| HCC is the most common malignant tumor in liver with a much poor prognosis.Although multiple treatments are available for HCC patients,the survival time is still unsatisfactory.About 350 to 400 thousand of cases of HCC were certified each year in China which cause a heavy burden to the whole society.Hypervascularized is an obvious character of HCC.Abundant vessel network can supply oxygen and nutrients to HCC and result in tumor development and metastasis.Therefor,inhibiton of tomor angiogensis is a key point in clinical treatment and experimental reaserch of HCC.FABP5,also named epidermical fatty acid binding protein,is a member of intracellular lipid protein binding family which plays a role in lipid transport and metabolism.FABP5 can be deteceted in many kinds of tissues and cells.FABP5 could bind with long chain fatty acids and transport them to intracellular space.This process will stimulate transcription of many genes.FABP5 has been found to be a regulator in inflammation reaction and tumoriogenesis.Since,FABP5 could be a target gene in many lipid metabolism related diseases.FABP5 has been shown to be overexpressed in many malignant tomor and promte tumor angiogensis.But little was known about the role ofFABP5 played in tumor angiogenesis.In HCC,FABP5 was detected to be frequently highly expressed,resulted in epithelial mesenchymal transition,and promted tumor development.However,the role of FABP5 in tomor angiogenesis in HCC is still unknown.Our study aims at the role of FABP5 in tumor angiogenesis of HCC and our work can be divided into 3 parts as follows.PART 1 FABP5 IS HIGHLY EXPRESSED IN HCC AND IT IS ASSOCIATED WITH ADVERSE HCC CHARACTERISTICSObjective: To detect the expression of FABP5 in HCC and the correlation of FABP5 and clinicopathological characteristics of HCC.Methods: The protein and mRNA expression levels of FABP5 in matched HCC and ANLTs from 43 patients were determined by immunohistochemical and real-time quantitative PCR,respectively.The correlation of FABP5 mRNA and VEGFA mRNA was detected.According to the immunohistochemical staining of FABP5 in HCC tissues,the patients was divided into FABP5 high expression group and FABP5 low expression group.The clinical data of these two groups was compared.Results: In 43 HCC patients,compared with ANLTs,the expression level of FABP5 mRNA(32/43,74.42%)and VEGFA mRNA(29/43,67.44%)were significantly higher in HCC tissues(p<0.05).And the expression of FABP5 mRNA was positively correlated with intratumoral VEGFA mRNA expression(R=0.6198,P<0.001).Compared with FABP5 low expression group(n=20),BCLC stage was more advaced(p<0.05),pathological differentiation(p<0.05)and microvascular invasion(p<0.05)were worse than those of FABP5 high expression group(n=23).Conclusion: FABP5 is highly expressed and correlates with poor clinicopathological characteristics of HCC.PART 2 THE EFFECTS OF FABP5 CONDITIONED MEDIUM OF HCC CELLS ON ANGIOGENESIS OF HUVECS IN VITROObjective: To detect the effect of FABP5 on the expression of VEGFA in HCC cells and the effect of FABP5 conditioned medium on angiogenesis of HUVECS in vitro.Methods: The expression of FABP5 were inhibited by FABP5 si RNA and increased by r FABP5 in two HCC cell lines,Huh7 and SMMC-7721.The protein and m RNA expression levels of FABP5 and VEGFA were detected by western-blot and qRT-PCR,respectively.FABP5 conditioned medium of HCC cells was collected.Human endothelial cells HUVECS was used to investigate the pro-angiogenic effect of FABP5 by tube formation,CCK8 and transwell migration assays.Results: The expression of VEGFA were down-regulated by FABP5 si RNA and up-regulated by r FABP5 in HCC cells.The number of branches formatted by HUVECs was increased(Huh7: 35.20±4.85 vs.23.00±1.95,p=0.0479;SMMC-7721: 30.00±4.72 vs.18.80±0.86,p<0.05),the cell viability of HUVECs was enhanced(Huh7:p<0.001;SMMC-7721: p<0.05)and the number of migration HUVECs was increased(Huh7:137.00±6.70 vs.95.60±6.74,p=0.0024;SMMC-7721: 100.00±4.22 vs.47.00±7.27,p<0.001)after treating with r FABP5 conditoned medium.On the contrary,all these effects could be decerased after treating with FABP5 si RNA conditioned medium(Number of baranches: Huh7: 13.60±1.54 vs.23.60±1.86,p=0.0032;SMMC-7721: 11.60±1.25 vs.19.00±0.45,p<0.001)(Cell viability: Huh7: p<0.01;SMMC-7721: p<0.001)(Number of migration cells: Huh7: 67.40±2.60 vs.91.60±3.30,p=0.0004;SMMC-7721:18.40±2.84 vs.52.20±2.74,p<0.001).Conclusion: FABP5 could increase the expression of VEGFA in HCC cells and increase angiogenic ability of HUVECs in vitro.PART3 FABP5 ACTIVATES THE IL6/STAT3/VEGFA PATHWAY IN HCCObjective: To detect the potential mechanism of FABP5 promotes tumor angiogenesis in HCC.Methods: The expression levels of FABP5 were inhibited by FABP5 si RNA and increased by r FABP5 in two HCC cell lines,Huh7 and SMMC-7721.The expression levels of FABP5,IL6,STAT3,P-STAT3,and VEGFA were detected by western-blotting.Two pro-angiogenic factors,IL6 and VEGFA,were detected by ELISA assay in FABP5 conditioned medium.Results: The expression of FABP5,IL6 and VEGFA,and phosphorylation of STAT3 were inhibited by FABP5 si RNA.On the contrary,the expression of FABP5,IL6 and VEGFA,and phosphorylation of STAT3 were enhanced by r FABP5.The similar results were found in the secretion of VEGFA and IL6 in HCC cell medium.Conclusion: FABP5 could activate IL6/STAT3/VEGFA pathway and promote the secretion of proangiogenic factors in HCC. |
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