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.
Highlights
Hepatocellular carcinoma (HCC) is the sixth most common cancer and third-leading cause of cancer-related death worldwide [1]
After modifying miR-26a expression levels in HepG2 and HCCLM3 cells, we established subclones with stable expression of miR-26a. We named those cells HepG2-wt, HepG2-control (HepG2 transfected with the negative control of precursor miRNA), and HepG2-miR-26a (HepG2 transfected with pre-miR-26a), or HCCLM3-wt, HCCLM3-control (HCCLM3 transfected with the negative control of anti-miRNALNAs), and HCCLM3-anti-miR-26a (HCCLM3 transfected with anti-miRNA-Locked nucleic acids (LNAs) against miR-26a)
We found Conditioned Medium (CM) from HepG2-miR-26a cells inhibited tube forming and migration of human umbilical vein endothelial cells (HUVECs), whereas CM from HCCLM3-anti-miR-26a cells promoted tube forming and migration of HUVECs, when compared with CM from their wild-type cells (Figure S1)
Summary
Hepatocellular carcinoma (HCC) is the sixth most common cancer and third-leading cause of cancer-related death worldwide [1]. MiRNAs down-regulate gene expression by either inducing degradation of target mRNAs or affecting their translation by binding to the 3′ untranslated regions (UTRs) of mRNAs. miRNAs are involved in many biological events, including cell growth, differentiation, apoptosis, fat metabolism, and viral infection [5]. They play an important role in many types of human cancers [6], and aberrant expression of specific miRNA is directly implicated in tumorigenesis including growth, apoptosis, metastasis, and especially angiogenesis [7,8,9,10]. 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). The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors
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