STX2 Promotes Colorectal Cancer Invasion And Metastasis By Activating NF-?B Signaling Pathway

BACKGROUND AND OBJECTIVE:Metastatic progression is the main cause leading to poor prognosis of colorectal cancer(CRC)patients.It is necessary to explore the mechanism of invasion and metastasis of CRC.STX2 belongs to highly conserved syntaxin protein family and functions in the transportion and secretion of intracellular vesicles.However,the relationship between STX2 and CRC,particularly the relationship between STX2 and the metastatic progression of CRC,and the regulation mechanisms of STX2 remain unclear.Therefore,in this study we will illustrate the function and associated molecular mechanisms of STX2 in the invasion and metastasis of CRC.METHODS:1.The public databases,qRT-PCR,Western blot and IHC were used to detect STX2 expression in CRC.Then,the relationship between the expression of STX2 and the clinical pathology parameters and the clinical prognosis were explored;2.The migratory and metastastic abilities of CRC cells were measured by soft agar colony formation assay,Wound healing assay,Transwell migration assay,Matrigel invasion assay,three-dimensional cell culture and surgical orthotropic implantation;3.GEO databases were used to analyze the enrichment of related signaling pathways in CRC with STX2 high expression.4.Dual luciferase reporter assay,Western blot,Nuclear and cytoplasmic separation experiment,Immunofluorescence technique and ELISA were used to verify the regulation of NF-?B signaling pathway by STX2;5.Liquid chromatography-tandem mass spectrometry(LC-MS-MS)was used to analyze the STX2 associated proteins.The interaction of STX2 and TRAF6 protein was further verified by CO-IP and IF;6.qRT-PCR and Western blot were used to verify the regulation of TRAF6 by STX2;7.Dual luciferase reporter assay was used to verify the inhibition of NF-?B signaling activity by TRAF6 siRNA and PDTC;The CRC cell lines with STX2 overexpression and TRAF6 shRNA knockdown were constructed;Cell-independent proliferation was measured by soft agar colony formation assay;the invasive,migratory and metastastic abilities were measured by Transwell migration assay,Matrigel invasion assay,3-D culture and surgical orthotropic implantation;8.The expression of STX2 and TRAF6 in CRC tissues were detected by qRT-PCR,Western blot and IHC.qRT-PCR was performed to detect the expression of STX2,TNFa and IL6 mRNA in CRC tissues;9.The binding site of NF-?B P50 on the STX2 promoter was measured by ChIP assay.RESULTS:1.The expression of STX2 was upregulated in CRC;there were positive correlations between STX2 expression and the clinicopathological parameters and the clinical prognosis of CRC patients(p<0.05);2.STX2 overexpression promoted the invasion and metastasis of CRC(p<0.05);STX2 knockdown inhibited the invasion and metastasis of CRC(p<0.01);3.NF-?B signaling pathway was enriched in GEO databases with STX2 high expression.4.NF-?B signaling pathway could be regulated by STX2;5.There were co-expression of STX2 and TRAF6;6.The expression of TRAF6 could be regulated by STX2(p<0.01);7.Inhibition of TRAF6 could abrogate the activation of NF-?B mediated by STX2(p<0.01);Inhibition of TRAF6 attenuated the invasive and metastatic potential of(?)STX2-overexpressing CRC cells(p<0.01);8.STX2 expression was positively correlated with the expression levels of TRAF6,TNFa and IL6(p<0.05);9.The results from CHIP assay identified that there was binding site of NF-?B P50 in the promoter of STX2.CONCLUSIONS:1.The expression of STX2 is upregulated in CRC;The upregulation of STX2 promotes the invasion and metastasis of CRC;2.STX2 regulates the activity of NF-?B signaling pathway by targeting TRAF6 and influnces the invasion and metastasis of CRC;3.The activition of NF-?B signaling pathway drives the transcription of STX2;4.STX2 activates NF-?B signaling pathway and forms a positive feedback loop in CRC;The study provides a possible target for the treatment and prognosis of CRC.