Research On The Mechanism Of CXCL8-related Signaling Pathways In Colorectal Cancer Invasion And Metastasis

The incidence rate and mortality rate of colorectal cancer are third and second respectively.Despite the progress in diagnosis and treatment in the past decade,the five-year survival rate is still about 65%,and about 50%of colorectal cancer mortality is related to metastasis.Tumor invasion and metastasis is a complex event,which involves the development process steps of initiating gene sequence,intermolecular multi-factors and multiple stages.In this study,we first investigated that overexpression of CXCL8 activates PI3K/AKT/NF-κB pathway by promoting the phosphorylation of PI3K,AKT and NF-κB in colorectal cancer cells.CXCL8 is the key cytokine in colorectal cancer invasion and metastasis;After CXCL8 interference vector was transfected into SW480 and Caco-2 cells,cell proliferation,migration and invasion were affected.Cell recovery test was conducted with si-cxcl8 and inhibitors to determine whether overexpression of CXCL8 could induce EMT like effect in Colorectal Cancer Cell SW480(Part Ⅰ).Secondly,erlotinib was used to target TrkB and ERK signaling pathways,and experiments were designed to verify the interaction between PI3K/AKT/ERK signaling pathway and erlotinib,so as to weaken the proliferation,invasion and metastasis of colorectal cancer cells(SW480 and Caco-2 cells)induced by CXCL8,and explore the antitumor activity of erlotinib in vitro Study on the signal pathway of inhibiting the growth activity and effect of transplanted tumor in vivo(Part Ⅱ).Thirdly,through transcriptome sequencing and differential expression gene analysis,it is demonstrated that erlotinib can inhibit the difference between Caco-2 cells and control cells under CXCL8 activity.It is analyzed that the functional signal pathways include PI3K/AKT,MAPK and genes closely related to CXCL8,and XIAP gene is selected as the key gene of PPI network,It is speculated that XIAP is also regulated by CXCL8 and participates in the proliferation and invasion of colorectal cancer cells.It is found that the expression level of XIAP mRNA and XIAP protein decreased in Caco-2 cells transfected with si-cxcl8(Part III).Through in vitro and in vivo studies,the mechanism of erlotinib inhibiting colorectal cancer by inhibiting CXCL8 and its mediated multidirectional cell signaling pathway was deeply discussed.This study aims to explore the molecular mechanism of tyrosine kinase inhibitor erlotinib participating in the proliferation and migration of colorectal cancer cells and finally affecting the invasion and metastasis of colorectal cancer by participating in the regulation of multidirectional signal pathways based on chemokine CXCL8:PI3K/AKT/ERK,TrkB and XIAP.Part Ⅰ:CXCL8 induces epithelial mesenchymal transformation of colorectal cancer cells through PI3K/AKT/NF-κB signaling pathway and its effect on invasion and metastasis of colorectal cancer cells[Objectives]To investigate the role of chemokine CXC ligand CXCL8 as one of the key cytokines in inducing EMT to activate PI3K/AKT/NF-κB signaling pathway and its role in promoting the growth and proliferation of colorectal cancer cells.[Methods]Human CRC cell lines SW480 and Caco-2 were cultured in vitro.The vector containing si-cxcl8 was transfected into SW480 cells with liposome 2000 reagent.The expression of CXCL8 was detected by real-time fluorescence quantitative PCR and Western blot.MTT assay,scratch assay and transmembrane assay were used to detect the proliferation,migration and invasion of SW480 and Caco-2 cells transfected with si-cxcl8.The changes of PI3K/Akt/NF-κB pathway protein phosphorylation in SW480 cells transfected with CXCL8 were detected by Western blot.Cells were transfected with si-cxcl8 and si-cxcl8+inhibitor,and the function of CXCL8 was verified by function recovery test.[Results]1.si-CXCL8 Interference can inhibit or reduce the proliferation,migration and invasive activity of colorectal cancer SW480 and Caco-2 cells.CXCL8 may be through PI3K/AKT/NF-κB signal axis induces epithelial mesenchymal transformation and stimulates tumor development.2.The expression levels of CXCL8 mRNA and protein in SW480 and Caco-2 cells transfected with si-cxcl8 were significantly lower than those in control and empty cell control(P<0.05).si-CXCL8 interference transfection reduced the proliferation of SW480 and Caco-2 cells.At 48 hours after transfection,the cell viability of si-cxcl8 SW480 and Caco-2 cells decreased significantly compared with the control group(P<0.05).After 72 and 96 hours of culture,the cell viability of si-CXCL8 SW480 and Caco-2 cells was further improved compared with the control group(P<0.05).3.After CXCL8 transfection,SW480 and Caco-2 cells showed EMT like characteristics.Compared with control cells and empty vector,the expression of E-cadherin decreased,and the expression of N-cadherin,vimentin and α-SMA expression increased.4,CXCL8 activates PI3K/AKT/NF-κB pathway by promoting the phosphorylation of PI3K,Akt and NF-κB.Compared with cells transfected with non-specific siRNA,silencing CXCL8 reduced the phosphorylation of PI3K and NF-κB,while the phosphorylation of AKT was not affected.5.si-CXCL8+inhibitor recovery test restored the proliferation,migration and invasion of SW480 and Caco-2 cells.[Conclusions]Silencing CXCL8 by si-cxcl8 interference inhibited the proliferation,migration and invasion of colorectal cancer SW480 and Caco-2 cells.After transfection with CXCL8,SW480 cells showed EMT like characteristics.CXCL8 may play a role in inducing EMT through PI3K/Akt/NF-κB signal axis.Part Ⅱ:CXCL8-related PI3K/AKT-ERK signaling pathway and the targeting mechanism of Erlotinib on EMT,invasion and metastasis of colorectal cancer cells and their effects on tumor formation in nude mice of human colorectal cancer cells[Objectives]Distant metastasis is the main cause of death in patients with colorectal cancer.Tyrosine receptor kinase B(TrkB)and ERK signals may be potential targets for the treatment of distant metastasis of colorectal cancer.The purpose of this study was to investigate whether erlotinib inhibits the metastasis of colorectal cancer cells by regulating TrkB and ERK signaling pathways under the induction of CXCL8.[Methods]Human colorectal cancer cell lines SW480 and Caco-2 were pretreated with exogenous C-X-C motif chemokine ligand 8(CXCL8)to observe the inhibitory effect of erlotinib on tumor metastasis,including anoikis,epithelial mesenchymal transformation,migration and invasion.To explore the role of TrkB,AKT and ERK in the inhibition of colorectal cancer cell metastasis induced by erlotinib.The apoptosis rate of erlotinib treated tumor cells was measured by flow cytometry,the expression of apoptosis related proteins was measured by WB,and erlotinib inhibited CXCL8 induced EMT in rectal cancer cells.An animal model of Caco-2 cell xenograft tumor was established to verify the inhibitory activity of erlotinib on the growth of transplanted tumor.[Results]1.Compared with CXCL8 alone,the apoptosis rate of SW480 cells and Caco-2 cells treated with CXCL8 plus erlotinib increased significantly.Erlotinib promoted the apoptosis of colorectal cancer cells;Erlotinib may inhibit the migration and invasion of colorectal cancer cells enhanced by CXCL8 by down regulating the levels of TrkB,p-ERK and p-AKT.2.Overexpression of TrkB in colorectal cancer cells attenuated the effects of CXCL8+erlotinib on anoikis apoptosis,epithelial mesenchymal transformation,migration and invasion,and down-regulation of p-ERK and p-Akt.It shows that erlotinib inhibits the migration and invasion of colorectal cancer cells by inhibiting CXCL8 and down regulating TrkB.3.In this study,it was found that the metastasis resistance of colorectal cancer cells induced by erlotinib mainly depended on the inhibition of ERK activation rather than AKT activation.4.Erlotinib has inhibitory activity on the tumorigenesis of colorectal cancer cells in nude mice,and its inhibitory activity is related to TrkB.Compared with the control group,the protein expression level of CXCL8 in the overexpression group increased significantly(P<0.05).[Conclusions]Erlotinib inhibits the anti-apoptosis,EMT,migration and invasion of colorectal cancer cells induced by CXCL8 by inhibiting TrkB dependent ERK signaling pathway.Part Ⅲ:Effect of interaction between CXCL8 and XIAP on proliferation and migration of colon cancer cells based on bioinformatics analysis[Objective]Transcriptome sequencing and genomic analysis were used to compare the differences of gene expression levels between Caco-2 cell lines silencing CXCL8 and Caco-2 cell lines in the control group,and to analyze which genes and pathways were differentially expressed.[Methods]Transcriptome sequencing was performed by stably transfecting sicxcl8 colorectal cancer cell line Caco-2 and control Caco-2 cell lines.The mRNA with significant difference in expression between different sample groups was found through differential expression analysis.The difference of gene expression level between the two groups was compared by using R software limma package.Taking P<0.05 and | log(foldchange)|>0.5 as the standard,the differentially expressed genes were enriched by go and KEGG to find the genes and cell signal pathways related to function.[Results]1.Through transcriptome sequencing analysis,969 differentially expressed genes were screened,including 259 up-regulated genes and 710 down-regulated genes.Go and KEGG enrichment analysis showed that these genes were mainly related to PI3K-Akt,MAPK,RAS signal pathway and cell adhesion molecules.2.Through go and KEGG enrichment analysis of differential genes,it is concluded that there are 23 differential genes involved in PI3K-AKT signaling pathway,including 5 up-regulated genes and 18 down-regulated genes.There are 4 differential genes involved in NF-κB signaling pathway,including 1 up-regulated gene and 3 down-regulated genes.3.The protein interaction of differential genes was studied in string database,and the key differential genes between sicxcl8 Caco-2 group simulating erlotinib treatment effect and control sinc Caco-2 group were deduced:MYC,BCL2L1(gene up regulation)and XIAP(gene down regulation).4.By detecting the expression of XIAP mRNA and protein,the expression levels of XIAP mRNA and XIAP protein in Caco-2 cells transfected with si-cxcl8 decreased significantly(P<0.05).[Conclusion]Through comparative transcriptome sequencing analysis,there were 969 differentially expressed genes in Caco-2 cells compared with normal Caco-2 cells under the condition that si-cxcl8 inhibited CXCL8 expression.Go and KEGG enrichment analysis showed that these genes were mainly related to PI3K-AKT,MAPK,RAS,XIAP signal pathway and cell adhesion molecules.CXCL8 interference resulted in the decrease of XIAP mRNA and protein expression in Caco-2 cells.