05).Morphologically,TGF-β1 promotes the transformation of BMECs from paving stone-like to slender morphology.Compared with the TGF-β1 group,the expressions of MMP9(p<0.01),Snail1(p<0.01)and Zeb1(p<0.05)were prominentl increased in the LPS and TGF-β1co-treated groups.Knockdowning the MMP9 gene of BMECs,compared with the TGF-β1group,the expressions of N-cadherin,MMP9 and Snail1 in the Si MMP9 and TGF-β1 cotreated groups were remarkably increased(p<0.05).Compared with the LPS group,the expressions of MMP9 and Snail1 in the Si MMP9 and LPS co-treated groups were significantly increased(p<0.05).In conclusion,this study is the first to explore the mechanism of TGF-β1 and IL-6induced epithelial-mesenchymal transformation in mouse breast tumor cells,to parse the molecular details of 6-bromo-indigo-3’-oxime inhibition of TGF-β1-induced epithelial mesenchymal transformation in 4T1 cells,to preliminary clarify the mechanism of IL-6-mediated TGF-β1/Smad pathway regulating epithelial-mesenchymal transformation,and to prove that 6-bromo-indigo-3’-oxime targeted IL-6 to inhibit TGF-β1 induced epithelial mesenchymal transformation,and further explored the biological phenomenon and mechanism of epithelial-mesenchymal transformation in bovine mammary epithelial cells. |
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