No abstract

Background & AimsmicroRNAs (miRNAs) have been reported to regulate angiogenesis by down-regulating the expression of pro-angiogenic or anti-angiogenic factors. The aims of this study were to investigate whether miR-26a inhibited angiogenesis by down-regulating vascular endothelial growth factor A (VEGFA) and its clinical relevance in hepatocellular carcinoma (HCC).MethodsThe expression of miR-26a was modified in HepG2 and HCCLM3 cell lines respectively, and a panel of angiogenic factors was measured by real-time PCR in the cells. A luciferase reporter assay was used to validate the target gene of miR-26a. Specific inhibitors of signal transduction pathway and siRNA approaches were used to explore the regulatory mechanism of miR-26a. Migration and tube forming assays were conducted to show the changes of angiogenesis induced by miR-26a and its target genes. Finally animal studies were used to further validate those findings.ResultsEctopic expression of miR-26a exhibited decreased levels of VEGFA in HepG2 cells. Migration and tube forming of human umbilical vein endothelial cells (HUVECs) were decreased in the conditioned medium from ectopic expression of miR-26a in HepG2 cells compared to control HepG2 cells. The pro-angiogenic effects of the conditioned medium of HepG2 cells on HUVECs were specifically decreased by LY294002, YC-1, and bevacizumab. Integrated analysis disclosed PIK3C2α as a downstream target gene of miR-26a. Ectopic expression of miR-26a suppressed ectopic and orthotopic tumor growth and vascularity in nude mice. The results in HCCLM3 were consistent with those in HepG2. miR-26a expression was inversely correlated with VEGFA expression in HCC patients.ConclusionsmiR-26a modulated angiogenesis of HCC through the PIK3C2α/Akt/HIF-1α/VEGFA pathway. The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors.

Our previous study has found that the abundance of peritumoral CD68+ macrophages was associated with poor prognosis in hepatocellular carcinoma (HCC) after resection. However, CD68 staining could not discriminate the protumoral or tumoricidal subpopulations from pan-macrophages. CD163 is a marker of alternatively activated macrophages. In this study, the clinical significance of CD163+ cells in tumors and peritumoral liver tissues was evaluated in a cohort of 295 patients with HCC after curative resection. We found that the density of CD163+ cells was well correlated with that of CD68+ cells in both tumors and peritumoral liver tissues but was much more. Immunostaining on consecutive sections and flow cytometry assay on surgical resected specimens further supported the findings that the CD163+ cells was more abundant than CD68+ cells. The density of peritumoral CD68+ cells was associated with poor recurrence-free survival (RFS) and poor overall survival (OS) (P = 0.004 and P = 0.001, respectively), whereas the CD163+ cells have no prognostic values either in tumors or in peritumoral liver tissues. In another cohort of 107 HCC patients, preoperative plasma concentration of soluble form of CD163 (sCD163) was associated with active hepatitis-related factors but not associated with the markers of tumor invasion. In conclusion, both the CD163+ cells local infiltration and plasma sCD163 were of limited significance in HCC, and they were more likely markers related to active hepatitis rather than tumor progression.