MicroRNA-519a promotes proliferation and inhibits apoptosis of hepatocellular carcinoma cells by targeting FOXF2.

Recent studies report that microRNA-519a (miR-519a) is a novel oncomir, which facilitates the onset and progression of human cancers. However, the clinical significance of miR-519a and its functional role and underlying mechanisms in hepatocellular carcinoma (HCC) are poorly investigated. In the present study, elevated expression of miR-519a was observed in HCC tissues compared with adjacent non-tumor tissues. The increased level of miR-519a expression was significantly correlated with adverse clinical features of HCC including hepatitis B virus (HBV) infection, large tumor size, cirrhosis and advanced tumor-node-metastasis tumor stage. Furthermore, high expression of miR-519a was prominently associated with a poorer 5-year overall survival and recurrence-free survival of HCC patients. Gain- and loss-of function experiments showed that miR-519a overexpression enhanced proliferation and reduced apoptosis of Huh7 cells. By contrast, miR-519a knockdown inhibited SMMC-7721 cell proliferation and induced apoptosis. Importantly, up-regulation of miR-519a reduced the expression of FOXF2 mRNA and protein in Huh7 cells, while down-regulation of miR-519a resulted in increased expression of FOXF2 in SMMC-7721 cells. An inverse correlation between mRNA levels of miR-519a and FOXF2 was observed in HCC tissues. Thus, Forkhead box F2 (FOXF2) was identified as a downstream target of miR-519a in HCC. Mechanistically, the effects of miR-519a knockdown on SMMC-7721 cells were abrogated by FOXF2 repression. In conclusion, miR-519a is a novel prognostic predictor for HCC patients and it may potentiate proliferation and inhibits apoptosis of HCC cells by targeting FOXF2.

Introduction

Hepatocellular carcinoma (HCC) is fifth common cancer in men and the seventh one in women, and it confers the third most frequent cause of cancer related death all over the world [1]. The 5-year survival rate of HCC patients is only 30%, although great progresses have been made in the surgical treatment and molecular-targeted therapy of HCC [2], [3]. The unsatisfactory prognosis of HCC patients largely results from the late diagnosis, frequent metastasis and recurrence of HCC [4]. It is of great clinical significance to identify novel biomarkers and therapeutic targets, which will improve the clinical outcomes of HCC.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules and act as important post-transcriptional regulators of targeted genes [5], [6]. They actively participate in various biological processes [5], [6], [7], [8] including embryogenesis, cell differentiation, cell proliferation, apoptosis, and cell movements. And the biological roles of miRNAs in human cancers have been widely recognized [9], [10]. Aberrant expression and/or dysfunction of miRNAs have been confirmed to be involved in the development and progression of HCC [11]. And miRNAs have been proposed as promising biomarkers and therapeutic targets of HCC [11].

Among numerous microRNAs, microRNA-519a (miR-519a) is identified as a novel cancer-related microRNA. The expression of miR-519a was found to be aberrantly elevated in ovarian cancers and was significantly correlated with advanced clinical stage and poor progression-free survival [12]. Study of ER+ breast cancer demonstrated that miR-519a served as an oncomir by regulating tamoxifen resistance and its elevated expression was correlated with poor survival of breast cancer patients [13]. Statistical analysis of Gene Expression Omnibus (GEO) database showed that miR-519a was involved in the progression of head and neck squamous cell carcinoma [14]. However, the clinical significance and functional role of miR-519a in HCC were still undefined.

In this study, we demonstrated that the expression of miR-519a was aberrantly increased in HCC tissues. High expression level of miR-519a was correlated with adverse clinicopathological characteristics and reduced survival of HCC patients. And the gain- and loss-of-function studies confirmed that miR-519a promoted HCC cell proliferation and inhibited apoptosis in vitro. Notably, miR-519a inversely regulated FOXF2 abundance in HCC. Furthermore, Forkhead box F2 (FOXF2) was identified as a downstream target of miR-519a. Taken together, miR-519a probably exerted its function role by inhibiting the expression of FOXF2 in HCC.

Materials and methods

Patients and tissue samples

A total of 80 human primary HCC and matched tumor-adjacent tissues were collected between January 2009 and December 2013 at Wuhan General Hospital of Guangzhou Military Command. All clinical samples were obtained and used after obtaining informed consent from all enrolled patients. Patients did not receive any preoperative chemotherapy or embolization. The demographic information and clinicopathological characteristics of these patients were presented in Table 1. This study was approved by the Medicine Ethics Committee of Wuhan General Hospital of Guangzhou Military Command according to the Declaration of Helsinki (as revised in Tokyo 2004).

Table 1

Clinicopathological correlation analyses of miR-519a expression in HCC.

Clinicopathologic features		Total no. of patients, n = 80	No. of patients		P
High miR-519a	Low miR-519a
Age (y)	⩽50	32	15	17	0.648
>50	48	25	23
Sex	Male	67	34	33	0.762
Female	13	6	7
HBsAg positive	No	20	4	16	0.002⁎
Yes	60	36	24
Serum AFP level (ng/mL)	⩽20	24	10	14	0.329
>20	56	30	26
Tumor size (cm)	⩽5	36	12	24	0.007⁎
>5	44	28	16
No. of tumor nodules	1	66	22	34	0.227
⩾2	14	8	6
Cirrhosis	Absent	18	5	13	0.032⁎
Present	62	35	27
Venous infiltration	Absent	49	22	27	0.251
Present	31	18	13
Edmondson–Steiner grading	I+II	60	28	32	0.302
III+IV	20	12	8
TNM tumor stage	I+II	61	25	36	0.004⁎
III+IV	19	15	4

HCC, hepatocellular carcinoma; HBV, hepatitis B virus; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis.

Statistically significant.

RNA isolation and quantitative real-time PCR

TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was employed to isolate the total RNA from frozen clinical specimens or cultured HCC cells. Quantification of the miR-519a was performed using the TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, USA). Relative expression of miR-519a was normalized to U6, which was used as an endogenous control. For FOXF2 mRNA quantification, complementary DNA (cDNA) was synthesized using the Taqman RT reagents (Applied Biosystems) and quantitative real-time PCR were performed using the SYBR® Premix Ex Taq™ ii (Perfect Real Time) Kit (Takara Bio, Shiga, Japan). The expression levels of FOXF2 mRNA were normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following primers were used: FOXF2, 5′-TGC ACT CCA GCA TGT CCT CCT A-3′, 5′-CGC TAG CTG AGG GAT GGA AAG A-3′; GAPDH, 5′-AAC TTT GGC ATT GTG GAA GG-3′ and 5′-ACA CAT TGG GGG TAG GAA CA-3′.

Cell culture and transfection

Human HCC cell lines, SMMC-7721 (the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) and Huh7 cells (the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China), were cultured in Dulbecco’s modified Eagle medium (DMEM, GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO), 100 U/ml penicillin (GIBCO), and 50 mg/ml streptomycin (GIBCO). HCC cells were maintained in the humidified containing of 5% CO2 incubator at 37 °C.

The targeted sequences for FOXF2 siRNA (Sense; 5′-GAA AAG AUU UCG UCC UCA Att-3′, Anti-sense; 5′-UUG AGG ACG AAA UCU UUU Ctg-3′) or a scrambled oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). MiRNAs vectors, including miR-519a mimics (HmiR0342-MR03), the control vector for miR-519a (CmiR0001-MR03), miR-519a inhibitor (HmiR-AN0588-AM03) and the negative control for the miR-519a inhibitor (CmiR-AN0001-AM03), were obtained from Genecopoeia (Guangzhou, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

Cell proliferation and apoptosis assays

The proliferative ability of HCC cells was measured using a Cell Proliferation ELISA, 5-Bromo-2-deoxyuridine (BrdU) (Roche, Indianapolis, IN, USA) 48 h after transfection. For apoptosis assay, the percentage of apoptotic HCC cells was assessed using the Annexin-V-FLUOS Staining Kit (Roche) based on the instructions from manufacturers 48 h after transfection.

Luciferase reporter assay

The sequence of FOXF2 3′-UTR predicted to interact with miR-519a or the mutated sequence within the predicted sites was inserted into the XbaI and FseI sites of the pGL3 control vector (Promega, Madison, WI, USA). These constructs were noted as wild-type (wt) FOXF2-3′UTR or mutant (mt) FOXF2-3′UTR, respectively. For the luciferase reporter assay, SMMC-7721 cells that were seeded in the 96-well plates were transfected with miRNAs vectors and the above constructs using Fugene (Promega). These cells were harvested 48 h after transfection, and the Dual Luciferase Assay System (Promega) were used to measure the Renilla and firefly luciferase activities. Results were obtained from three independent experiments performed in triplicate.

Western blot

Total protein of HCC cells was collected using the Total Protein Extraction Kit (KeyGen, Nanjing, China). 30 μg of the protein samples per lane were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The separated protein were transferred onto a nitrocellulose membrane, and the blots were incubated with the primary antibodies for FOXF2 (1:300, Santa Cruz biotechnology, Inc., CA, USA) and GAPDH (1:1500, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. The blots were then incubated with anti-mouse or anti-rabbit secondary antibodies (1:5000–1:10000, Bio-Rad, USA) and detected using the Bio-Rad Gel imaging system.

Statistical analysis

The SPSS statistical package 19 (SPSS, IBM Corporation, Armonk, NY USA) and GraphPad Prism 5 software (GraphPad Software, Inc, USA) were used to conduct statistical analysis. The quantitative data were compared between groups using the Student’s t-test. The Pearson chi-squared test or Fisher’s exact test were used to compare the categorical variables. Kaplan–Meier plot and a log-rank test were used to analyze overall survival and recurrence-free survival rates. All P-values were two-sided, and P < 0.05 was considered as significant difference.

Results

The clinical significance of miR-519a expression in HCC tissues

To clarify the clinical significance of miR-519a in HCC, we first examined the levels of miR-519a expression in the clinical specimens from 80 HCC patients. Our data found that the expression levels of miR-519a were significantly elevated in HCC tissues compared to adjacent non-tumor tissues (P < 0.05, Fig. 1A). Then, we investigated the correlations between miR-519a expression and the clinical features of HCC patients. The expression of miR-519a was considered as either low (n = 40) or high (n = 40) according to its cutoff value, which was defined as the median value of the cohort of patients tested. As summarized in Table 1, high miR-519a expression were significantly correlated with positive HBsAg (P = 0.002), large tumor size (P = 0.007), cirrhosis (P = 0.032) and advanced tumor-node-metastasis (TNM) tumor stage (P = 0.004). Furthermore, we performed Kaplan–Meier and log-rank analysis to examine the prognostic value of miR-519a. As shown in Fig. 1B and C, high level of miR-519a was significantly correlated with a poorer overall survival (P = 0.014) and recurrence-free survival (P = 0.019). These results indicate an oncogenic role of miR-519a in the development of HCC, and suggest that miR-519a can potentially serve as a valuable prognostic predictor of HCC patients.

Fig. 1

The expression level of miR-519a and its prognostic significance in HCC. (A) Comparing expression of miR-519a between HCC tissues and matched tumor-adjacent tissues. n = 80, ∗P < 0.05. (B) and (C) High expression of miR-519a was associated with reduced overall survival and (C) recurrence-free survival rates of HCC patients.

MiR-519a potentiated proliferation and inhibited apoptosis of HCC cells

To elucidate the functional role of miR-519a in HCC, we further examined the proliferation and apoptosis of HCC cells after miR-519a alteration through gain- and loss-of-function assays. Huh7 cells that were transfected with miR-519a mimics showed a significant up-regulation of miR-519a expression compared to control cells (P < 0.05, Fig. 2A). Then, we performed BrdU incorporation assays and found that miR-519a overexpression in Huh7 cells resulted in significant increased proliferative ability (P < 0.05, Fig. 2B). At the same time, decreased apoptosis of Huh7 cells were observed after miR-519a overexpression (P < 0.05, Fig. 2C). On the other hand, SMMC-7721 cells that were transfected with miR-519a inhibitors led to an obvious down-regulation of miR-519a expression (P < 0.05, Fig. 3A). And down-regulation of miR-519a significantly inhibited cell proliferation (P < 0.05, Fig. 3B) and induced apoptosis (P < 0.05, Fig. 3C) in SMMC-7721 cells. These results indicate that miR-519a can potentiate proliferation and inhibit apoptosis of HCC cells in vitro.

Fig. 2

Overexpression of miR-519a enhanced proliferation and reduced apoptosis of Huh7 cells. (A) Transfection of miR-519a mimics significantly increased the expression level of miR-519a in Huh7 cells. n = three independent experiments, ∗P < 0.05. (B) Cell proliferation as measured by BrdU incorporation assays was increased after miR-519a overexpression in Huh7 cells. n = 3 repeats with similar results, ∗P < 0.05. (C) Overexpression of miR-519a significantly decreased the percentage of apoptotic Huh7 cells. n = 3 repeats with similar results, ∗P < 0.05.

Fig. 3

Inhibition of miR-519a expression inhibited proliferation and increased apoptosis of SMMC-7721 cells. (A) Transfection of miR-519a inhibitors significantly down-regulated the expression level of miR-519a in SMMC-7721 cells. n = three independent experiments, ∗P < 0.05. (B) Cell proliferation was decreased after suppression of miR-519a in SMMC-7721 cells. n = 3 repeats with similar results, ∗P < 0.05. (C) Inhibition of miR-519a in SMMC-7721 cells significantly increased the percentage of apoptotic cells. n = 3 repeats with similar results, ∗P < 0.05.

FOXF2 was identified as a downstream target of miR-519a in HCC cells

To further dig out the molecular mechanisms by which miR-519a exerts its functional role in HCC cells, we used the publicly available databases (TargetScan 6.2 and MiRanDa) to search for potential downstream target of miR-519a. FOXF2, which was recently identified as an important regulator of the proliferation and apoptosis of HCC cells, was predicted to be a downstream target of miR-519a. As shown in Fig. 4A, overexpression of miR-519a in Huh7 cells significantly decreased the level of FOXF2 mRNA (P < 0.05). And the results of Western blot analysis showed that the level of FOXF2 protein was also significantly reduced after forced expression of miR-519a (P < 0.05, Fig. 4B). In contrast, inhibition of miR-519a in SMMC-7721 cells led to an obvious increased expression level of FOXF2 mRNA (P < 0.05, Fig. 4C) and protein (P < 0.05, Fig. 4D). Furthermore, HCC specimens were subjected to qRT-PCR for FOXF2 mRNA. A statistically significant inverse correlation was revealed by Spearman’s correlation analysis between mRNA levels of miR-519a and FOXF2 (r = −0.683, P < 0.001, Fig. 4E). Next, the complementary sequence of miR-519a was found in the 3′-UTR of FOXF2 mRNA (Fig. 5A), suggesting that miR-519a could bind to the 3′-UTR of FOXF2 mRNA. Then, we determined whether miR-519a could directly interact with the 3′-UTR of FOXF2 mRNA as predicted. The results of luciferase reporter assays demonstrated that overexpression of miR-519a in SMMC-7721 cells significantly inhibited the luciferase activity of FOXF2 with the wild-type (wt) 3′-UTR (P < 0.05, Fig. 5B). No obvious effect was observed in the context of mutant-type (mt) 3′-UTR. Accordingly, down-regulation of miR-519a in SMMC-7721 led to significant elevated luciferase activity of wt FOXF2 3′-UTR (P < 0.05, Fig. 5B) and had no influence on that of mt FOXF2 3′-UTR. Taken together, these results indicate that FOXF2 is a direct downstream target of miR-519a in HCC.

Fig. 4

MiR-519a regulates the expression of FOXF2 in HCC cells. (A) qRT-PCR and (B) Western blot analysis of FOXF2 expression in Huh7 cells after miR-519a overexpression. Overexpression of miR-519a significantly reduced the levels of FOXF2 mRNA and protein in Huh7 cells. n = three independent experiments; ∗P < 0.05. (C) qRT-PCR and (D) Western blot analysis of FOXF2 expression in SMMC-7721 cells after miR-519a knockdown. Inhibition of miR-519a expression significantly increased the levels of FOXF2 mRNA and protein in SMMC-7721 cells; ∗P < 0.05. (E) Spearman’s correlation analysis demonstrated that miR-519a expression level was inversely correlated with FOXF2 mRNA level in HCC tissues.

Fig. 5

FOXF2 is a downstream target of miR-519a in HCC cells. (A) The 3′-UTR of FOXF2 mRNA was found to contain the complementary sequence of miR-519a. (B) Overexpression of miR-519a significantly suppressed the luciferase activity of wt 3′-UTR of FOXF2 but not mt 3′-UTR of FOXF2. Down-regulation of miR-519a resulted in an obvious increase in luciferase activity of wt 3′-UTR of FOXF2. n = 3 repeats with similar results; ∗P < 0.05.

FOXF2 knockdown influence the effects of miR-519a on HCC cells

To further confirm that FOXF2 is a functional target of miR-519a, a specific FOXF2 siRNA was transfected into miR-519a down-regulating SMMC-7721 cells. FOXF2 knockdown was confirmed by Western blot analysis (P < 0.05, Fig. 6A). Furthermore, we found that cell proliferation was remarkably increased after FOXF2 knockdown in miR-519a down-regulating SMMC-7721 cells (P < 0.05, Fig. 6B). Moreover, FOXF2 knockdown significantly rescued down-regulation of miR-519a-induced apoptosis of SMMC-7721 cells (P < 0.05, Fig. 6C). Thus, these results provide further evidence supporting FOXF2 as a downstream mediator of miR-519a.

Fig. 6

FOXF2 knockdown rescue the effects of miR-519a repression on SMCC-7721 cells. (A) miR-519a down-regulating SMMC-7721 cells that were transfected with scrambled siRNA or FOXF2 siRNA were subjected to immunoblotting for FOXF2. n = 3 repeats with similar results; ∗P < 0.05. (B) BrdU incorporation assays indicated that FOXF2 knockdown prominently increased cell proliferation in miR-519a down-regulating SMMC-7721 cells. n = 3 repeats with similar results; ∗P < 0.05. (C) FOXF2 knockdown significantly rescued miR-519a down-regulating-induced apoptosis of SMMC-7721 cells. n = 3 repeats with similar results; ∗P < 0.05.

Discussion

MiRNAs, which are critical post-transcriptional regulators of gene expression, have been confirmed to actively participate in the initiation and progression of human cancers by exerting either tumor promotive or suppressive effects [7], [9], [10], [15]. And the fundamental roles of miRNAs in HCC have made them become valuable biomarkers and attractive therapeutic targets [3], [16], [17]. MiR-519a was recently identified as a novel cancer related miRNA. The oncogenic roles of miR-519a has been confirmed in ovarian cancer [12] and ER+ breast cancer [13], and elevated expression of miR-519a was correlated with adverse clinicopathological characteristics and poor survival of cancer patients [12], [13]. In this study, we confirmed that the expression level of miR-519a was significantly elevated in HCC tissues compared to matched tumor-adjacent tissues. This result was in accordance with the other studies of miRNAs expression profiles in HCC [18], [19]. Moreover, our study also confirmed that high expression of miR-519a was correlated with unfavorable clinical features and poor prognosis of HCC patients. These results indicate that miR-519a is an onco-miRNA in HCC and can serve as a prognostic predictor of HCC patients.

It has been widely accepted that sustained proliferation and resistance to apoptosis are two important hallmarks of human cancers [20]. In this study, we found that miR-519a could potentiate proliferation and inhibit apoptosis of HCC cells through the gain- and loss-of-function experiments. Overexpression of miR-519a promoted proliferation and inhibited apoptosis of Huh7 cells, while miR-519a down-regulation in SMMC-7721 cells led to decreased proliferation and increased apoptosis. These data indicate that miR-519a promotes the progression of HCC by influencing cell proliferation and apoptosis. Interestingly, Ward et al. found that miR-519a could increase the viability, promote cell cycle progression and enhance tamoxifen resistance of breast cancer cells [13]. Therefore, the exact role of miR-519a in human cancers seems to be cancer-type specific.

FOXF2, which is a member of FOX family [21], is recognized as an important tumor suppressor in human cancers. It was found to inhibit the metastasis and epithelial-mesenchymal transition of basal-like breast cancer [22], [23] and lung cancer [24]. And the study of prostate cancer demonstrated that FOXF2 was a direct target of miR-182-5p, which promoted cell invasion and proliferation [25]. In this study, we confirmed that miR-519a inversely regulated FOXF2 abundance in HCC. Notably, FOXF2 was a direct downstream target of miR-519a using luciferase report assay. Importantly, FOXF2 knockdown abrogated down-regulation of miR-519a’s proliferation inhibition and apoptosis-inducing effects on SMMC-7721 cells. Recently, a study of HCC showed that FOXF2 acted as a tumor suppressor and prognostic marker in HCC [26]. And FOXF2 inhibited the proliferation and induced apoptosis of HCC cells [26]. These indicate that miR-519a probably exerts the regulatory effects on HCC cell proliferation and apoptosis by targeting FOXF2.

In conclusion, this study demonstrates that the expression level of miR-519a is aberrantly elevated in HCC tissues. Elevated miR-519a expression is correlated with adverse clinical features and poor prognosis of HCC patients. Functionally, miR-519a promotes the tumor growth of HCC by enhancing proliferation and inhibiting apoptosis. And we confirm that FOXF2 is a direct functional target of miR-519a. In summary, this study suggests that miR-519a acts as a potent prognostic predictor, and may potentially become an effective therapeutic target of HCC in the future.

Conclusions

In summary, our data indicates that the expressions of miR-519a in HCC tissues are significantly higher than those in matched tumor-adjacent tissues. Clinical association analyses disclose that miR-519a is expressed at obvious higher levels in HCC tissues arising from patients with hepatitis B virus (HBV) infection, large tumor size, cirrhosis and advanced TNM tumor stage. Importantly, high expression of miR-519a confers a poorer 5-year overall survival and recurrence-free survival rates for HCC patients. Gain- and loss-of function studies demonstrate that miR-519a facilitates HCC cell proliferation and prevents apoptosis in vitro. Furthermore, miR-519a inversely regulates FOXF2 abundance in HCC. Luciferase activity assays identify that FOXF2 is a direct downstream target of miR-519a. Notably, FOXF2 knockdown significantly rescued down-regulation of miR-519a-induced proliferation inhibition and apoptosis in SMMC-7721 cells. Our study suggests that miR-519a functions as an oncomir and may be implicated in the tumor growth of HCC.

Competing interest

The authors declare that they have no competing interests.

Author contribution statement

Junwei Shao, Jun Cao, Yong Liu, Hongliang Mei and Yang Zhang carried out the cell biology and molecular biology experiments, participated in the sequence alignment and drafted the manuscript. Junwei Shao, Jun Cao and Weitian Xu participated in the design of the study and performed the statistical analysis. Weitian Xu conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.