IL-8/CXCR2 System Enhances Migration And Invasion Of Tongue Squamous Cell Carcinoma Via Regulating NF-?B-mediated Epithelial-mesenchymal Transition

Objective: Tongue squamous cell carcinoma(TSCC)is one of the most common incident oral cancers,which is accompanied by high rate of metastasis and recurrence.Although TSCC possesses the highest mortality rate in multiple oral cancers,surgical resection,radiotherapy and chemotherapy are still the most common therapeutic methods in the present situation.The process of epithelial mesenchymal transition(EMT)is essential to the metastasis and recurrence of the tumor.During the EMT progress,the original tight junctions between cells and cells are disturbed,the loosely arranged cells acquire the characteristics of mesenchymal cells,namely enhanced migration and invasion ability.Therefore,blocking the EMT process of tumors has become a hot research topic in the treatment of tongue squamous cell carcinoma.The IL-8 is one of the inflammatory factors participated in the pathogenesis of TSCC.IL-8,also known as CXCL8,is one of vital members in the C-X-C chemokine family,and it is involved in multiple biological functions via binding to cell-surface G protein-coupled receptors,such as CXCR2.Previous studies have found that IL-8 is closely relevant to the inflammatory reaction in a regional tumor microenvironment.Accumulated IL-8 contributes to carcinoma progression by means of accelerating angiogenesis and promoting the migration and invasion capacity,suggesting that IL-8 in the tumor microenvironment may function as a possible biomarker for tumorigenesis.However,whether IL-8/CXCR2 system could promote migration and invasion of TSCC cells by regulating EMT process has yet to be illustrated.NF-?B(nuclear factor-kappa B),also known as nuclear transcription factor,is a group of proteins that can specifically bind to the nucleotide sequences of promoters on certain specific genes in cells to initiate gene transcription and expression functions.Recently,ample studies indicated that activation of NF-?B is an essential requirement for EMT course during tumor metastasis.Recent researches supported that the transcriptional capacity of NF-?B could be reinforced that was accompanied by IL-8 stimulation in a variety of cancer cell lines.Accumulated IL-8 increased the expressions and phosphorylation of Akt,induced the nuclear migration of NF-?B,thereby promoting the process of epithelial mesenchymal transition(EMT)in cells.Notably,the effects of IL-8/CXCR2 system on promoting EMT progress through regulating NF-?B activity in TSCC cell lines have not been well illustrated.Hence,we detected the changes in migration and invasion ability with IL-8 stimulation in human TSCC cell line Tscca and Tca8113.Moreover,in order to investigate the potential mechanisms by which IL-8 involved in the EMT progress,we also silenced IL-8 receptor CXCR2 or inhibited NF-?B activity,and thereby measuring the expressions of EMT-associated proteins,including E-cadherin,N-cadherin and Twist.Methods: 1.For the experiments,the TSCC cells(Tscca and Tca8113)were exposed to 10 ng/mL human recombinant IL-8 for 24 h before the following assays.The cells were randomized into two groups:(a)blank group(The cells without transfection);(b)IL-8 control group(The un-transfected cells treated with 10 ng/mL human recombinant IL-8 for 24 h).The wound healing assay was performed to detected every group TSCC cells migration capacity.The transwell assay was to investigate the invasion ability of every group TSCC cells.Western blotting assay was used to detect the EMT markers' expression levels of every group of TSCC cells(E-cadherin,N-cadherin and Twist).2.CXCR2 were silenced by specific small interfering RNA(siRNA)in human TSCC line Tscca and Tca8113.The cells were randomized into five groups:(a)blank group(The cells without transfection);(b)IL-8 control group(The un-transfected cells treated with 10 ng/mL human recombinant IL-8 for 24 h);(c)IL-8+siControl group(24 hours after siControl transfection,TSCC cells were stimulated with 10 ng/mL IL-8 for 24 h);(d)IL-8+siCXCR2-1 group(24 hours after siCXCR2-1 transfection,TSCC cells were stimulated with 10 ng/mL IL-8 for 24 h);(e)IL-8+siCXCR2-2 group(24 hours after siCXCR2-2 transfection,TSCC cells were stimulated with 10 ng/mL IL-8 for 24 h).Real-time polymerase chain reaction(RT-PCR)analysis was conducted to measure the mRNA expression levels of CXCR2 when CXCR2 were silenced of every group of TSCC cells.Western blotting assay was used to detect CXCR2 expression levels of every group of TSCC cells.The wound healing assay was performed to detected every group TSCC cells migration capacity.The transwell assay was to investigate the invasion ability of every group TSCC cells.The cell immunofluoresence assay was used to detect the expression of NF-?B p65 of every group TSCC cells.3.PDTC treatment,TSCCA and Tca8113 cells were divided into three groups:(a)blank group(Cells without any treatment);(b)IL-8 group(TSCC cells treated with 10 ng/mL IL-8 for 24 h);(c)IL-8+PDTC group(TSCC cells co-treated with 10 ng/mL IL-8 and 100 ?M PDTC(Beyotime,China)for 24 h).The transwell assay was to investigate the invasion ability of TSCC cells after PDTC treatment.Electrophoretic mobility shift assay(EMSA)assay was detected the activity of NF-?B of TSCC cells after PDTC treatment.Western blotting assay was used to detect the expression levels of protein of E-cadherin,N-cadherin and Twist of TSCC cells after PDTC treatment.Results: 1.Would healing assay and transwell assay were conducted to detect the migration and invasion ability of TSCC cells.Stimulation with IL-8 significantly enhanced cell migration of TSCCA and Tca8113 cell lines.Besides,IL-8 treatment increased the number of invasive cells compared with parallel cells.Western blotting assay was performed to evaluate the expression levels of EMT markers in TSCC cells.The data indicated that stimulation with IL-8 down-regulated the E-cadherin,and up-regulated the N-cadherin and Twist.2.Real-time PCR analysis was conducted to measure the mRNA expression levels of CXCR2 when CXCR2 were silenced.the mRNA levels of CXCR2 were observably down-regulated in Tscca and Tca8113 cells after the transfection steps were completed.Likewise,the protein levels of CXCR2 protein were also obviously decreased by Western blot.Using would healing assay and transwell assay,the migration and invasion ability of TSCC cells of transfection with siCXCR2 attenuated the IL-8-mediated increased.3.the expression levels of p65 and p-I?B? in TSCC cells were elevated after IL-8 stimulation,while the expression level of I?B? was decreased.Furthermore,results of immunofluorescence staining indicated that knockdown of CXCR2 attenuated the IL-8-induced translocation of p65 to nucleus.Western blotting assay was performed to evaluate the expression levels of EMT markers in TSCC cells.The data indicated that stimulation with IL-8 down-regulated the E-cadherin,and up-regulated the N-cadherin and Twist.siCXCR2-1 and siCXCR2 reversed IL-8-induced decreased expression level of E-cadherin.The siCXCR2 also attenuated the increased expression of N-cadherin and Twistmediated by IL-8.4.Transwell assay were conducted to detect the migration and ability of TSCC cells after PDTC treatment,the ability of migration of TSCC cells is increased comparallel cells.Through Western blotting assay,PDTC treatment significantly up-regulated the expressions of E-cadherin and down-regulated the expressions of N-cadherin and Twist.Conclusion: 1.IL-8 significantly enhanced cell migration of TSCC(TSCCA and Tca8113)cell lines.Transfection with siCXCR2 attenuated the IL-8-mediated increased migration and invasion ability of TSCC cells.2.Transfection with siCXCR2 decreased the expression level of NF-?B,and down-regulated the E-cadherin,and reversed IL-8-induced decreased expression level of E-cadherin,also attenuated the increased expression of N-cadherin and Twist mediated by IL-8.3.PDTC,an inhibitor of NF-?B,also significantly inhibited the EMT process and metastasis of Tscca and Tca8113 cells.In conclusion,data from present work demonstrates that IL-8/CXCR2 system enhances migration and invasion of tongue squamous cell carcinoma via regulating NF-?B-mediated epithelial-mesenchymal transition(EMT).