Research On The Effect Of CXCL8/CXCR1 In The Proliferation、invasion And Migration Of Human Hepatocellular Carcinoma

Interleukin-8(IL-8), also known as CXC chemokines 8(CXCL8) is a small molecular secreted inflammatory cytokine which belong to the CXC chemokine family, and participate in mediating the inflammatory response. Recent studies have found that CXCL8 could be produced by monocytes and other cell types such as macrophages, endothelial cells, keratinocytes and tumor cells. Its cognate receptor CXCR1 of the surface membrane is capable to bind CXCL8 which mediate the activation and migration of neutrophils into tissue from peripheral blood, and play a vital role in pathogenesis of tumor by promoting angiogenesis, proliferation, survival and movement of tumor cells. Studies have showed that CXCL8 and its receptor could activate or inhibit proliferation of keratinocyte or endothelial cells, angiogenesis and migration, and participate in repairing tissue damage in wound healing, ischemia-reperfusion injury(RI), psoriasis and cancer. This study aim to elucidate the possible molecular mechanism mediated by CXCL8/CXCR1 signaling axis in hepatocellular carcinoma by transfecting CXCR1 gene specific targeting small interfering RNA, detecting the potential change of proliferation and migration of HCC cells and the expression variation of related protein in signaling pathways. In addition, clinical pathology data was statistical analyzed by meaning of providing new strategies and theoretical basis for the treatment of liver cancer.Part I The identification of expression of CXCR1 and interference efficiency of CXCR1-siRNA【Objective】 To identificate the expression level of CXCR1 in different HCC cell lines and normal liver cell line and interference efficiency of CXCR-siRNAs.【Methods 】 Western blotting assay was used to analyze the expression level of CXCR1 in WRL68 and 5 different HCC cell lines; the same method was used to detect theinterference efficiency of three CXCR1-siRNAs.【 Results 】 Western blot assay showed that the expression level of CXCR1 is significantly higher in 5 different HCC cell lines than that in normal liver cell. HCC cell line HepG2 presented the highest expression of CXCR1. The second CXCR1-siRNA presented the strongest interference efficiency and will be used for subsequent experiments.【Conclusions】 The expression of CXCR1 is significantly increased in HCC cells.We successfully selected a CXCR1-siRNA which presented strongest interference efficiency and could be used for subsequent experiments.Part II The effect and molecular mechanism of CXCL8/CXCR1 signaling axis on HCC cell proliferation, migration and invasion ability【Objective】To explore the biological behavior changes of HCC cell lines caused by CXCL8/CXCR1 signaling axis and possible molecular mechanism.【Methods】 The biological behavior changes of HCC cells such as proliferation,migration, invasion, apoptosis ability were detected by MTT, colony formation, transwell,wounding-healing and flow cytometry assays after stimulating by exogenous CXCL8and/or CXCR1-siRNA. Western blotting assay was used to analyze related signaling pathway proteins changes.【 Results 】 MTT and colony formation assays showed that exogenous CXCL8 contribute to the proliferation ability of HCC cells, and this effect could be inhibit by CXCR1-siRNA. Transwell and wounding-healing assays showed that exogenous CXCL8 will enhance cell migration and invasion ability which could be interfered by CXCR1-siRNA. Flow cytometry and Hochest assay results revealed that CXCR1-siRNA could induce apoptosis of HCC cells. Western blot assay showed that phosphorylation level of PI3 K, Akt and NF-kB is related to CXCL8/CXCR1 signaling axis. After transfection with CXCR1-siRNA, the phosphorylation level of related signaling pathway proteins were significantly inhibited.【Conclusions】 CXCL8/CXCR1 signaling axis could effect the biological behaviorof HCC cells by activating PI3K/Akt/NF-kB signaling pathway and promoting survival,invasion and metastasis.Part III The clinical significance of CXCL8/CXCR1 signaling axis in HCC samples【Objective】Discuss sensitive indicators of hepatocarcinogenesis, development and metastasis of hepatocellular carcinoma, providing objective basis for early diagnosis,treatment and course monitoring.【 Methods 】 To detect the expression of CXCR1 in HCC tissues and matching adjacent normal liver 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 HCC patients by Logistic regression model.【Results 】The positive expression rate of CXCR1 is significantly higher in HCC tissues comparing to the corresponding adjacent tissues; further studies found that the expression of CXCR1 has no correlation with age, gender, virus infection, serum AFP levels, cirrhosis, tumor size and numbers, and capsule integrity(P> 0.05), and significantly related with BCLC stage(P = 0.006), TNM classification(P = 0.017), and vascular invasion(P = 0.035). CXCR1 positive patients have lower overall survival rate(P< 0.05)and shorter disease-free survival rate(P< 0.05).【Conclusions】The proliferation and metastasis have related with tumor angiogenesis,CXCR1 might be a promising indicator of clinical prognosis.