Regulation Of COX2 On Metastasis,invasion And Migration Of Osteosarcoma MG63 Cells And Its Molecular Mechanism

ObjectiveTo explore the effects of COX2 on osteosarcoma cell metastasis and EMT based on PI3K/AKT/NOTCH/Wnt axis,and provide molecular basis and potential molecular targets for anti-osteosarcoma drug development.Methods1 The COX2 high expression MG63 cell line was constructed by Ad Easy adenovirus transfection system;and grouped into(1)Blank group: normal cultured MG63 cells without any treatment;(2)plvx-Ds Red group: infected empty vector adenovirus plvx-Ds Red MG63 cells;(3)plvx-COX2-Ds Red group: MG63 cells infected with recombinant adenovirus plvx-COX2-Ds Red;cell immunofluorescence assay to detect whether the vector was successfully transfected;Wetern blotting and q PCR assay for each group of cells Expression of COX2.2 cell grouping:(1)Blank group: normal cultured MG63 cells without any treatment;(2)plvx-Ds Red group: MG63 cells infected with empty vector adenovirus plvx-Ds Red;(3)plvx-COX2-Ds Red group : MG63 cells infected with recombinant adenovirus plvx-COX2-Ds Red;(4)COX2+NS398 group: cells treated with COX2 inhibitor NS398 5μM for 24 h in plvx-COX2-Ds Red group;(5)COX2+EP4A group: plvx-COX2-Cells in the Ds Red group were treated with type 4 prostaglandin E2 receptor antagonist GW627368X(EP4A)at 10 μM for 24 h.MTT assay was used to detect cell proliferation;Transwell cell migration assay was used to detect the migration ability of each group;Western blotting was used to detect the expression of β-Catenin,Snail1,vimentin and E-cadherin in each group;q PCR assay was used to detect vimentin in each group of cells.The expressions of E-cadherin,β-Catenin and Snail1 m RNA were detected by ELISA.The contents of MMP2,MMP9 and VEGF in the supernatant of each group were detected by ELISA.3 cell grouping:(1)Blank group: normal cultured MG63 cells without any treatment;(2)plvx-Ds Red group: MG63 cells infected with empty vector adenovirus plvx-Ds Red;(3)plvx-COX2-Ds Red group Cells: MG63 cells infected with recombinant adenovirus plvx-COX2-Ds Red;(4)COX2+NS398 group: cells treatedwith COX2 inhibitor NS398 5μM for 24 h in plvx-COX2-Ds Red group;(5)COX2+PI3K inhibitor group: plvx-COX2-Ds Red group treated with PI3 K inhibitor for 24h;(6)COX2+Notch inhibitor group: plvx-COX2-Ds Red group treated with Notch inhibitor for 24h;(7)COX2+Wnt inhibitor group Cells: cells treated with Wnt inhibitor for 24 h in plvx-COX2-Ds Red group;cell migration and migration assays were detected by Tranwell cell migration and invasion assay;Elisa assay was used to detect changes in MMP2,MMP9 and VEGF levels in cell supernatant;Wstern blotting assay The changes of β-Catenin,Snail1,Vimentin,E-cadherin,PI3 K,p-PI3 K,AKT,p-AKT,IKK,p-IKK,Notch,Wnt and GSK-3β protein levels were detected in the cells;Western blotting assay for detection of NF-κB(P65),β-Catenin nuclear transcription changes;q PCR assay to detect vimentin,E-cadherin,β-Catenin,Snail1,Notch1,Wnt3 a,Gsk-3β,β-Catenin m RNA content changes.Results1 Compared with the Blank group,the red fluorescence in the plvx-Ds Red group and the plvx-COX2-Ds Red group was significantly enhanced;the COX2 protein and its m RNA in the plvx-Ds Red group were not significantly changed,and the COX2 in the plvx-COX2-Ds Red group was COX2.Both protein and its m RNA were significantly increased(P<0.05).2 Compared with the Blank group,the cell survival rate,migration number,β-Catenin,Snail1,vimentin,E-cadherin protein and m RNA expression in the plvx-Ds Red group and MMP2,MMP9 and VEGF levels in the supernatant were not significant.Sexual difference(P>0.05),cell survival rate,migration cell number,β-Catenin,Snail1,vimentin protein and m RNA expression in the plvx-COX2-Ds Red group and MMP2,MMP9 and VEGF in the supernatant were significantly increased,E-cadherin The protein and m RNA expression levels were significantly decreased(P<0.05).Compared with the plvx-COX2-Ds Red group,COX2+NS398 group,COX2+EP4A group,COX2+PI3K inhibitor group,COX2+Notch inhibitor The cell viability,migration cells,β-Catenin,Snail1,vimentin protein and m RNA expression in the COX2+Wnt inhibitor group and MMP2,MMP9 and VEGF in the supernatant were significantly decreased,and the expression levels of E-cadherin protein and m RNA were significant.The difference was statistically significant(P<0.05).Cell viability,β-Catenin,COX2+NS398 group,COX2+EP4A group,COX2+PI3K inhibitor group,COX2+Notch inhibitor group,COX2+Wnt inhibitor group Snail1,vimentin,E-cadherin protein There was no significant difference in the expression ofm RNA and MMP2,MMP9 and VEGF in the supernatant,and the difference was not statistically significant(P>0.05).3 Compared with the Blank group,there was no significant difference in PI3 K,p-PI3 K,Notch1/2/3,Wnt protein content in the plvx-Ds Red group(P>0.05),and PI3 K in the plvx-COX2-Ds Red group.The contents of p-PI3 K,Notch1/2/3 and Wnt protein increased significantly,the difference was statistically significant(P<0.05).Compared with the cells of plvx-COX2-Ds Red group,PI3 K in COX2+NS398 group and COX2+PI3K inhibitor group The contents of p-PI3 K,Notch1/2/3 and Wnt protein decreased significantly,and the difference was statistically significant(P<0.05).There was no significant difference in the content of PI3 K and p-PI3 K protein in COX2+Notch inhibitor group(P>0.05),Notch1/2/3,Wnt protein content decreased significantly,the difference was statistically significant(P<0.05),COX2+Wnt inhibitor group COX2,PI3 K,Notch protein content did not change significantly(P>0.05)The Wnt protein content decreased significantly,and the difference was statistically significant(P<0.05).4 Compared with the Blank group,there was no significant difference in NFT,p-AKT,IKK,p-IKK,NF-κB protein and Notch1 m RNA and protein content in the cells of the plvx-Ds Red group(P>0.05),RKT,p-AKT,IKK,p-IKK,NF-κB protein and Notch1 m RNA and protein levels in the plvx-COX2-Ds Red group increased significantly,the difference was statistically significant(P<0.05);NKT,p-AKT,IKK,p-IKK,NF in the nucleus were inhibited by the cells in the plvx-COX2-Ds Red group,respectively,compared with the plvx-COX2-Ds Red group.The changes of κB protein and Notch1 m RNA and protein content were significantly decreased,and the difference was statistically significant(P<0.05).There was no significant difference between the two proteins(P>0.05).The above suggests that COX2 promotes Notch expression via the PI3K/AKT/NF-κB pathway and activates the Notch signaling pathway.5 Compared with the Blank group,there were no significant differences in Wnt,GSK-3β and β-Catenin protein levels in the cells of the plvx-Ds Red group(P>0.05);the nuclei of the plvx-COX2-Ds Red group with high COX2 expression The content of β-Catenin in Wnt and nucleus increased significantly,and the content of GSK-3β protein decreased significantly(P<0.05).Compared with plvx-COX2-Ds Red group,plvx-COX2-After the inhibition of COX2 and Notch protein activity in Ds Red group,the content of Wnt and β-Catenin protein in the nucleus of the nucleus decreasedsignificantly,and the content of GSK-3β protein increased significantly,the difference was statistically significant(P<0.05).These suggest that high expression of COX2 in MG63 cells can activate the Wnt/β-Catenin pathway by activating the Notch signaling pathway to promote β-Catenin nucleation and promote the expression of related proteins.Conclusions1 A recombinant MG63 cell line highly expressing COX2 protein was successfully constructed.2 COX2 promotes EMT of MG63 cells by activating PGE2 receptor EP4 and induces MG63 cell metastasis.3 COX2 can promote the migration of MG63 cells by activating PI3K/AKT/NF-κB pathway,Notch1 signaling pathway and Wnt signaling pathway.Inhibition of any of these three pathways can inhibit MG63 cells caused by high COX2 expression.Increased migration capacity.4 COX2,which is highly expressed in MG63 cells,promotes high expression of Notch receptor by activating PI3 K pathway,while activation of Notch protein promotes activation of Wnt signaling pathway,affecting the stability of β-Catenin in MG63 cells,and up-regulated Expression of MMP2,MMP9,VEGF and Vimentin in MG63 cells inhibits E-cadherin protein expression and promotes EM63 cell EMT.