|Other Abstract||Aim: To investigate the role of IL-6 involves in regulating hepatocarcinoma angiogenesis and related mechanisms, and hopefully our study can provide new therapeutic strategy for anti-angiogenetic of HCC.
In vivo study, we build mouse HCC xenograft animal model, and overexpressed IL-6 in the xenograft by transfecting plasmid IL-6, first of all, we need to confirm if IL-6 has been successfully overpressed through testing serum IL-6 by enzyme-linked immunosorbent assay (ELISA), after that, we used both RT-PCR and WB to to detect the gene and protein expression level of IL-6 and angiogenesis related factors and correlation rate between them in mouse HCC xenograft. In vitro study, we picked up several strong invation and metastatic HCC cell lines: HUH7, SNU182, SNU423 and PLC/PRF/5, and overexpressed IL-6 in these cells by thansfecting plasmid IL-6, after trasfection has been confirmed, we used pIL-6 conditioned medium (CM) of HCC cell lines to culture human umbilical vein endothelial cells (HUVEC), and observe HUVEC cell viability, cell proliferation, cell migration and angiogenetic function in pIL-6 CM through cell counting, bromodeoxyuridine (BrdU) assay, cell migration assay, and in vitro angiogenesis assay. After overexpression IL-6, we also inhibited IL-6 expression level in HCC cell lines by small interfering RNA (siRNA), and after the transfection has been confirmed, we detected HUVEC cell viability, cell proliferation, cell migration and angiogenetic function in siIL-6 CM through above assays. At last, we generated HUH7 sorafenib resistant cell lines, and test cell viability and IL-6/STAT3 signaling pathway expression level in sorafenib resistant cell lines and normal HUH7 cells.
Results: both gene and protein expression level of IL-6/STAT3 was significantly higher in C57 DEN induced HCC tumor tissue compared with non-tumor. Meanwhile, angiogenetic factors (VEGF, VEGFR-2 and CD31) were also much higher in tumor tissue. According to ELISA and WB results, the correlation rate of IL-6 and VEGF protein expression level in human HCC tumor tissue was 0.928, from IHC results, we can tell that the correlation rate of IL-6 and vascular endothelium marker CD31 in human HCC tumor tissue was 0.951. After mouse HCC xenograft animal model was built up, and plasmid IL-6 was successfully transfected, the weight and the volume of the tumors were both significantly increased compared with control vector transfected tumor. Angiogenetic factors’ gene and protein e...|