| Background:Hepatocellular carcinoma (HCC) is one of the most common human cancers in the world, particularly in China. Although the improvement of the comprehensive treatment of HCC, the mortality of HCC remains still high. Active angiogenesis and frequent metastasis are two major characteristics of HCC, which obviously lead to poor prognosis. However the mechanism underlying the metastasis and angiogenesis of HCC still remains unclear.MicroRNAs (miRNAs) are a class of highly conserved short RNAs. They regulate diverse cellular processes by binding to the 3’untranslated region (3‘UTR) of target messenger RNAs (mRNAs). Increasing numbers of studies have reported that miRNAs play important roles in HCC development, such as apoptosis, proliferation, autophagy, EMT. Till now, few studies have suggested that miRNAs possess both metastasis and angiogenesis functions in HCC.MiR-126 has been reported to play a vital role in cancer development. But the potential role of miR-126 in HCC metastasis and angiogenesis still remains unclear. Therefore, exploring the relevance of miR-126 with metastasis and angiogenesis in HCC might be a promising strategy for HCC therapy.Aim:The aim of this study is to explore the expression of miR-126 in HCC tissues and cells, and then analyze the correlation of miR-126 with clinicopathological features of HCC patients. Further, we sought to study the functions of miR-126 on metastasis and angiogenesis, as well as its potential mechanism.Methods:1. The expression levels of miR-126 in HCC tissues and cells were detected by RT-PCR.2. MiR-126 mimics and miR-126 inhibitors are used to increase or reduce the level of miR-126 in vitro.3. Transwell assay and capillary tube formation assay were applied to assess the metastasis and angiogenesis.4. Nude mice subcutaneous tumor model was used to perform in vivo study.5. Bio-information Webs and dual-luciferase reporter assay was conducted to confirm the direct binding of miR-126 and target genes.6. IHC staining of CD34 was used to detect the angiogenesis level in vivo.Results:1. In the 70 HCC cases, we observed that the miR-126 expression levels were obviously down-regulated. However no significant correlation between miR-126 and clinicopathological features were found. Compared to the normal liver cells L02 and Changliver, the levels of miR-126 in 6 out of 7 HCC cells were reduced.2. In vitro, we found that the metastasis and angiogenesis abilities were suppressed after miR-126 over-expression and the opposite result was found while miR-126 was inhibited.3. In vivo study with nude mice subcutaneous tumor model showed that the tumors in miR-126 stably over-expression group are smaller.4. LRP6 and PIK3R2 were predicted as the potential targets of miR-126. And this prediction was confirmed by dual-luciferase reporter assay and western-blot assay. In the 70 HCC cases, the level of miR-126 was inversely correlated with LRP6 and PIK3R2.5. After suppression of LRP6 and PIK3R2, the metastasis and angiogenesis were inhibited, respectively. The "rescue" study proved that co-transfection of LRP6 could weaken the effects of miR-126 on metastasis, while the co-transfection of PIK3R2 could attenuated the effects of miR-126 on angiogenesis.6. We found the the level of CD34 was inversely correlated with miR-126 in HCC cases. And the miR-126 over-expression mice tumors showed lower level of CD34.Conclusion:1. Compared to the normal liver tissue and normal liver cells, the level of miR-126 in HCC tissues and HCC cells are significantly reduced.2. MiR-126 inhibits the metastasis and angiogenesis of HCC cells, and suppresses the tumor growth of nude mice.3. LRP6 and PIK3R2 are proved to the target genes of miR-126 in HCC cells and they mediate the effects of miR-126 on metastasis and angiogenesis. |
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