PI3K/AKT/mTOR/NF-?B Signaling Pathway Regulates The Mechanism Of MMP9 Expression In BMECs

The mammary gland is a dynamic developmental organ,and its morphological structure and physiological function will be periodically remodeled with the development of the body under the fine regulation of extracellular matrix(ECM).The process of mammary g land remodeling is of great significance to improve milk yield.Matrix metalloproteinase 9(MMP9)is a member of the matrix metalloproteinase(MMP)family.MMP9 is considered to be a key enzyme in periodic mammary gland remodeling.There are many studies on MMP9 gene in breast cancer.Studies have shown that the expression of MMP9 can be regulated by signal pathways that regulate important metabolic processes in the mammary gland,but the mechanism of PI3K/AKT/mTOR/NF-?B signal pathway regulating MMP9 expression has not been reported,and there is a lack of related research in bovine.In order to explore the mechanism of PI3K/AKT/mTOR/NF-?B signal pathway regulating the expression of MMP9 in bovine mammary epithelial cells(BMECs),this study first confirmed the expression and localization of MMP9 in bovine mammary epithelial cells,and then used BMECs experimental materials.LY-294002,Rapamycin or Celastrol were added to BMECs to detect the expression of MMP9 and related signal pathway proteins by Western b lotting.Then P65 overexpression vector and MMP9 promoter dual luciferase reporter vectors with different length were constructed.The changes of p-P65,P65 and MMP9 proteins during P65 overexpression were detected by Western blotting,the promoter activity was detected by dual luciferase experiment,the core promoter region of MMP9 gene was determined,and Celastrol was added to confirm the effect of P65 on promoter activity.Finally,cell scratch test was used to detect the migration of BMECs after treatment with different signal pathway inhibitors.The results are as follows:(1)The results of confocal laser scanning showed that MMP9 was mainly located on the basal side of mammary gland and expressed in acinar epithelial cells during lactation,and the expression of MMP9 in mammary gland tissue during lactation was significantly higher than that in puberty mammary gland tissue(P< 0.01).(2)After adding different signal pathway inhibitors to BMECs,LY-294002 significantly decreased the expression of p-AKT(P< 0.05),p-mTOR(P< 0.01),MMP9(P< 0.05)and nuclear P65(P< 0.01)protein,and up-regulated the expression of P65(P< 0.01)protein in cytoplasm.Rapamycin significantly decreased the expression of p-mTOR(P< 0.01),MMP9(P< 0.05)and nuclear P65(P< 0.01),and up-regulated the expression of P65(P< 0.01)in cytoplasm,while Celastrol significantly decreased the expression of MMP9(P< 0.01)and nuclear P65(P< 0.01),and up-regulated the expression of P65(P< 0.01)in cytoplasm.(3)The expression of p-P65(P< 0.05),P65(P< 0.01)and MMP9(P< 0.05)proteins could significantly increase after treatment with P65 overexpression,.The results of dual luciferase assay showed that the binding site of transcription factor NF-?B on MMP9 promoter of bovine was in-420?-80 bp region,and the activity of MMP9 promoter of bovine was significantly decreased by adding NF-?B inhibitor.(4)BMECs scratch test showed that the addition of three inhibitors could up-regulate the expression of E-Cadherin(P< 0.01)protein and inhibit cell migration.In conclusion,MMP9 is involved in the regulation of mammary lactation in dairy cows;PI3K/AKT/mTOR/NF-?B signaling pathway can regulate the transcription of MMP9,and then regulate the expression of E-Cadherin,and affecting the migration of BMECs.