Abstract: 【Objective】 The aim of this study was to investigate the effect and mechanism of Runt-associated transcription factor 1 (RUNX1) on epithelial mesenchymal transition (EMT) in bovine mammary epithelial cells (MAC-T) induced by Staphylococcus aureus (S. aureus).【Method】 CCK-8 method was used to detect the effect of different multiplicity of infection of heat-inactivated S. aureus on the viability of MAC-T cells.The morphological changes of MAC-T cells treated by S. aureus were observed by inverted microscope.The expression changes of EMT marker molecules (E-cadherin, N-cadherin, α-SMA and Vimentin), TGF/Smad pathway signal molecules (TGF-β1, Smad2, Smad3 and Smad4)and RUNX1 in MAC-T cells treated by S. aureus were detected by Real-time quantitative PCR and Western blotting.The expression levels of EMT markers, TGF/Smad pathway signaling molecules and RUNX1 were compared before and after treatment with RUNX1 and TGF-β-specific inhibitors.【Result】 After 72 h of treatment in different concentration groups, there was extremely significantly decreased in cell viability compared with the 24 and 48 h (P＜0.01), and the observation by microscope revealed that the cells appeared to float and die at this time.Therefore, the time settings for subsequent observation of cell morphology changes were 12, 24, 36 and 48 h.When MAC-T cells were treated with heat inactivated S. aureus (MOI=100) for 48 h, the morphology of MAC-T cells changed from the typical 'pebble’ shape of epithelial cells to the 'long spindle’ shape of interstitial cells.The results of Real-time quantitative PCR and Western blotting detection showed that, compared with normal cells, the mRNA expression of EMT marker E-cadherin was extremely significantly down-regulated (P＜0.01), and the mRNA expression levels of Vimentin, α-SMA, N-cadherin, TGF-β1, Smad2, Smad3, and Smad4 and RUNX1 were extremely significantly or significantly up-regulated (P＜0.01), and the protein expressions of Vimentin, α-SMA, P-Smad2 and RUNX1 obviously up-regulated.After treatment with the LY2109761 inhibitor, compared to the infection group, the mRNA expression of Smad2, Smad3, Smad4, N-cadherin, Vimentin, α-SMA, and RUNX1 was extremely significantly down-regulated in MAC-T cells in the two inhibitor groups (P＜0.01), E-cadherin mRNA expression was extremely significantly up-regulated (P＜0.01).After treatment with the inhibitor Ro5-3335, there was no significant interstitial cell-like change in the cell morphology of MAC-T cells.Compared to the infection group, the mRNA expression of E-cadherin was extremely significantly up-regulated in the cells of the two inhibitor Ro5-3335 groups (P＜0.01), and N-cadherin, Vimentin, α-SMA (except α-SMA in the 10 μmol/L Ro5-3335 inhibitor group), TGF-β1, Smad2, Smad3 and Smad4 mRNA expression levels were all extremely significantly down-regulated (P＜0.01).【Conclusion】 Heat-inactivated S. aureus treatment of MAC-T cells for 48 h could activate the TGF/Smad pathway to induce EMT changes in MAC-T cells.RUNX1 could regulate MAC-T cells EMT process induced by S. aureus by mediating TGF/Smad pathway.

Key words: Staphylococcus aureus; epithelial mesenchymal transition (EMT); Runt-associated transcription factor 1 (RUNX1)