RUNX1对奶牛乳腺上皮细胞间质转化的调节作用

doi:10.16431/j.cnki.1671-7236.2024.02.007

摘要/Abstract

摘要： 【目的】探究Runt相关转录因子1(RUNX1)对金黄色葡萄球菌(Staphylococcus aureus,S.aureus)诱导的奶牛乳腺上皮细胞(MAC-T)发生上皮细胞间质转化(EMT)的作用及机制。【方法】通过CCK-8法检测不同感染复数的热灭活金黄色葡萄球菌处理后对MAC-T细胞活力的影响;通过倒置显微镜观察经金黄色葡萄球菌处理后MAC-T细胞的形态变化。通过实时荧光定量PCR和Western blotting技术检测金黄色葡萄球菌处理MAC-T细胞后,细胞中EMT标志分子(E钙黏蛋白(E-cadherin)、N钙黏蛋白(N-cadherin)、α平滑肌动蛋白(α-SMA)、波形蛋白(Vimentin))、TGF/Smad通路信号分子(TGF-β1、Smad2、Smad3、Smad4)和RUNX1的表达量变化情况;并比较了分别使用RUNX1和TGF-β特异性抑制剂前后细胞EMT标志分子、TGF/Smad通路信号分子和RUNX1的表达水平变化。【结果】不同浓度热灭活金黄色葡萄球菌处理72 h后,MAC-T细胞活力较24和48 h组均极显著下降(P＜0.01),通过显微镜观察发现此时细胞出现漂浮死亡现象。因此后续观察细胞形态变化时设置时间为12、24、36和48 h。当用感染复数(MOI)为100热灭活金黄色葡萄球菌处理MAC-T 细胞48 h后,细胞形态由上皮细胞典型的"鹅卵石"样转变为间质细胞的"长梭状";实时荧光定量PCR和Western blotting检测结果发现,与正常细胞相比,EMT标志分子E-cadherin mRNA表达量极显著下调(P＜0.01),Vimentin、α-SMA、N-cadherin、TGF-β1、Smad2、Smad3、Smad4和RUNX1 mRNA表达量显著或极显著上调(P＜0.01);且Vimentin、α-SMA、P-Smad2和RUNX1蛋白表达量明显上调。经LY2109761抑制剂处理后,与感染组相比,2个抑制剂组MAC-T细胞中Smad2、Smad3、Smad4、N-cadherin、Vimentin、α-SMA和RUNX1的mRNA表达量均极显著下调(P＜0.01),E-cadherin mRNA表达量极显著上调(P＜0.01);经抑制剂Ro5-3335处理后,MAC-T细胞形态未出现显著的间质细胞样变化;与感染组相比,2个抑制剂Ro5-3335组细胞中E-cadherin的mRNA表达量极显著上调(P＜0.01),N-cadherin、Vimentin、α-SMA(除10 μmol/L Ro5-3335抑制剂组α-SMA外)、TGF-β1、Smad2、Smad3和Smad4的mRNA表达水平均极显著降低(P＜0.01)。【结论】热灭活金黄色葡萄球菌处理MAC-T细胞48 h可激活TGF/Smad通路诱导MAC-T细胞发生EMT变化;RUNX1可通过介导TGF/Smad通路调节金黄色葡萄球菌诱导的MAC-T细胞EMT过程。

关键词: 金黄色葡萄球菌; 上皮细胞间质转化(EMT); Runt相关转录因子1(RUNX1)

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)

中图分类号:

S852.61

王宽, 杨博文, 王菊玉, 邓健明, 许慧, 杨阳, 陈洪剑, 代飞燕, 顾小龙, 曲伟杰, 张立梅. RUNX1对奶牛乳腺上皮细胞间质转化的调节作用[J]. 中国畜牧兽医, 2024, 51(2): 500-512.

WANG Kuan, YANG Bowen, WANG Juyu, DENG Jianming, XU Hui, YANG Yang, CHEN Hongjian, DAI Feiyan, GU Xiaolong, QU Weijie, ZHANG Limei. Regulation of Epithelial Mesenchymal Transition in Mammary Epithelial Cells of Dairy Cow by RUNX1[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 500-512.

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