MiR-486-5p Suppresses Proliferation and Migration of Hepatocellular Carcinoma Cells through Downregulation of the E3 Ubiquitin Ligase CBL

MicroRNAs have been broadly implicated in cancer, but precise functions and mechanisms in carcinogenesis vary among cancer types and in many cases remain poorly understood. Hepatocellular carcinoma (HCC) is among the most frequent and lethal cancers. The aim of the present study was to investigate the role of miR-486-5p in HCC and identify its specific target. MiR-486-5p was significantly downregulated in HCC tissues and cell lines compared with noncancerous tissues and, respectively, although expression level was not correlated with the degree of infiltration or tumor stage. However, miR-486-5p overexpression in HCC cells inhibited proliferation and migration as evidenced by CCK-8 cell counting, wound healing, and transwell assays, indicating that miR-486-5p is an HCC suppressor. We employed four miRNA databases to predict the target genes of miR-486-5p and verified retrieved genes using qPCR and western blotting. The E3 ubiquitin ligase CBL was significantly downregulated by miR-486-5p overexpression in HCC cell lines at both mRNA and protein level, and overexpression of CBL counteracted the inhibitory effects of miR-486-5p on HCC cell proliferation and migration. Moreover, CBL expression was negatively correlated with miR-486-5p expression in HCC tissues. Collectively, our results suggest that miR-486-5p may act as a tumor suppressor gene in HCC by downregulating CBL expression.


Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancerrelated mortality [1]. Although improvements in diagnostic techniques have increased early detection and decreased mortality over the past decade, the incidence of HCC continues to increase and overall outcomes remain poor, with 5-year overall survival (OS) rate of only 3%-5% [2]. Early diagnosis and treatment is critical for improved prognosis. Therefore, new targets for preventing the initiation and progression of HCC are urgently required.
MicroRNAs (miRNAs) are a class of 20-25 nt small RNAs that silence the transcription of specific genes participating in diverse physiological and pathological processes, including carcinogenesis [3]. Evidence is accumulating that miRNAs are dysregulated in various human cancers, including HCC. These dysregulated miRNAs are often involved in processes relevant to tumorigenesis, tumor growth, and metastasis, such as cell proliferation, apoptosis, angiogenesis, and migration, thereby acting as oncogenes or tumor suppressors [4][5][6][7]. MiR-486-5p, encoded by the 40th intron of the ankyrin-1 gene, was first discovered in fetal liver and subsequently implicated in the development of many diseases including tumors. It has been reported that miR-486 relieves particulate matter-induced injury of human lung alveolar epithelial A549 cells by targeting PTEN and FOXO1 [8]. Mimics of miR-486-5p also inhibited the progression of colorectal cancer (CRC) by inhibiting the AKT signaling pathway through PIK3R1 downregulation [9]. However, the biological functions and downstream targets of miR-486-5p in HCC have remained elusive.
CBL was discovered as a cellular homologue of the v-Cbl oncogene [10]. The CBL family is composed of Cbl, Cbl-b, and Cbl-c/Cbl-3, all of which structurally resemble E3 ubiquitin ligases. Recent studies have shown that E3 ubiquitin ligases regulate the development of neuropathic pain by attenuating the production of IL-2 [11]. CBL also regulates the proliferation, differentiation, and survival of human mesenchymal-derived osteoblasts [12]. Silencing Cbl-b expression in breast cancer cells enhanced the risk of lung metastasis in nude mice, and it was concluded that Cblb reduces RANK protein expression and inhibits RANKLinduced breast cancer cell migration through negative regulation of the Src-AKT/ERK pathway [13]. Therefore, Cbl appears to have multiple and often divergent effects on different cancer types, presumably by interacting with distinct partners or due to differential regulation by upstream factors, possibly including miRNAs.
In the present study, the functions of miR-486-5p in HCC cells were explored. Results demonstrate that miR-486-5p inhibits the proliferation and migration of HCC cells through downregulation of CBL. The miR-486-5p-CBL regulatory pathway may thus be a promising therapeutic target for the treatment of HCC.  in HCC Tissues. The miRNA Seq data, mRNA Seq data, and corresponding HCC clinical data were downloaded from the TCGA database (http://tcga-data.nci.nih.gov/). A total of 422 samples were included in the miRNA Seq data, 372 of which were tumor samples and the remaining 50 normal tumor-adjacent samples. We found that miR-486-5p was downregulated in tumor samples compared with tumor-adjacent samples (P < 0.0001; Figure 1(a)). We then investigated the expression of miR-486-5p in different tumor stages and various degrees of infiltration from the clinical data but found no significant difference between T1-2 (n � 279) and T3-4 (n � 94) (P � 0.29; Figure 1(b)) or between Stage I-II (n � 261) and Stage III-IV (n � 91) (P � 0.09; Figure 1(c)).

Overexpression of MiR-486-5p Inhibits HCC Cell Proliferation and Migration.
To examine miR-486-5p functions in tumorigenesis, we first compared miR-486-5p expression in three HCC cell lines (SMMC-7721, HepG2, and Huh-7) with noncancerous cells by qRT-PCR. MiR-486-5p was significantly downregulated in all three HCC lines compared with the noncancerous human fetal hepatocyte cell line L-02 (P < 0.05) (Figure 2(a)), suggesting that loss of miR-486-5p function (target gene regulation) is involved in hepatic tumorigenesis. We chose SMMC-7721, the HCC cell exhibiting the lowest miR-486-5p expression, to construct a recombinant cell line stably overexpressing miR-486-5p ( Figure 2(b)) and conducted CCK-8 assays to examine the effects of miR-486-5p on cell proliferation and migration. As shown in Figure 2(d), SMMC-7721 cells overexpressing miR-486-5p proliferated more slowly than control SMMC-7721 (P < 0.001). Wound-healing assay and transwell assay also revealed that miR-486-5p overexpression significantly reduced the migratory capacity of SMMC-7721 cells (Figure 2(e)) (P < 0.001). Collectively, these results suggest that miR-486-5p suppresses tumor activity by reducing growth and metastasis. . The mechanisms underlying inhibition of tumor growth by miR-486-5p were then examined. We predicted the target genes of miR-486-5p using four miRNA databases: TargetScan, miRWalk, RNA22, and miRanda. A total of 22 candidate target genes were found by all 4 databases (Figure 3(a)). According to KEGG pathway annotation, these 22 genes were enriched in signal transduction and immune system categories ( Figure 3(b)). GO analysis indicated that these 22 genes were widely distributed across a variety of cellular component groups and biological processes (Figure 3(c)). To verify whether the expression levels of these 22 genes could be regulated by miR-486-5p, mRNA expression levels were measured by qPCR in SMMC-7721 cells stably overexpressing miR-486-5p. As shown in Figure 3(d), the expression of CBL was downregulated to the greatest extent by miR-486-5p (P < 0.001) among the 22 genes. The expression levels of TPA2 and PIGT were also inhibited by miR-486-5p but to a lesser extent, and F2R and F9 were upregulated by miR-486-5p overexpression. We thus focused on CBL as a potential mediator of miR-486-5p effects on HCC cell line proliferation and migration. We first compared the expression of CBL among cell lines and found upregulation in HCC lines compared with control cells (P < 0.05) (Figure 3(e)). Conversely, CBL was downregulated in SMMC-7721 cells overexpressing miR-486-5p (P < 0.05), suggesting that miR-486-5p inhibits CBL expression ( Figure 3(e)).

Discussion
Although the overall survival rate of HCC patients has increased in recent years, metastasis and recurrence are still common. Factors controlling these processes are thus potentially promising targets for life-extending therapies. MicroRNAs are novel molecular markers for many diseases and are gaining increased importance for diagnosis and treatment. It is expected that miRNAs will also be identified that contribute to HCC and thus provide new targets for screening and individualized HCC treatment. In the present study, we demonstrate that miR-486-5p inhibits the proliferation and migration of HCC cells, at least in part by downregulating expression and activity of the E3 ubiquitin ligase CBL. The miR-486 encoding site on human chromosome 8 yields three alternative species, miR-486-3p, miR-486-5p, and miR-486, all of which have been shown to be aberrantly expressed in human cancers [14]. Thus, miR-486 may serve as a biological marker for diagnosis [15]. MiR-486 has been shown to interfere with gastric cancer cell apoptosis by regulating PTEN and PIM-1 [16,17], and elevated expression may have a more general antiapoptotic effect as it is associated with reduced expression of the tumor suppressor p53 and the apoptosis effector caspase-3 [18]. Conversely, miR-486 was found to suppress the growth and metastasis of esophageal cancer by targeting CDK4/BCAS2 [19]. Low miR-486 expression has been observed in esophageal squamous cell carcinoma and gastric adenocarcinoma, suggesting utility as an independent marker to evaluate prognosis [20][21][22]. MiR-486-5p may also act as a tumor suppressor in lung and gastric cancers, but other studies have shown that miR-486-5p plays a procarcinogenic role in Down's syndrome-associated myeloid leukemia and chronic myelogenous leukemia [20]. Thus, miR-486 exhibits complex and sometimes opposing effects on malignancy.
In the present study, we found lower miR-486-5p expression in HCC tissues and HCC cell lines compared with surrounding noncancerous tissues and noncancerous hepatocytes, respectively, consistent with a tumor-suppressor function. Indeed, overexpression inhibited both the proliferation and migration of HCC cells, suggesting that miR-486-5p upregulation (or downregulation of its target) may be a novel treatment strategy.
CBL is an E3 ubiquitin ligase and multifunctional adaptor that regulates cell growth, invasion, apoptosis, and angiogenesis in multiple malignancies [23]. For example, CBL generally targets receptor tyrosine kinases and other nonreceptor tyrosine kinases, such as Src family kinases, and mutations in the components of this pathway may result in tumorigenesis [24]. Previous studies have confirmed that Cbl increases the chemosensitivity of gastric cancer cells by enhancing the epidermal growth factor receptor (EGFR) and mitochondrial signaling pathways [25]. High CBL expression has actually been recognized as linked with the development of several types of cancer, including gliomas, gastric carcinoma, colorectal cancer, prostate cancer, breast cancer and non-small-cell lung cancer [26]. A previous study has shown that CBL might be a prognostic indicator of HCC [27]; however, the biological effect of CBL in HCC has not been reported. In the current study, CBL was demonstrated as a downstream target of miR-486-5p, and CBL overexpression reversed the antitumor functions of miR-486-5p in HCC.
In summary, we found that higher miR-486-5p is associated with better prognosis of HCC and that over-expression can inhibit HCC cell proliferation and migration in vitro. These effects may stem from downregulation of its target CBL. These findings provide a targeted treatment strategy for HCC.

Statistical
Analysis. Group means were compared by one-way ANOVA or Student's t-test with Welch's correction for unequal variance. All statistical analyses were conducted using GraphPad 5 software. A P < 0.05 (two tailed) was considered statistically significant.

Data Availability
The data used to support the findings of this study are included within the article.