The Mechanism Of Upregualted Tie1 Promoting Cervical Cancer Cell Migration And Invision Through Basigin/MMPs

ObjectiveThe prognosis of locally advanced and metastatic cervical cancer is relatively poor due to the limited ways of treatment,which poses a great threat to women's life and health.With the development of molec?lar targeted therapy,this situation has been improved and alleviated.Our previous studies found that Tie1(Tyrosine kinase with Ig and EGF homology domains 1)was significantly increased in cervical cancer epithelial cells,suggesting that it may play an important role in the malignant progression of cervical cancer.The purpose of this project is to study the expression and function of Tie1 in cervical cancer,to explore the molec?lar mechanisms that affect the malignant biological behavior of cervical cancer,and the possible upstream reg?latory pathways of Tie1,which may provide new clues and theoretical basis for molec?lar treatment of cervical cancer.Methods1.Using the public databases of GEO and TCGA,we analyzed the ch Anges of Tie1 at gene and RNA levels in cervical cancer,and studied their relationship with the survival prognosis of patients.2.Western Blot was used to examine the expression of Tie1 in cervical cancer cell lines SiHa,HeLa,Ca Ski and C33A.Immunofluorescence was applied to confirm the subcell?lar localization of Tie1 in cervical cancer cells.3.We used Lenti-virus to construct cell lines stably overexpressing Tie1 in SiHa and HeLa,namely SiHa^LV-TIE1/Ctrl HeLa^LV-TIE1/Ctrl.Western Blot was used to detect the Tie1 expression.4.In the SiHa^LV-TIE1/Ctrl HeLa^LV-TIE1/Ctrl cells,clony formation assay and Ed U assay were used to assess cell proliferation;the cell stemness was evaluated by sphere formation assay;the migration and invasion ability of cells was detected by Transwell invasionassay and wound healing assay.5.Immunofluorescence was used to examine the expression and subcell?lar localization of phosphorylated Tie1 in cervical cancer.6.Cervical cancer cell lines SiHa and HeLa were stim?lated with recombinant human Angiopoietin-1 protein at different time points.Western Blot was used to detect the level of phosphorylated Tie1 and Tie1.7.We treated the SiHa^LV-TIE1/Ctrl HeLa^LV-TIE1/Ctrl cells with Tie2-specific kinase inhibitors. The Transwell invasion assay and wound healing assay were applied to see whether the increase in cell migration and invasion ability induced by Tie1 upreg?lation co?ld be reversed by the inhibitor.8.Tie1 with FLAG-tagged protein was overexpressed in cervical cancer cell HeLa. FLAG antibody is used for protein immunoprecipitation to p?ll down the protein complex binding to Tie1.Coomassie blue staining and Western Blot were used to verify the efficiency and specificity of immunoprecipitation.Tie1 binding proteins were quantitatively identified by liquid-phase secondary mass spectrometry.Based on mass spectrometry quality control and differential expression analysis,the potential binding proteins of Tie1 were screened for further study.Bioinformatics was used to analyze the pathway enrichment in GO and KEGG databases.9.Immunoprecipitation was used to detect the binding between endogenous Tie1 and Basigin in cervical cancer cell HeLa.10.In the HeLa^LV-TIE1/Ctrl cells,Western Blot was used to detect the expression levels of Basigin and its downstream Matrix Metallo Proteinases(MMPs).11.The HeLa^LV-TIE1/Ctrl cells were treated with Cycloheximide(CHX)at different time points.Western Blot was used to detect the expression of Basigin.The correlation between Tie1 and MMP family at the RNA level was analyzed in the TCGA database.12.We treated the SiHa^LV-TIE1/Ctrl HeLa^LV-TIE1/Ctrl cells with Basigin blocking antibody.The Transwell invasion assay and wound healing assay were used to observe whether the increase in cell migration and invasion ability induced by Tie1 upreg?lation co?ld be reversed by the inhibitor.13.We collected surgical specimens from patients with cervical cancer,and use laser capture microdissection(LCM)to obtain 35 cases of cervical cancer epithelium and 19 cases of normal cervical epithelium.Real-time quantitative PCR was used to detect the level of Tie1 antisense long non-coding RNA(lncRNA)Tie1-AS in 35 cervical cancer epithelial samples.The relationship between lncRNA Tie1-AS levels and clinicopathological characteristics was analyzed by one-way ANOVA.14.We used the normal cervical smear samples as a control,and detected the levels of lncRNA Tie1-AS in three cervical cancer cell lines SiHa,HeLa,C33A and HUVEC by real-time quantitative PCR.15.After separating the nucleus and cytoplasmic components,the levels of lncRNA Tie1-AS were measured to determine their subcell?lar localization.16.Plasmid was used to overexpress lncRNA Tie1-AS in cervical cancer cell lines SiHa and HeLa.Transwell invasion assay and wound healing assay were used to detect the cell invasion and migration.Ed U assay was used to evaluate the cell proliferation.17.HeLa cells were transfected with plasmid or small interfering RNA(si RNA)to overexpress or knockdown lncRNA Tie1-AS respectively.Real-time quantitative PCR was used to detect Tie1 ch Anges at messenger level;Western Blot and immunofluorescence were used to detect Tie1 ch Anges at protein level.18.HeLa cells transfected with plasmid/small interfering RNA(si RNA)were treated with ?-Amanitin at different time points.Tie1 m RNA levels were detected by real-time quantitative PCR.19.Tie1 m RNA levels were detected by real-time quantitative PCR in cervical cancer tissue specimens from LCM,and the correlation between lncRNA Tie1-AS and Tie1 m RNA levels was analyzed.20.In HeLa cells transfected with lncRNA Tie1-AS plasmid,real-time quantitative PCR was used to detect the m RNA levels of six neighbor genes,KDM4A,CDC20,YBX1, EDN2,PRPRF and SLC2A1.21.The m RNA levels of six neighbor genes were measured in cervical cancer tissue specimens from LCM,and the correlation between lncRNA Tie1-AS and neighbor genes m RNA levels was analyzed.22.We used plasmids to sim?ltaneously upreg?late lncRNA Tie1-AS and Tie1 in SiHa and HeLa.Transwell invasion assay and wound healing assay were used to observe whether the decrease in cell invasion and migration induced by lncRNA Tie1-AS upreg ?lation can be reversed by overexpressed Tie1.Res?lts1.Compared to normal cervical epithelium,Tie1 is highly expressed in cervical cancer epithelial tissue.In terms of subcell?lar location,it is mainly distributed in the cell membrane and cytoplasm.Public database analysis showed that high Tie1 expression is significantly correlated with the poor prognosis of cervical cancer.Cell experiments in vitro show that Tie1 can significantly promote the migration and invasion of cervical cancer cells,but has no obvious effect on cell proliferation and stemness.2.Although being a receptor tyrosine kinase,Tie1 exhibits a lower level of phosphorylation in cervical cancer cells.Diifferent form endothelial cells,it does not rely on the classic Tie2 pathway for its cancer-promoting effects. 3.1128 Tie1 binding proteins were identified by mass spectrometry,and 169 target proteins were obtained after differential expression analysis.Overall,Tie1 binding proteins are significantly enriched in biological processes such as energy metabolism, cytoskeleton,protein degradation,and cell adhesion.Consistent with our findings,Tie1 binding proteins are significantly enriched in the positive reg?lation of cell igrationpathway from GO database.In the KEGG database,Tie1 binding proteins are gnificantly enriched in HPV infection and a variety of signaling pathways activated n tumors,such as MAPK and PI3K/AKT pathway.4.There is a direct binding between Tie1 and Basigin,and Tie1 overexpression can reduce the degradation of Basigin,thereby promoting the expression of its downstream gene MMP2 and MMP9.High level of Tie1 enhances cervical cancer cells migration nd invision through the reg?lation of Basigin at protein level.5.The tie1 antisense lncRNA Tie1-AS is lowly expressed in cervical cancer epithelial tissues and is mainly distributed in the nucleus.In vitro cell experiments show that lncRNA Tie1-AS can greatly inhibit the migration and invasion of cervical cancer cells, but has no significant effect on cell proliferation.6.LncRNA Tie1-AS can negatively reg?late Tie1 by reducing its RNA stability.Although lncRNA Tie1-AS is mainly located in the nucleus,it does not significantly reg?late the expression levels of its neighboring genes.7.LncRNA Tie1-AS overexpression weaken cervical cancer cells migration and invision through decreasing Tie1 m RNA stability.ConclusionDecreased levels of lncRNA Tie1-AS in cervical cancer epithelial cells,relieved the inhibition of Tie1,and enhanced the expression of Tie1 in cervical cancer.Tie1 can bind to the membrane protein Basigin,which positively reg?lates its protein level by reducing Basisin degradation,stably enhances the downstream signaling pathway of Basisin and the expression of MMP family proteins,thereby promoting the migration and invasion of cervical cancer cells.In summary,this topic revealed a new mechanism for the activation of the Tie1-Basigin-MMPs pathway mediated by the low-expression lncRNA Tie1-AS to promote the migration and invasion of cervical cancer cells.