MicroRNA-212-3p inhibits the Proliferation and Invasion of Human Hepatocellular Carcinoma Cells by Suppressing CTGF expression

MicroRNA-212-3p inhibits several human cancers but its effects on hepatocellular carcinoma (HCC) remain unclear. In this study, we show that miR-212-3p is down-regulated in HCC cell lines and tissues, and correlates with vascular invasion (p = 0.001), and the absence of capsule formation (p = 0.009). We found that miR-212-3p influenced the epithelial to mesenchymal transition (EMT) of HCCLM3 and Huh7 cells. Mechanistically, miR-212-3p repressed cell invasion through the suppression of connective tissue growth factor (CTGF). We therefore validate the anti-HCC effects of miR-212-3p through its ability to suppress CTGF and subsequent EMT.


Materials and Methods
Clinical samples and ethics statement. This study was approved by the ethic committee of Eastern Hepatobiliary Surgery Hospital. This included 83 HCC patients, diagnosed pathologically between January 2010 and December 2013, in the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). The patients received no preoperative therapy. Samples were collected, immediately frozen in liquid nitrogen, and stored at −80 °C. All HCC and adjacent non-cancerous tissues (ANT) were simultaneously harvested. The hospital's Institutional Review Board approved the study in strict adherence to the Declaration of Helsinki protocol. Written informed consent was requested from each patient. Post-surgical follow-up of all patients involved procuring survival information through telephone contact.
Transwell migration and invasion assays. Transwell migration and invasion assays were performed as previously described (20).
Western blot analysis. Cells were lysed in RIPA buffer and proteins were separated on SDS-PAGE gels.
Cell counting kit-8 (CCK-8) proliferation assays. CCK-8 kits (Solarbio Biotechnology, Beijing, China) were used to measure Huh7 or HCCLM3 cells proliferation. Cells were seeded at a density of 5 × 10 3 cells/well into 96-well plates and incubated for 48 h. CCK-8 reagent (10 µl) was added to each well for 1 hr. Miniature microplate readers were used to determine the OD values at 450 nm. Experiments were performed in triplicate.
Luciferase reporter assays. TargetScan (http://www.tar getscan.or g/v ert_72/) was used to identify CTGF as a target gene for luciferase reporter assays and miR-212-3p correlation analysis. Mutated (MUT) or wild-type  www.nature.com/scientificreports www.nature.com/scientificreports/ (WT) CTGF 3′-UTRs were ligated into pGL3 (Promega, Madison, WI, USA). The luciferase reporter comprised CTGF 3′-UTR regions with miR-212-3p targets or mutant site indicators, to determine whether miR-212-3p directly targeted CTGF. Cells were seeded in triplicate into six-well plates, and left to settle for 12 h. Cells were transfected with miR-212-3p or NC controls (Thermo Fisher Scientific) and Dual-Luciferase Reporter Assays (Promega Corporation, Fitchburg, WI, USA) were employed 24 h post-transfection. www.nature.com/scientificreports www.nature.com/scientificreports/ Statistical analyses. SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. Data are shown as the mean ± SD. A student's t-test was used to statistically differentiate the groups. A one-way ANOVA was employed for multiple tests. The correlation of clinic pathological characteristics with miR-212-3p expression was analyzed via the chi-square test. The Kaplan Meier method was used to plot survival curves, which were compared using the log-rank test. Spearman correlation analysis evaluated the correlation between miR-212-3p and CTGF in HCC tissues. A P < 0.05 was deemed significant. All experiments were performed in triplicate.

HCC cell lines and tissues display reduced miR-212-3p expression. The expression of miR-212-3p
in the HCC cell lines Huh7, HCCLM3, HepG2 and Hep3B was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and compared to the immortalized human hepatic cell line LO2. The expression of miR-212-3p was considerably lower in HCC cell lines (Fig. 1A). Subsequently, HCC tissues and ANT were assessed for miR-212-3p expression. The levels of miR-212-3p were considerably lower in 83 paired HCC tissues, in comparison to the corresponding ANT tissue (Fig. 1B). In addition, qRT-PCR assays were used to substantiate vascular invasion and tumor encapsulation deficiency following miR-212-3p down-regulation (Fig. 1C,D). This indicated that miR-212-3p could suppress HCC tumorigenesis and invasion.  www.nature.com/scientificreports www.nature.com/scientificreports/ Low miR-212-3p expression indicates poor prognosis in HCC. The correlation between miR-212-3p expression and the clinic pathologic aspects of HCC were further studied. The relative median values of the 83 HCC samples were classed as low or high miR-212-3p expression using defined cut-off values. As per results, low miR-212-3p expression was linked to a lack of capsule formation (p = 0.009), and the presence of vascular invasion (p = 0.001), (Table 2). Recurrence-free survival (RFS) and overall survival (OS) were comparatively higher in the high miR-212-3p expression group (Fig. 1E,F) assessed via Kaplan-Meier survival analysis. This suggested that miR-212-3p expression is related to poor HCC prognosis, and is a potential prognostic marker for HCC stratification.
We next assessed the migration and invasion of miR-212-3p expressing HCC cells via transwell assays. As shown in Fig. 2C,D, enhanced miR-212-3p expression reduced the invasiveness of Huh7 and HCCLM3 cells compared to NC groups. These data confirm that miR-212-3p inhibits HCC proliferation, migration, and invasion.

Up-regulation of miR-212-3p alters the expression of EMT markers in HCC cells. We next
assessed the effects of miR-212-3p on EMT. We measured the expression of epithelial ZO-1 and E-cadherin 15,16 , mesenchymal N-cadherin 17 and fibronectin markers in Huh7 and HCCLM3 cells via qRT-PCR or western blotting. Figure 3A,B show that the upregulation of miR-212-3p led to substantially higher levels of E-cadherin and ZO-1 at the mRNA and protein level, whilst N-cadherin and fibronectin expression levels decreased in comparison to the control groups. We thus concluded that miR-212-3p inhibited cell invasion by modifying EMT markers in HCC cells.

Up-regulation of CTGF partially rescues the expression of EMT markers in HCC cells.
The upregulation of CTGF in Huh7 or HCCLM3 cells partially rescued EMT. We found that E-cadherin and ZO-1 were considerably down-regulated at both the mRNA and protein level in HCCLM3 miR-212-3p+CTGF , compared to HCCLM3 CTGF cells, whilst N-cadherin and fibronectin levels appreciably recovered in HCCLM3 miR-212-3p+CTGF , compared to HCCLM3 CTGF (Fig. 6A). These results were comparable in Huh7 cells (Fig. 6B). These results indicate that miR-212-3p influences the expression of EMT markers through its ability to downregulate CTGF. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Recurrence and metastasis contribute to the mortality of HCC patients following post-surgical resection. Understanding the metastatic mechanisms of HCC are required for effective HCC therapeutics. Numerous analyses have validated the role of miRNAs in HCC progression and metastasis [17][18][19][20] yet their primary molecular mechanisms remain poorly understood. Further studies on the regulatory characteristics of miRNAs during HCC progression and metastasis are required to assess their potential as prospective clinical diagnostic and prognostic biomarkers.
We found that miR-212-3p is downregulated in HCC cells and tissues, and correlates with vascular invasion (p = 0.001) and the absence of capsule formation (p = 0.009). Low miR-212-3p expression was related to lower survival rates and miR-212-3p was found to target CTGF. We found that miR-212-3p could repress HCC cell www.nature.com/scientificreports www.nature.com/scientificreports/ proliferation, migration, and invasion, by inhibiting CTGF and reducing EMT. We therefore established miR-212-3p as a HCC biomarker and potential therapeutic target.
A series of complex events occur during tumor metastasis 21 . The invasive-metastatic cascade involves EMT endowing non-invasive tumor cells with metastatic ability, enabling their migration into the extracellular matrix, the ability to cross the basal membranes of vessels, to persist in the bloodstream, and initiate clonal metastatic growth on other organs via angiogenesis 22 . EMT promotes hepatic fibrosis in a pre-metastatic microenvironment 23 . Several analyses have confirmed the influence of miRNAs in the EMT process 24,25 . In this study, miR-212-3p positively correlated with E-cadherin and ZO-1, and negatively correlated with N-cadherin and fibronectin. In vitro trials also established that miR-212-3p can reverse the metastatic capabilities of HCC cells and the EMT phenotype.
CTGF is vital to both angiogenesis and EMT due to its role as a transcriptional TGF-β target and ability to interact with extracellular matrix and cell surface proteins, in addition to other growth factors 26 . TGF-β is