Tumor-suppressive miR-3650 inhibits tumor metastasis by directly targeting NFASC in hepatocellular carcinoma

In recent years, a growing body of evidence has provided support for the important role of microRNAs (miRNAs) in the progression of human cancers. A recent study showed that a novel miRNA miR-3650 expression was significantly decreased in hepatocellular carcinoma (HCC). However, the precise role of miR-3650 in HCC have remained poorly understood. In this study, we found that miR-3650 expression was frequently decreased in HCC tissues. Low expression of miR-3650 is positively associated with tumor metastasis and poor survival of HCC patients. Forced expression of miR-3650 significantly inhibited the migration and epithelial-mesenchymal transition (EMT) of HCC cells. Through bioinformatic analysis and luciferase assays, we confirmed that neurofascin (NFASC) is a directly target mRNA of miR-3650. Rescue experiment demonstrated that NAFSC overexpression could partially counteracted the inhibitory effect of miR-3650 in HCC metastasis and EMT. In conclusion, our findings are the first time to demonstrate that reduced expression of miR-3650 in HCC was correlated with tumor metastasis and poor survival. MiR-3650 repressed HCC migration and EMT by directly targeting NFASC. Our findings suggested that miR-3650 may serve as a potential prognostic marker and promising application in HCC therapy.


INTRODUCTION
Hepatocellular carcinoma (HCC) is the sixth most frequent cancer and the fourth most common cause of cancer-related death worldwide in 2018 [1]. About 80% HCC cases occur in East Asia (including China) and Sub-Saharan Africa, where the main risk factors are chronic hepatitis B and aflatoxin B1 exposure [2]. Although great developments in the diagnosis and therapy of this disease in the past decades, the prognosis of HCC patients is still dismal [3,4]. Moreover, most newly diagnosed HCC patients are unresectable distant AGING metastasis. The median overall survival (OS) of these patients treated with transcatheter arterial chemoembolization or sorafenib was less than one year because of the high rate of recurrence and metastasis [5,6]. In clinical practice, many HCC patients with same clinical stage have obviously different outcome, suggesting the inherent heterogeneity of the biological behavior of cancer cells [7]. Therefore, a better understanding of the underlying mechanisms involved in HCC metastasis will provide potential molecular targets for treatment of HCC metastasis and improve the prognosis of HCC patients.
As the development of high-throughput transcriptome analysis and next generation sequencing, it has been recognized that up to 90% of genomic DNA are not transcribed and translated into proteins [8], but they can be transcribed into noncoding RNAs [9]. MiRNAs are a class of 18-22 nucleotide small noncoding RNAs that can negatively regulate target genes expression by partially base-pairing with the 3 untranslated regions (3'-UTR) of target mRNAs [10]. Up to date, over 38,000 miRNAs have been identified and play key roles in a large range of biological processes including regulation of cell proliferation, apoptosis, stress resistance, epithelial-mesenchymal transition (EMT) and metastasis [11,12]. Recent studies have shown the alterations in miRNAs expression during the progression of HCC, highlighting their potentials for the diagnostic and prognostic applications, and targeted therapy for this malignancy [13]. For instance, it has been found that miR-214-5p overexpression inhibited the migration and EMT by targeting Wiskott-Aldrich Syndrome Like in HCC cells [14]. And miR-135a AGING promoted the metastasis of HCC cells by inhibiting tumor suppressor forkhead box O1 (FOXO1) [15].
A recent study showed that compared with non-side population (SP) cells, miR-3650 expression was significantly reduced in HCC SP cells [16]. This finding suggested that miR-3650 may play critical role in the progression of HCC. However, the biological functions and molecular mechanisms of miR-3650 in HCC remain mysterious. In this study, we investigated the expression and prognostic value of miR-3650 in HCC patients. We further assessed the effect of miR-3650 on HCC migration and EMT in vitro. Finally, we explored the underlying mechanism of miR-3650 in HCC metastasis.

The expression of miR-3650 is significantly decreased and correlated with clinical stage in HCC patients
To determine the importance of miR-3650 in HCC patients, we first detected the expression level of miR-3650 in 120 paired HCC samples by quantitative RT-PCR. The results showed that compared with adjacent normal liver tissues, miR-3650 expression in HCC tissues was significantly decreased (P < 0.0001, Fig.  1A). Moreover, miR-3650 expression was negatively correlated with tumor size, TNM stage (tumor-nodemetastasis stage) and Barcelona Clinic Liver Cancer . Nuclei were counterstained with DAPI (blue). Scale bars represent 50 μm. For all quantitative results, the data are presented as the mean ± SD from three independent experiments. * P < 0.05 and ** P < 0.01. AGING (BCLC) stage (all P < 0.05, Fig. 1B-1D). To evaluate the clinical relevance of miR-3650 in HCC patients, the median value of miR-3650 expression (0.315) was defined as a cutoff value to divide HCC patients into high-or low-expression groups. The relationship between clinicopathological parameters and miR-3650 expression was summarized in Table 1. Reduced expression of miR-3650 was remarkably associated with tumor size (P=0.010), TNM stage (P=0.037) and BCLC stage (P=0.017). In contrast, miR-3650 expression displayed no correlation with other clinicopathological variables (Table 1). These results suggested that reduced expression of miR-3650 might be involved in the progression and metastasis of HCC.

Low expression of miR-3650 predicts poor prognosis in HCC patients
Since miR-3650 was negatively associated with tumor size and clinical stage of HC patients, we reasoned that miR-3650 is correlated with patients' outcome. As expected, survival analysis indicated that patients with miR-3650 low-expression have apparently shorter 5year OS (29.7% vs. 74.8%) and 5-year disease-free survival (DFS) (20.4% vs. 61.5%) than those with highexpression (all P < 0.0001, Fig. 1E and 1F). To figure out the prognostic value of miR-3650 in this disease, univariate and multivariate analyses were performed on clinicopathological variables and patient survival. The univariate Cox proportional analysis indicated that tumor size, satellite nodule, vascular invasion and miR-3650 expression are significantly prognostic predictors for OS and DFS (Table 2). Multivariate Cox proportional analysis further demonstrated that miR-3650 was an independent protective predictor for OS and DFS in HCC patients (all P < 0.0001, Table 2). In addition, vascular invasion also was an independent risk factor for OS and DFS (all P < 0.0001), and AFP level also was an independent prognostic factor only for OS (P=0.009). These above results suggested that miR-3650 may serve as a potential prognostic biomarker for HCC patients.

Overexpression of miR-3650 inhibits HCC cells metastasis and EMT in vitro
We also performed RT-PCR to determine the expression of miR-3650 in seven HCC cell lines and one immortalized liver cell line LO2. The result showed that miR-3650 expression was generally lower in HCC cell lines than LO2 ( Fig. 2A). The significant decrease of miR-3650 expression in HCC tissues and cells prompted us to observe its biological significance in HCC. In order to manipulate miR-3650 expression, Hep3B and MHCC-97H cells with the lowest level of miR-3650 were transfected with miR-3650 mimic, and effective overexpression in both cell lines were verified by RT-PCR (Fig. 2B). Transwell assay were subsequently conducted, and the result indicated that enforced expression of miR-3650 significantly inhibited HCC cell migratory capacity ( Fig. 2C and 2D). Moreover, we did immunofluorescence (IF) assay to investigate whether miR-3650 could affect epithelialmesenchymal transition (EMT) in HCC cells. To our interest, miR-3650 overexpression increased the expression of epithelial marker E-cadherin while decreased the expression of mesenchymal marker Fibronectin (Fig. 2E). We also found that miR-3650 overexpression promoted the morphology of MHCC-97H from the dispersed type into the condensed type (Fig. 2E). Taken together, our data demonstrated that miR-3650 overexpression could suppress HCC migration and EMT in vitro.

NFASC is identified as a directly target mRNA of miR-3650 in HCC
It is well known that miRNAs directly bind to the 3'-UTR region of protein coding genes, and result in either transcriptional inhibition or mRNA degradation [17]. Thus, we predicted potential target mRNAs of miR-3650 using four online publicly available algorithms (TargetScan, RNA22, miRPathDB and TargetMiner). The result showed 19 mRNAs were the predicted targets of miR-3650 (Fig. 3A). We further predicted the signaling pathway of 19 potential targeted mRNAs using the online database DAVID 6.7. As shown in Fig.  S1, gene Ontology KEGG pathway enrichment analysis indicated that these predicted mRNAs were only enriched in cell adhesion molecules, which included netrin G1 (NTNG1), neurofascin (NFASC) and neuronal growth regulator 1 (NEGR1). Then we further confirmed that NFASC expression was significantly increased in 20 paired HCC tissues compared with adjacent normal tissues (Fig. 3B). Figure 3C displayed tow putative binding sites within miR-3650 and 3'-UTR of NFASC predicted using TargetScan 7.2 algorithm. In addition, we also found that the expression of NFASC was significantly higher in HCC tissues than in adjacent normal tissues (Fig. 3D).

MiR-3650 inhibits HCC cells migration and EMT via targeting NFASC
To explore whether the anti-metastatic effect of miR-3650 were mediated through targeting NFASC, gain-offunction rescue experiments were performed. Firstly, we found that NFASC mRNA expression was significantly reduced or enhanced after Hep3B and MHCC-97H cells were transfected with miR-3650 mimic or inhibitor, respectively (Fig. 4A). Secondly, MHCC-97H cells co-transfected with miR-3650 mimic and pcDNA3.1-NFASC partially restored the inhibitory effect of miR-3650 mimic only (Fig. 4B). Similarly, transwell assay indicated that the suppressive effect of miR-3650 on HCC migration could be partially rescued by co-transfected with pcDNA3.1-NFASC, which contained the coding sequences but lacked the 3'-UTR regions of NFASC ( Fig. 4C and 4D). As EMT plays critical roles in cancer metastasis [18], we further performed western blot assay to observe the expression of EMT-related proteins. The results showed that miR-3650 led to decreased expression of E-cadherin and elevated expression of Fibronectin, Vimentin and MMP-9 (Fig. 4E), which was consistent with the result of Fig. 2E. As anticipated, the abnormal expression of EMT-related proteins induced by miR-3650 mimic was contracted after cells co-transfected with pcDNA3.1-NFASC (Fig. 4E). Collectively, these data suggest that miR-3650 inhibits HCC cells migration and EMT in a NFASC-dependent manner.

DISCUSSION
The progression of HCC is a complex multistep process that involves huge genomic alterations and epigenetic modifications [19]. Unfortunately, none of the molecular classification of HCC so far can predict the prognosis and metastasis of this malignancy in clinical practice [20]. Considerable evidences demonstrated the complicated functional involvement of dysregulated miRNAs in the carcinogenesis, progression, recurrence, metastasis and survival of HCC cases [21]. However, the exact molecular mechanism of dysregulated miRNAs in HCC metastasis has remained elusive.
As a novel miRNA, the precursor of miR-3650 gene located in chromosome 5. Recently, miR-3650 has been reported to be involved in few cancer types. Two studies showed that miR-3650 expression was reduced in laryngeal carcinoma tissues and colorectal cancer cell [22,23]. More recently, Xie et al. indicated that miR-3650 was significantly decreased in HCC SP cells compared with non-SP cells [16], suggesting that miR-3650 may play an important role in HCC stemness and progression. Whereas no studies have reported the expression pattern, biological function and underlying mechanism of miR-3650 in HCC. In the current study, miR-3650 expression was significantly decreased in AGING HCC tissues, which was similar to the previous study [16]. Low expression of miR-3650 was positively associated with adverse prognosis of HCC patients and could be used as an independent prognostic predictor. Moreover, our study is the first report that miR-3650 overexpression inhibited HCC migration and EMT processes in vitro. Our results provide a more comprehensive understanding of the tumor suppressor role of miR-3650 in HCC metastasis.
It has been demonstrated that the interaction between miRNAs and 3'-UTR of their target mRNAs through complementary base pairing exerts their translational repression and target mRNAs degradation, then influences cell fate and biological functions [24,25]. Previous study has demonstrated that miR-487 enhanced HCC cells metastasis by directly binding to sprouty-related EVH1 domain containing 2 induced MAPK pathway [26]. This prompted us to investigate the downstream target mRNAs of miR-3650 in HCC. In this study, we confirmed that NFASC is the directly target of miR-3650. Overexpression of NAFSC could partially counteracted the inhibitory effect of miR-3650 in HCC metastasis and EMT. Actually, Samulin Erdem et al. showed that knockdown of NFASC could decrease the migration of non-small cell lung cancer cells, rearrange the actin cytoskeleton and inhibit Factin networks [27], which was consistent with our current results. This implying that miR-3650 inhibit the migration and EMT abilities of HCC cells at least in part by directly binding and inhibiting NFASC expression (Fig. 4F).
Interestingly, mounting reports have verified that many miRNAs expression were directly regulated by long noncoding RNAs (lncRNAs) as upstream regulators in several human cancers. LncRNAs usually function as competitive endogenous RNA (ceRNA) or molecular sponges for miRNAs [28]. A recent study showed that LINC01133 inhibited the EMT and metastasis of gastric cancer cells by acting as a ceRNA for miR-106a-3p to inhibit the adenomatous polyposis coli gene expression [29]. However, the upstream regulator of miR-3650 in HCC remains unanswered, and whether miR-3650/NFASC axis inhibits HCC metastasis by regulating certain signaling pathways has not yet been unraveled. Further investigations were made to solve these issues in our future studies.
In conclusion, our study reported the following new findings: (1) miR-3650 expression is significantly reduced in HCC tissues and cells; (2) low expression of miR-3650 is positively associated with poor survival of HCC patients and could be used as an independent prognostic predictor; (3) miR-3650 functions as a tumor suppressor in the migration and EMT abilities of HCC; (4) NFASC is a directly target mRNA of miR-3650; (5) miR-3650 inhibits HCC cells migration and EMT by binding and inhibiting NFASC. Our study underscores the inhibitory role of miR-3650 in HCC metastasis and facilitate the development of miRNA-direct prognosis and therapeutics against HCC patients.

RNA extraction and quantitative real-time polymerase chain reaction analyses (RT-PCR)
Total RNA was isolated from fresh-frozen tissues and HCC cells using TRIzol reagent (Invitrogen, NY, USA) as per the manufacturer's instructions. The quantity of RNA samples was measured by a NanoDropTM 1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). 1 μg total RNA and All-in-one TM First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, USA) were used to generate cDNA, which was subjected to RT-PCR using All-in-One™ miRNA qRT-PCR Detection Kit (Gene-Copoeia, Carlsbad, CA, USA). PCR was performed on a Roche Light Cycler 480 instrument (Roche, Basel, Switzerland) according to a standard method as described previously [30]. The relative expression of miR-3650 was calculated using the 2 −ΔΔCt method and U6 was used as the endogenous control. The primer sequences used in this study are shown in Supplementary Table S1.

Lentivirus and cell transfection
Lentivirus particles containing miR-3650 mimic, inhibitor and scramble sequences were purchased from GenePharma (Shanghai, China). The Hep3B and MHCC-97H cells were transfected with these lentivirus particles. At 48h after transfection, cells were treated with puromycin (2 μg/ml) for 7 days to construct cell AGING lines with stable miR-3650 knockdown or overexpression. NFASC ORF cDNA but lacked the 3'-UTR were amplified by PCR and then sub-cloned into the eukaryotic expression vector pcDNA3.1 (+), and the constructs were called pcDNA3.1-NFASC. The cell transfection was performed with Lipofectamine 3000 to construct HCC cells with stable NFASC overexpression.

In vitro migration assay
The in vitro migration assay was performed with the Corning Polycarbonate Membrane Insert transwell chambers (Corning Costar Corp, Cambridge, MA, USA). The migration assay was conducted according to a previous report [31]. Briefly, HCC cells (5~6 × 10 5 ) in serum-free media were seeded into the upper chamber (8-μm pore size). 600 μl medium containing 20% FBS was added to the lower chamber as the chemoattractant. After incubation at 37°C for 24h, cells were fixed in methanol, stained with 0.1% crystal violet. After scraping of cells remaining on the upper chamber by cotton swab, those migrated into the lower chamber were imaged and counted under the microscope.

Immunofluorescence (IF) assay
HCC cells were seeded into 24-well culture plate with a glass coverslip over each well and allowed to attach overnight. The cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.25% Triton X-100 (Sigma) for 10 min. After blocked with 4% bovine serum albumin, the cells were incubated overnight at 4°C with rabbit anti-Fibronectin or anti-E-cadherin antibody (Cell Signaling Technology, Danvers, MA, USA). On the following day, the cells were incubated with secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Sigma-Aldrich, Poole, UK) for 1 h. HCC cells were then incubated with 4',6-diamidin-2phenylindole (DAPI; Life Technologies, Carlsbad, CA, USA) for 15 min to stain the nuclei. Images of the cells were acquired using a fluorescence microscope (FV1000; Olympus, Tokyo, Japan).
Luciferase activity levels were measured using the Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Western blot analysis
The procedure of Western blot was performed as previously described [33]. Briefly, HCC cells were collected and lysed in RIPA lysis buffer with protease inhibitor (Selleck, Houston, USA) on the ice. After quantification, 30 μg of lysate protein were separated by SDS-PAGE gel and then transferred onto PVDF membrane. After blocking nonspecific binding sites with 5% non-fat milk, the membranes were incubated with primary antibodies at 4℃ overnight. The proteins in the membranes were visualized with the SuperSignal® ECL Kit (Pierce, USA). The primary antibodies used in this study included GAPDH, Ecadherin, Vimentin, Fibronectin (Cell Signaling Technology, USA), NFASC (Proteintech, Wuhan, China) and MMP-9 (Santa Cruz, USA). HRPconjugated goat anti-rabbit or anti-mouse IgG antibody (Abcam, Cambridge, UK) was used as the secondary antibody. GAPDH was used as a loading control.

Statistical analyses
Statistical analyses were performed using the SPSS 24.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism V7 (GraphPad Prism, Inc., USA). For the experimental studies, our data are presented as mean ± standard deviation (SD). Comparisons between groups were analyzed by the two-tailed unpaired Student's t test or one-way ANOVA. Kaplan-Meier plots and logrank test were used to estimate cancer specific survival curves. P value of < 0.05 was considered to be statistically significant. Each experiment was carried out in repeated triplicates.