| Objective Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality, with the increase of incidence year by year, and approximately750,000new cases are diagnosed with HCC each year worldwide. Surgical intervention is the most effective method to treat patients with early-stage HCC. However, patients with HCC usually present at advanced stages and do not benefit from surgical resection. Traditional chemotherapy agents, such as doxorubicin and cisplatin, frequently encounter important problems such as low specificity and non-selective biodistribution. Up to now, due to our limited knowledge in molecular pathogenesis of HCC, the outcomes of HCC patients are still dismal. Therefore, studies aiming at the definition of the mechanisms associated with hepatocarcinogenesi are urgently needed. HCC displays marked vascular abnormalities and active metastasis which significantly promote the initiation and progression of HCC. Hence, strategies based on mechanisms of anti-angiogenic and anti-metastasis may offer breakthrough for HCC treatment. MicroRNAs (miRNAs) are evolutionarily conserved, small noncoding RNAs, and they play fundamental roles in various biological processes through regulation of targeting gene expression at the post-transcription level. Alterations of miRNAs expression involves in key cellular processes of tumorigenesis. In our previous study, we searched for the differential expressed miRNAs between cancerous hepatocytes and normal primary human hepatocytes using miRNA expression profiling. MiR-497was one of the aberrantly expressed miRNAs involved in HCC. In this study, we systematically studied the function and the mechanism of miR-497in HCC angiogenesis and metastasis.Methods SYBR Green qRT-PCR was performed to analyze the expression level of miR-497in36paired HCC and adjacent nontumor liver tissues. Next, the expression level of miR-497was further examined in eight human liver cancer cell lines as well as in two human normal liver cell lines. To explore the effect of miR-497on HCC angiogenesis and metastasis, in vitro capillary tube formation, endothelial cell recruitment assays and transwell migration and invasion assays were performed with two HCC cell lines, Huh7and PLC/PRF/5. Then, miRNA target prediction softwares such as miRBase, miRNA.ORG, PicTar and TargetScan were used to predict target genes of miR-497. Next, we applied SYBR Green qRT-PCR, western blot, ELISA and Luciferase assays to validate these predicted genes. Finally, we explored the effect of miR-497on HCC growth and metastasis by using xenograft model in nude mice.Results In comparison with the corresponding control noncancerous tissues, miR-497was downregulated in HCC samples. Consistent with this, significant reduction in the expression of miR-497was observed in cancer cell lines examined compared with normal cell lines. Overexpression of miR-497in HCC cells could not only repressed the ability of HCC cells to promote the migration and capillary tube formation of endothelial cells, but also suppress the migration and invasion abilities of HCC cells. MiR-497suppresses angiogenesis and metastasis of HCC by directly targeting VEGFA and AEG-1. In vivo analysis also indicated that miR-497could significantly inhibit the growth and metastasis of hepatoma xenografts in nude mice.Conclusions Our study indicates that miR-497could suppresse the angiogenesis and metastasis of hepatocellular carcinoma by inhibiting the expression of VEGFA and AEG-1both in vivo and in vitro, which partialy contribute to the molecular mechanism of miR-497in HCC. Furthermore, our study establishes the theoretical foundation for new therapeutic target for HCC treatment using miR-497 and its target genes. |
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