Role And Mechanism Of CXCL8/CXCR1 Induced Epithelial Mesenchymal Transition In Human Gastric Cancer Progression,Metastasis And Clinical Significance

Gastric cancer(GC)is one of the most common malignant tumors in humansand remains the third leading cause of cancer death worldwide,following lung and liver cancers.Surgical resection is still the main way to cure early GC.However,the postoperative metastasis seriously affects the prognosis and long-term survival of the patients.In the tumor microenvironment,tumor cells and tumor associated stromal cells can secrete various cytokines which play a crucial role in tumor cell proliferation,stem cell phenotype,invasion and metastasis,chemotherapy resistance and survival signal.There are a large number of cytokines and chemokines in tumor microenvironment,which plays an important role in the survival,invasion and metastasis of tumor cells and the maintenance of survival signals.Chemokines,as one of the important cytokines in tumor microenvironment,play an important role in the growth and metastasis of gastric cancer cells.Interleukin-8(also known as CXCL8)is one of the first chemokines to be discovered,and its biological function is to induce neutrophil chemotaxis to the inflammatory site.CXCL8 hastwo specific cell surface receptors CXCR1 and CXCR2 which belong to the CXCR family.They are mainly expressed in neutrophils,CD8+T cells,CD56+NK cells,vascular endothelial cells,intestinal nerve epidermal cells.When CXCL8 is combined with CXCR1/2,CXCR1/2 activates the downstream cascade signaling pathway by combination with the G protein,andparticipate in local and systemic inflammatory responses.Recent studies have shown that CXCL8 and its receptor CXCR1/2 play an important role in the genesis and development of various tumors.This study aim to elucidate the possible molecular mechanism mediated by CXCL8/CXCR1 signaling axis in GC by transfecting CXCR1 gene specific targeting short hairpin RNA through EMT process,detecting the potential change of proliferation and migration of GC cells and the expression variation of related protein in signaling pathways.Finally,combining the clinicopathological data and survival analysis of patients with GC,to evaluate the value of CXCL8/CXCR1 signal axis in the prognosis of GC patients,and to provide new strategies and theoretical basis for the treatment of GC.Part ? The expression of CXCL8/CXCR1 signal axis in gastric cancercells and its influence on cell growth[Objective] To identify the expression level of CXCR1 in different human GC cell lines and normal human gastric epithelial cell line and interference efficiency of CXCR1-sh RNAs;gastric cancer cells were cultured in vitro to observe the effect of CXCL8 on cell growth.[Methods] Western blotting assay was used to analyze the expression level of CXCR1 in different GC cell lines(MKN45,SGC7901 and MKN28)and normal gastric epithelial cell line GES1;the same method was used to detect the interference efficiency of three CXCR1-sh RNAs.The morphological changes of the cells after CXCL8 induction were observed under microscope,and the effect of CXCL8 on the proliferation of gastric cancer cells was detected by MTT.[Results] Western blot assay showed that the expression level of CXCR1 is significantly higher in different GC cell lines than that in normal gastric epithelial cell line.GC cell line MKN45 presented the highest expression of CXCR1.The CXCR1-sh RNA1 presented the strongest interference efficiency and will be used for subsequent experiments.CXCL8 can induce EMT like changes in gastric cancer cells and promote the proliferation of gastric cancer cells in vitro.[Conclusions] The expression of CXCR1 is significantly increased in GC cells.We successfully selected a CXCR1-sh RNA which presented strongest interference efficiency and could be used for subsequent experiments.CXCL8 can induce EMT like changes in gastric cancer cells and promote the proliferation of gastric cancer cells in vitro.Part ? The effect and molecular mechanism of CXCL8/CXCR1signaling axis on GC cell proliferation,migration and invasion abilitythrough EMT[Objective] To explore the biological behavior changes of GC cell lines caused by CXCL8/CXCR1 signaling axis and possible molecular mechanism.[Methods] The biological behavior changes of GC cells such as proliferation,migration,invasion ability were detected by MTT,colony formation,transwell and wound-healing assays after stimulating by exogenous CXCL8 and/or CXCR1-sh RNA.The morphological changes of the cells were observed under light microscope.Western blotting assay was used to analyze related signaling pathway proteins and EMT related marker changes.[Results] MTT and colony formation results showed that exogenous CXCL8 contribute to the proliferation ability of GC cells,and this effect could be inhibit by CXCR1-sh RNA.Transwell and wound-healing assays showed that exogenous CXCL8 will enhance cell migration and invasion ability which could be interfered by CXCR1-sh RNA.Under light microscopy,CXCL8 can induce EMT like changes in gastric cancer cells,while CXCR1-sh RNA can inhibit this induction process.Western blot assay showed that after treated with CXCL8 the expression of epithelial marker E-cadherin was down-regulated and mesenchymal marker Vimentin was up-regulated as the same time;phosphorylation level of PI3 K,AKTand PTEN is related to CXCL8/CXCR1 signaling axis.After transfection with CXCR1-sh RNA,the phosphorylation level of related signaling pathway proteins were significantly inhibited.[Conclusions] CXCL8/CXCR1 signaling axis could effect the biological behavior of GC cells through EMT process by activating PTEN/PI3K/AKT signaling pathway and promoting survival,invasion and metastasis.Part ? The clinical significance of CXCL8/CXCR1 signaling axis inGC samples[Objective] Discuss sensitive indicators of carcinogenesis,development and metastasis of GC,providing objective basis for early diagnosis,treatment and course monitoring.[Methods] To detect the expression of CXCR1 in GC tissues and matching adjacent normal tissues;to collect patients' clinical data and analyze the correlation between the expression of CXCR1 and clinical pathological data;to analyze 5-year survival rate of 108 GC patients by Logistic regression model.[Results] The positive expression rate of CXCR1 is significantly higher in GC tissues comparing to the corresponding adjacent tissues;further studies found that the expression of CXCR1 has no correlation with age,gender,tumor size,hepatic metastasis,serum CA19-9and CEA(P> 0.05),and significantly related with tumor differentiation(P = 0.010),TNM classification(P = 0.006),lymph node metastasis(P = 0.035).CXCR1 positive patients have lower overall survival rate(P = 0.043)and shorter disease-free survival rate(P = 0.029).[Conclusions] The expression of CXCR1 in gastric cancer tissues is up-regulated,which is significantly related to tumor differentiation,TNM classificationand lymph node metastasis.CXCR1 positive is significantly related to the poor prognosis of patients,and may be one of the indicators of clinical prognosis.