Clinicopathological Significance Of Allograft Inflammatory Factor-1 (AIF-1) In Hepatocellular Carcinoma And The Effect Of AIF-1 In Cell Biological Behavior Of Hepatocellular Carcinoma Cells

Hepatocellular carcinoma(HCC)is the main type of primary liver cancer.Although available treatment options for HCC continue to improve,most current therapeutic strategies have only limited efficacy,and the mortality of patients with HCC remains persistently high.Therefore,it is important to continue to investigate new,and potentially more reliable,diagnostic and prognostic biomarkers and the molecular mechanisms involved hepatic carcinogenesis to provide new targets for treatment of patients with HCC.Molecular links between inflammation and carcinogenesis are continuing to be discovered.Inflammatory cytokines involved in liver carcinogenesis and the progression of malignant tumors of the liver include interleukin 1(IL-1),interleukin 6(IL-6),and tumor necrosis factor-alpha(TNF-a).AIF-1 is a inflammation-responsive scaffold protein,which is mainly produced by circulating monocytes and tissue macrophages.Recent studies have shown that AIF-1 participates in the modulation of the inflammatory response in rheumatoid arthritis,inflammatory skin disorders,and systemic sclerosis.Recently,data from several studies have shown that AIF-1 is involved in promoting the proliferation and migration of breast cancer cells in vitro,which may indicate a role for AIF-1 in the progression of malignant tumors.Also,AIF-1 has been shown to be expressed in activated Kupffer cells lining the walls of liver sinusoids.Therefore,we hypothesized that AIF-1 may be involved in the progression of human HCC.However,the role of AIF-1 expression in the development and progression of HCC and the significance of the expression of AIF-1 for patients with HCC remain unknown.The aim of this study was to evaluate the expression of AIF-1 in paired HCC and adjacent non-cancerous liver tissues,using immunohistochemistry and western blotting,and to determine whether AIF-1 expression was correlated with clinicopathological features in patients with HCC.Furthermore,we further evaluated the effect of AIF-1 expression on the cell proliferation and migration in HCC cell lines in vitro.The major results of this study as follows:1.Increased expression of allograft inflammatory factor-1(AIF-1)in human hepatocellular carcinoma(HCC)tissuesTo explore the possible role of allograft inflammatory factor-1(AIF-1)in the progression of hepatocellular carcinoma(HCC),we detected increased expression of AIF-1 protein in HCC in paired tumor tissue and adjacent non-cancerous liver tissues from patients with HCC by immunohistochemistry staining.The expression level of the AIF-1 protein was detected in paired HCC tissues and corresponding adjacent non-cancerous liver tissue by western blot and quantitative real-time polymerase chain reaction.Consistent with the results of immunohistochemistry analysis,HCC tissues expressed significantly increased levels of AIF-1 protein and mRNA when compared with matched non-tumor control tissue.These results indicated that AIF-1 was up regulated in HCC tissues.2.Correlation between AIF-1 expression and clinicopathological characteristics of patients with HCCTo evaluate the clinical significance of AIF-1 expression in HCC,analysis of the correlation between AIF-1 protein expression and clinicopathological patient parameters was performed.There was no significant relationship found between up regulation of AIF-1 expression and patient clinicopathological parameters,including age,gender,hepatitis B surface antigen(HBsAg),serum alpha fetoprotein(AFP)levels,the presence of liver cirrhosis,and tumor encapsulation.However,we found that the AIF-1 expression levels were significantly increased in.the patient group with a large primary liver tumor diameter(diameter>5 cm)and with multiple tumor nodules(number ? 2).Increased expression of AIF-1 was also detected in the group of patients with advanced Barcelona Clinic Liver Cancer(BCLC)stage(stage B and C).Also,elevated AIF-1 expression was associated with the presence of portal vein tumor thrombus(PVTT).These findings suggest that AIF-1 expression may be involved in the progression of human HCC.3.Effect of AIF-1 on the cell proliferation in HCC cell linesTo characterize the functional significance of AIF-1 on cell proliferation,a transfection experiment included a specific short interfering RNA(siRNA),performed to silence endogenous AIF-1 expression.We further investigated the proliferation rate of AIF-1 silenced cells using the Cell Counting Kit-8(CCK-8)assay.Data indicated that the proliferation rate was inhibited in liver cancer cells infected with AIF-1 siRNA when compared with the proliferation rate in control cells.These findings indicated that AIF-1 might have a role in the proliferation of liver cancer cells.4.Effect of AIF-1 on the migration of HCC cell linesThe wound healing assay showed that two liver cancer cell lines transfected with siRNA stimulated reduced wound closure ability,compared with control cells.Furthermore,the trans-well migration assay also showed that AIF-1 silenced liver cancer cells reduced cell migration properties into the lower trans-well chamber when compared with control cells.Taken together,these data suggested that increased expression of AIF-1 may be associated with the metastatic potential of human HCC cells.