Elevated levels of miR-99b predicted poor overall survival as well as disease-free survival of patients with HCC. Moreover, miR-99b expression levels correlated with capsule formation

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, mainly due to its high rates of postoperative recurrence and metastasis. Moreover, there is no widely accepted prognostic marker of recurrence. Therefore, the aim of the present study was to determine whether such a marker could be provided by a microRNA (miRNA), since recent evidence indicates that miRNAs are important contributors to the metastatic phenotype. In the present study, we showed that miR-99b was expressed at high levels in tissues of patients with HCC and in cell lines derived from HCCs. Elevated levels of miR-99b predicted poor overall survival as well as disease-free survival of patients with HCC. Moreover, miR-99b expression levels correlated with capsule formation and microvascular invasion, which are required for postoperative recurrence. Overexpression or knockdown of miR-99b expression increased or inhibited, respectively, the metastasis of HCC cells in vitro. Furthermore, using a dual-luciferase assay, we demonstrated that miR-99b inhibited the expression of claudin 11 (CLDN11), a component of tight junction strands by directly targeting the 3'-untranslated region of CLDN11 mRNA. In addition, CLDN11 expression was increased or decreased when miR-99b expression was inhibited or elevated in the HCC cells, respectively. Moreover, the expression of miR-99b was inversely correlated with CLDN11 mRNA or CLDN11 levels in the HCC tissues. These findings suggest that a high level of miR-99b expression is an independent prognostic factor and correlates with poor survival of patients with HCC. Therefore, inhibition of miR-99b expression may serve as a therapeutic approach for inhibiting the metastatic phenotype of HCC. Introduction Hepatocellular carcinoma (HCC) is the fifth most common malignant disease worldwide and is the second leading cause of cancer-related deaths (~700,000 annually) (1-3). Although some patients will benefit from surgery (4), the long-term survival of most is unsatisfactory due to the high incidence of postoperative recurrence and metastasis (5). Unfortunately, effective measures to predict or prevent recurrence and metastasis of HCC are unavailable, mainly since the detailed mechanisms of postoperative metastasis and relapse remain to be determined. Due to the absence of widely accepted nucleic acid and protein markers for predicting the prognosis of HCC, we focused our research on microRNAs (miRNAs), which are highly conserved RNAs, comprised of ~19-22 nucleotides. miRNAs inhibit gene expression at the posttranscriptional level by binding to the 3' untranslated region (3'UTR) of target mRNAs (6). Different miRNAs are involved in the regulation of diverse physiological processes or contribute to pathological processes. Among these miRNAs, more than 50% are encoded by cancer-associated genomic regions or fragile sites (7), indicating that dysregulation of miRNA expression plays important roles in oncogenesis. The epithelial-mesenchymal transition (EMT), which is implicated in pathologies such as organ fibrosis, tumor progression and metastasis (8,9), is an important step in HCC metastasis (10). Among numerous miRNAs, miR-99b, which is encoded by chromosome 19, has attracted our research interest, since it promotes the EMT of normal murine mammary gland cells and increases their ability to migrate (11). Moreover, the expression of miR-99b is upregulated significantly in tumors such as multiple myeloma (12,13) and esophageal cancer (14) and is associated with lymph node metastasis (15). These findings led us to propose the hypothesis that miR-99b functions as an oncogene and contributes to the metastatic phenotype of HCC, although we are unaware of any studies indicating a role of miR-99b in HCC. To test this hypothesis, we conducted experiments to detect the expression of miR-99b in HCC samples. We uncovered compelling evidence that miR-99b may serve as a prognostic marker for overall survival (OS) and disease-free survival (DFS) of patients with HCC. We analyzed the role of miR-99b in promoting the migration and metastasis of HCC cells. miR-99b promotes metastasis of hepatocellular carcinoma through inhibition of claudin 11 expression and may serve as a prognostic marker JIANHUI YANG, XIWU LIU, XIAOHUA YUAN and ZHIMING WANG Department of General Surgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China Received April 22, 2015; Accepted May 18, 2015 DOI: 10.3892/or.2015.4104 Correspondence to: Dr Zhiming Wang, Department of General Surgery, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, P.R. China E-mail: [email protected]