Tumor metastasis is a complex series of steps that involve a number of influential factors. Additionally, due to tumor heterogeneity, the mechanisms underlying distal metastasis could be totally different even though primary tumors possess similar clinical manifestations and histological types. Thus, the advance of detecting biomarker indicative of high-risk tumor metastasis may immensely benefit the approach of personalized cancer treatment. Lately, miRNA has been regarded as an excellent biomarker owing to several unique features: an average 22 nucleotides in length, more stable expression compared with mRNA and more likely to be detected in samples with mRNA and protein degraded. Furthermore, the convenience of synthesizing an overexpressed or an interfering sequence also made miRNA a potential candidate for novel therapeutic strategies.
The expression level of miR-1246 in HCC cells is quite high . Our previous study analyzed the differential expression of miRNAs between Hep11 and Hep12 using microRNA microarray  and discovered differential expression of miR-1246. To define its role, we carried out several biological function assays in different HCC cell lines and found that inhibition of miR-1246 in SMMC7721 and BEL7402 which have relatively high expression levels of miR-1246 significantly reduce migration and invasion while overexpression of miR-1246 had little effects. We speculate that this may be attributed to the abundant expression levels of miR-1246 in these HCC cell lines and that regulation of target genes by miR-1246 may have reached the maximal extent. For Hep11 which has low levels of miR-1246, transfection with miR-1246 mimic could significantly promote migration and invasion. Variation of miR-1246 levels had no effect on HCC cell proliferation.
According to the prediction of biological databases, CADM1 might be a target gene of miR-1246. CADM1, also known as TSLC1, is a well-defined tumor suppressor gene that has been discovered recently. CADM1 gene encodes a 442-amino acid protein which contains an extracellular domain, a transmembrane domain and a cytoplasmic domain. Extracellular domain of CADM1 of 373 amino acids includes three Ig-like C2-type domain connected by disulfide bonds. This structure is also existed in other immunoglobulin superfamily cell adhesion molecule (IgCAM) members, which are refered to as nectins [15–17]. Therefore, CADM1 is considered to be involved in cell-cell interactions. Expression of CADM1 is lost or reduced in a variety of cancers, including non-small cell lung cancer (NSCLC) [18, 19], breast cancer , cervix cancer [21, 22], and HCC [23, 24]. This reduction has been associated with enhanced metastasis potential and poor prognosis. So far, it has been postulated that loss of heterozygosity (LOH) , promoter hypermethylation [18, 19, 24] and miRNA mediated regulation might be mechanisms underlying the loss of CADM1 expression. miR-10b and miR-216a are two microRNAs implicated in regulation of CADM1 in HCC [23, 25]. Li et al. reported that miR-10b enhances tumor invasion and metastasis through targeting CADM1. Moreover, patients with higher miR-10b expression had significantly poorer overall survival.
Although higher expression of miR-1246 has been reported in the serum of tumor carrying mice  and oesophageal squamous cell carcinoma , few studies are available for interpreting miR-1246’s function in tumor. Our study is the first to report miR-1246 could regulate invasion and migration of HCC cell via targeting CADM1. There is no doubt that CADM1 functions as a tumor suppressor gene in HCC [23, 28]. In this study, we also demonstrated that CADM1 RNA interference results in up-regulation of invasion and migration in HCC cell lines. However, the mechanism underlying tumor suppression by CADM1 remains to be fully elucidated.
We confirmed that miR-1246 could promote migration and invasion through CADM1 in HCC cell lines. Whether miR-1246 and CADM1 expression are correlated in tumor tissues is not investigated before. Here, using clinical samples from 38 patients of liver cancer, we analyzed miR-1246 and CADM1 expression by RT-PCR and immunohistochemisty, respectively and found that miR-1246 expression was negatively correlated to CADM1, which was of statistical significance. We also analyzed the influence of multiple factors on DFS such as ECOG score, serum AFP, TNM, pathological differentiation and miR-1246 and CADM1 expression and found only TNM and pathological differentiation were statistically significantly correlated with DFS. In 25 patients who were classified as stage 1 according to TNM, those who were CADM1 positive had long DFS while patients were CADM1 negative had short DFS and the difference was statistically significant. Patients who had high miR-1246 expression had short DFS while those with low miR-1246 expression had long DFS, although the difference was not statistically significant. When we analyze miR-1246 expression, we use the total RNA extracted from the tumor tissues which contain not only epithelial cancer tissues, but also meschymal cancer tissues. Since the proportion of epithelial cancer tissues in tumors differs between patients, analyzing miR-1246 expression in total RNA might influence the results. CADM1 expression is detected by immunohistochemistry which is more accurate because pathologists can directly determine the expression of CADM1 in tumor tissues. In our study, miR-1246 expression is negatively correlated with CADM1. So although the correlation between miR-1246 and DFS is not statistically significant, high miR-1246 expression combined with low CADM1 could still serve as a risk factor for DFS.