Up-regulation of miR-9 expression predicate advanced clinicopathological features and poor prognosis in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has become the third leading cause of cancer-related deaths globally [1]. The incidence of HCC is still increasing in the developing countries, especially in East and South-East Asia, with the highest reported primary liver cancer rates. Annually, more than 700,000 new cases are diagnosed worldwide and unfortunately more than 600,000 cases dead due to this cancer [2]. The five-year survival rate of this cancer is merely 7% [3]. The development and progression of HCC is a multistage process involving the deregulation of genes that are crucial to cellular processes, such as cell cycle control, cell growth, apoptosis and cell migration. HCC is highly lethal because of its aggressive metastasis and an advanced stage at the time of diagnosis. The occurrence of HCC is a multi-factor and multi-stage process, including both hereditary and environmental factors. Since the diagnosis at early stage of HCC offers the only hope for curative therapies, it is of utmost importance to screen high-risk patients effectively. With the increasing understanding of tumor biology of HCC, recent studies have identified more and more molecular markers with high sensitivity and specificity for diagnosis and prognosis in patients with HCC.

MicroRNA (miRNA) belongs to a class of endogenously expressed, non-coding small RNA and contains about 22 nucleotides, which exhibits a high degree conservation of structure and function in metazoa. They exist in two forms of pre-miRNAs and mature miRNAs, and only the mature miRNAs mediated by the two RNase III endonucleases Dicer and Drosha play a key biological role [4]. The mature miRNAs inhibit protein translation through binding the 3′-untranslated region (3′-UTR) of target mRNA partly, while they induce target mRNA cleavage through binding mRNA with perfect complementarity [5],[6]. At present, approximately 450 miRNAs have been cloned in mammalian cells, and it is believed that up to 1,000 miRNAs genes exist [7],[8]. Moreover, it is estimated that 30% genes of the human genome are regulated by miRNAs [9]. Though the biological functions of a small amount of identified miRNAs are elucidated, miRNAs have been proved to be important for cell growth, differentiation, and apoptosis [10],[11]. miRNAs also regulate oncogenesis because they are both oncogenic and tumor suppressors. miRNAs regulate posttranscriptional expression of target genes involved in any biological processes including development, differentiation, cell proliferation, apoptosis, and the stress response [12]. In addition, miRNAs are involved in cancer development and progression and are differentially expressed in normal tissues and cancers.

MiR-9 is a regulator of neuronal progenitor cell fate during neurogenesis [13],[14], which has recently been implicated in cancer [15]. Although most studies indicate a tumour-suppressor activity for miR-9 in cancer cells [16], conflicting data exist, and the outcome of miR-9 function appears to be tumour specific [17]. miR-9 is downregulated in breast cancer, renal cell carcinoma, and gastric cancer due to promoter methylation [18]-[20]. In contrast, miR-9 has been found to be upregulated in gliomas and in colorectal cancer [21],[22]. Recent studies also report that miR-9 is heterogeneously expressed within a given tissue [23]. miR-9 has also recently been shown to be associated with metastasis formation in several cancer types, such as breast, colon, ovarian, cervix, liver, and gastric cancer [16],[22],[24]-[26]. Recent studies have shown that miR-9 promotes metastasis formation [22],[24],[26], however, in contrast, other studies have suggested that increased expression of miR-9 suppresses metastasis formation [16],[25] and that miR-9 inhibits tumor growth through inhibition of NFkappa B1 [27],[28]. Therefore, miR-9 might have different functions in different cancers. In this study, we investigated the miR-9 expression in HCC tissues to evaluate their value in prognosis of this tumor.

Although with the development of early diagnosis and surgical techniques, the patients with hepatocellular carcinoma still results with a relatively bad prognosis. The molecular biomarkers for early diagnosis and predictor of prognosis are desperately required now. A lot of work has been conducted in an attempt to identify biomarkers with diagnostic and prognostic implications for HCC. MiRNAs have been demonstrated to be critical regulators of carcinogenesis and tumor progression in HCC. The role of miR-9 as a prognostic factor has been recently described in different tumours, such as acute lymphocytic leukemia [30], acute myeloid leukemia [31], and colon cancer [32]. Similarly in the present study, we observed that miR-9 expression was up-regulated in HCC tissues compared with noncancerous liver tissues. The up-regulation of miR-9 in HCC cancer tissues was also significantly correlated with aggressive clinicopathological features. We found that the patients with high miR-9 expression have an advanced tumor staging and are in higher risk of venous infiltration. Moreover, the results of Kaplan–Meier analyses showed that HCC patients with the high miR-9 expression tend to have shorter overall survival and progression free survival. The multivariate analysis clearly indicated that the high miR-9 expression in biopsy samples may be considered as an independent prognostic factor in HCC for decreased survival.

MiR-9 is a regulator of neuronal progenitor cell fate during neurogenesis [13],[14], which has recently been implicated in cancer [15]. Although most studies indicate a tumour-suppressor activity for miR-9 in cancer cells [16], conflicting data exist, and the outcome of miR-9 function appears to be tumour specific [17]. Recent studies also report that miR-9 is heterogeneously expressed within a given tissue [23]. miR-9 is downregulated in breast cancer, renal cell carcinoma, and gastric cancer due to promoter methylation [18]-[20]. The transcriptional activity of miR-9 seems to be related to gene silencing by DNA methylation. Previous studies have demonstrated that the high frequency of hypermethylated CpG islands at miR-9 genes resulted in down-regulation of miR-9 in human cancer [20]. In addition, microRNA-9 is a methylation-silenced tumour suppressor that could be a potential candidate predictive marker for poor prognosis of medulloblastoma. Loss of microRNA-9 may confer a proliferative advantage to tumour cells, and it could possibly contribute to disease pathogenesis [33]. On the other hand, the down-expression of miR-9 may be introduced by hypoxic stress, resulting in alternative splicing shift to proangiogenic isoforms of VEGF165 [34]. However, in contrast, miR-9 has been found to be upregulated in various cancer tissues like HCC, gliomas, and colorectal cancer [21],[22],[35]. Previous study reported miR-9 may possibly promote HCC migration and invasion through regulation of KLF17 [35]. In the present study, the up-expression of the miR-9 demonstrated aggressive clinicopathological features, with the mechanism still unknown. Ma et al. have suggested a regulatory pathway of miR-9 as a metastasis-promoting miRNA [24]. They demonstrated that miR-9 directly targets CDH1, the E-cadherin- encoding mRNA, leading to down-regulation of E-cadherin and increase in cancer cell motility and invasiveness [24]. Besides induction of Epithelial-mesenchymal transition (EMT), miR-9 was found to contribute to tumor angiogenesis, another important mechanism in metastasis, through activation of beta-catenin signaling pathway induced by E-cadherin down-regulation [24]. Recently, they also showed that miR-9 can function as a metastasis-promoting miRNA even in the E-cadherin-negative breast cancer cells through downregulation of leukemia inhibitory factor receptor [36].

In conclusion, our results have demonstrated that the levels of miR-9 are higher in HCC tissues than those in matched normal liver tissues and correlated with disease stage and the presence of venous infiltration. These findings enhance our understanding of the role of miR-9 in HCC progression and suggest that miR-9 may function as microtumor promoter genes in HCC. These findings suggest the potential clinical use of microRNA measurements, particularly in estimating prognosis for patients with HCC. Large well-designed studies with diverse populations and functional evaluations are warranted to confirm and extend our findings. Examining new targets and other biological experiments will clarify the functions and roles of microRNAs in HCC.