Introduction
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the second leading cause of death from cancer worldwide [
1]. Currently, the prognosis for HCC patients remains poor, with a 5-year survival rate of approximately 30 % after liver resection, which is considered to be the best therapeutic strategy to treat HCC [
2]. Local and systemic metastases are the main reasons for the unsatisfactory prognosis of HCC patients [
3]. Elucidating the underlying molecular mechanisms for HCC metastasis is critical for identifying novel therapeutic targets of HCC.
Epithelial-to-mesenchymal transformation (EMT) has been widely accepted as a key mechanism underlying the metastatic process of HCC [
4]. During the development of EMT, the expressions of epithelial markers such as E-cadherin, zonula occludens-1, and claudins decrease while the expressions of mesenchymal markers such as vimentin, N-cadherin, and fibronectin increase [
5]. The EMT process in HCC cells can be regulated by various factors, including hypoxia [
6], cytokines [
7], long non-coding RNAs (lncRNAs) [
8], microRNAs [
9], and so on, and targeting the EMT process has been found to be an attractive and promising strategy to prevent the metastasis of HCC [
7].
lncRNAs are RNA molecules over 200 nucleotides in length with little protein-coding potential [
10]. Previous studies have shown that aberrant lncRNA expression is observed in human cancers, including those in the liver [
11], breast [
12], colon [
13], ovary [
14], pancreas [
15], and bladder [
16]. lncRNAs have been identified with oncogenic properties (
KRASP,
HULC,
HOTAIR,
MALAT1,
HOTTIP,
ANRIL, and
RICTOR) or oncosuppressive properties (
MEG3,
GAS5,
LincRNA-p21,
PTENP1,
TERRA,
CCND1/
CyclinD1, and
TUG1) or both (
CCAT1 and
XIST) [
17,
18]. Tumor suppressor candidate 7 (
TUSC7), also called
LOC285194 or
LSAMP antisense RNA3, is an lncRNA consisting of four exons of more than 2 kb in length and is located at 3q13.31 [
19]. Recent studies indicated that lncRNA
TUSC7 is downregulated in cancers including gastric cancer [
20], osteosarcoma [
21], colorectal cancer (CRC) [
22], esophageal squamous cell carcinoma (ESCC) [
23], and so on. In gastric cancer,
TUSC7 is a p53-regulated tumor suppressor that acts in part by repressing miR-23b to suppress tumor cell growth in vitro and in vivo [
20]. In osteosarcoma, depleting
TUSC7 promoted proliferation of normal osteoblasts by regulating apoptotic and cell cycle transcripts as well as the vascular endothelial growth factor (VEGF) receptor 1 [
21]. In human pancreatic ductal adenocarcinoma (PDAC) and CRC, by analyzing the association of
TUSC7 expression with clinicopathologic features, it was found that low
TUSC7 expression was closely correlated with lymph node metastasis, liver metastasis, and more distant metastases [
19,
22]. These data validated that
TUSC7 is a tumor suppressor by regulating cell proliferation, apoptosis, migration, invasion, cell cycle, and tumor growth. However, the exact role of
TUSC7 in HCC progression and the underlying mechanisms remain unknown.
MicroRNAs (miRNAs) are an abundant group of endogenous non-coding single-strand RNAs, and it is known that aberrant miRNA expression profiles are causally connected to tumor progression [
24]. Recently, the competing endogenous RNA (ceRNA) hypothesis proposed that a large number of non-coding RNAs might function as molecular sponges for miRNAs and, hence, functionally liberate other RNA transcripts targeted by the aforementioned active miRNAs [
25]. For example, lncRNA-
UCA1 has been reported to play an oncogenic role in breast cancer through directly interacting with miR-143 to lower its expression and affect its downstream regulation [
26]. miR-222 could be downregulated by lncRNA-Gas5 in glioma, thereby suppressing the tumor malignancy [
27,
28], and has been reported to play critical roles in the development of a variety of human cancers [
29‐
31], including HCC [
28,
32]. In HCC, acting as a tumor promoter, the expression of miR-10a has been shown to be upregulated, which accelerates the cell migration, invasion, and EMT [
32,
33]. Additionally, Eph tyrosine kinase receptor A4 (
EphA4), a member of the Eph receptor tyrosine kinase family, has been identified as an EMT suppressor in cancers [
34‐
36]. It is reported that miR-10a could regulate the EMT process in HCC through directly binding the 3′-untranslated region (UTR) of the
EphA4 transcript [
32]. However, limited knowledge is available concerning whether
TUSC7 could act as a sponge for miR-10a to affect the biological processes of HCC and the potential primary mechanism among
TUSC7, miR-10a, and
EphA4 in HCC progression remains unknown.
In this study, we found that the expression of TUSC7 was decreased in HCC and that TUSC7 may be a promising prognostic or progression marker for HCC. Additionally, TUSC7 suppressed cell migration, invasion, and EMT of HCC cells. Moreover, mechanistic analysis revealed that TUSC7 may function as a ceRNA for miR-10a to regulate the expression of EphA4 to suppress EMT in HCC, thus playing an oncosuppressive role in HCC pathogenesis. Here, we provide the first evidence for the TUSC7-miR-10a-EphA4 axis, shedding new light on the mechanism of HCC.
Materials and methods
Clinical samples
HCC samples were collected from 75 patients including 51 males and 24 females, who underwent resection of their primary HCC in the Department of Hepatobiliary Surgery at the First Affiliated Hospital of Xi’an Jiaotong University during January 2009 to December 2011. Patients did not receive any preoperative chemotherapy or embolization.
Patients’ demographic and clinicopathologic data were obtained through a review of hospital records. And disease recurrence and survival information was updated at each follow-up visit. The time between the surgery date and first disease recurrence date was calculated as disease-free survival (DFS). The time between the diagnostic biopsy and surgery date to death or last follow-up was determined as overall survival (OS) duration.
Cell culture
The human immortalized normal hepatocyte cell line (LO2) and six HCC cell lines (HepG2, MHCC97L, Hep3B, SMMC-7721, MHCC97H, and Huh7) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. All cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum (FBS; Gibco) with 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma, St. Louis, MO, USA) in a humidified incubator containing 5 % CO2 at 37 °C.
Cell transfection
Three
TUSC7-specific small interfering RNAs (siRNAs), the
TUSC7-siControl (Table
1), pcDNA3.1-
TUSC7 (pcDNA/
TUSC7), and pcDNA3.1-Control (pcDNA/Control), were purchased from Invitrogen (Carlsbad, CA, USA). Four miRNA vectors, including anti-miR-10a, anti-Control, miR-10a, and miR-10a-Control, were purchased from GeneCopoeia (Guangzhou, China). All cell transfections were performed according to the manufacturer’s protocol.
Table 1
TUSC7-siRNAs and TUSC7-siControl sequences
TUSC7-siRNA1 | Sense: 5′-GGCCAAACCCUCAAUGAAUtt-3′ Antisense: 5′-AUUCAUUGAGGGUUUGGCCtg-3′ |
TUSC7-siRNA2 | Sense: 5′-GCGCAUUUCUCUUAAACAATT-3′ Antisense: 5′-UUGUUUAAGAGAAAUGCGCTT-3′ |
TUSC7-siRNA3 | Sense: 5′-CUGCCCUCCAUUCUAUCUATT-3′ Antisense: 5′-UAGAUAGAAUGGAGGGCAGTT-3′ |
TUSC7-siRNA4 | Sense: 5′-GGAGAGAGAUAUGCUAAGUTT-3′ Antisense: 5′-ACUUAGCAUAUCUCUCUCCTT-3′ |
TUSC7-siControl | Sense: 5′- UUCUCCGAACGUGUCACGUTT-3′ Antisense: 5′-ACGUGACACGUUCGGAGAATT-3′ |
Luciferase reporter assay
To search for the miR-10a binding site of TUSC7, we used a number of bioinformatics tools (MicroRNA, Mircode, Starbase v2.0, and RNAhybrid). The putative miR-10a target binding sequence in TUSC7 and its binding site mutant were synthesized and cloned downstream of the luciferase gene in the pmirGLO luciferase vector (Promega, Madison, WI, USA). Hep3B cells were co-transfected with wild-type or mutated pmirGLO-miR-10a reporter plasmid and pcDNA/Control or pcDNA/TUSC7 using Lipofectamine 2000 (Invitrogen). After 48 h, the cells were harvested and luciferase activity was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to the Renilla luciferase activity. Results were obtained from three independent experiments performed in triplicate.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from HCC tissues and cell lines using TRIzol (Invitrogen) following the manufacturer’s instructions. The RNA levels of
TUSC7 and
EphA4 were determined by quantitative real-time PCR (qRT-PCR) and calculated using the 2
−ΔΔCt method, with the Ct values normalized using GAPDH as an internal control. The primers are listed in Table
2. miRNAs were obtained using the mirVana MiRNA Isolation Kit (Ambion, Austin, TX, USA). Mature miR-10a and
U6 snRNA were reversely transcribed using Stem-loop RT Primer with miScript II RT Kit (Qiagen, Valencia, CA, USA). qRT-PCR was performed using SYBR Green PCR Master Mix (Qiagen) in an ABI 7500 system (Applied Biosystems, USA).
Table 2
Primers used in qRT-PCR
GAPDH | Forward: 5′-CCGGGAAACTGTGGCGTGATGG-3′ Reverse: 5′-AGGTGGAGGAGTGGGTGTCGCTGTT-3′ |
TUSC7
| Forward: 5′- CACTGCCTATGTGCACGACT-3′ Reverse: 5′- AGAGTCCGGCAAGAAGAACA-3′ |
E-cadherin | Forward: 5′- GCCGCTGGCGTCTGTAGGAA -3′ Reverse: 5′- TGACCACCGCTCTCCTCCGA -3′ |
Vimentin | Forward: 5′-GAGAACTTTGCCGTTGAAGC-3′ Reverse: 5′-GCTTCCTGTAGGTGGCAATC-3′ |
EphA4
| Forward: 5′ - ATGGATCCTGTTGCCCTCAC -3′ Reverse: 5′- CAGAATTCCTCCTACCCTTACC -3′ |
Western blot
Western blot analysis was performed using standard techniques. The following antibodies were used: E-cadherin (3195S, Cell Signaling, Beverly, MA, USA), vimentin (sc-6260, Santa Cruz Biotechnology, Santa Cruz, CA, USA), EphA4 (SRP00347b, Saierbio, Tianjin, China), and β-actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Wound healing assays
To determine cell motility, HCC cells were seeded into six-well plates and grown to 80–90 % confluence. A 200-μL sterile plastic tip was used to create a wound line across the surface of plates, and cellular debris was removed by washing with phosphate-buffered saline (PBS). Cells were cultured in DMEM in a humidified incubator with 5 % CO2 at 37 °C for 48 h, and then images were taken with a phase-contrast microscope.
Transwell assays
The 8 μM pore-size transwell inserts (Nalge Nunc, Penfield, New York, NY, USA) were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at 1:8 dilution on the inner layer. Hep3B and MHCC97H cells were resuspended with reduced serum DMEM, and the density was adjusted to 2.5 × 105/mL 48 h after transfection. A 200-μL cell suspension was added into the upper chamber, and 750 μL DMEM containing 10 % FBS was added into the lower chamber and then incubated for 24 h.
Cells were fixed in 4 % paraformaldehyde for 2 min and then permeabilized in 100 % methanol for 20 min. The cells on the inner layer were softly removed with a cotton swab, and the adherent cells on the undersurface of the insert were stained with 0.3 % crystal violet dye for 15 min. The filters were washed with PBS, and images were taken. Cells on undersurface were counted under a light microscope.
Immunohistochemistry
Immunohistochemistry staining was performed on paraformaldehyde-fixed paraffin sections. The sections were dewaxed and dehydrated. Following rehydration and antigen retrieval in citrate buffer, endogenous peroxidase activity was blocked for 10 min using 3.0 % hydrogen peroxide. The sections were blocked for 30 min using 10 % goat plasma and then separately incubated with the primary antibodies directed against E-cadherin (1:400) and vimentin (1:200) at 4 °C overnight. The primary antibody was detected using biotinylated secondary antibodies (Golden Bridge Biotechnology, Zhongshan, China) according to the manufacturer’s recommendations. The sections were visualized with diaminobenzidine and counterstained with hematoxylin and then dehydrated in alcohol and xylene and mounted onto glass slides.
Statistical analysis
Results are presented as mean ± SD. The SPSS statistical package for Windows version 13 (SPSS, Chicago, IL, USA) and GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA) were used for the Pearson chi-square test, a two-tailed Student’s t test, a Kaplan-Meier plot, a log-rank test, or an ANOVA where appropriate. Differences were considered to be significant when p < 0.05.
Discussion
HCC patients currently have a poor prognosis, and it is without doubt that early detection and treatment could significantly increase their chances of survival. Recently, lncRNAs have shown great therapeutic potential for human diseases, including HCC [
39]. For example, studies from Yuan SX et al. have revealed that
DANCR increases stemness and offers a potential prognostic marker, and a therapeutic target, for HCC [
40]. Research from Chen CL et al. unveiled the molecular mechanisms of how
PTENP1 repressed the tumorigenic properties of HCC cells and demonstrated the potential of the
SB-BV hybrid vector for
PTENP1 lncRNA modulation and HCC therapy [
41]. Accordingly,
TUSC7 was identified as a robust suppressor of cancer [
21]. In this study, we found that
TUSC7 expression in HCC was significantly downregulated.
TUSC7 expression in HCC tissues was negatively associated with more tumor nodes, more venous infiltration, advanced Edmondson-Steiner grading, and advanced TNM tumor stage. Moreover, comparison of Kaplan-Meier survival curves indicated that patients with lower
TUSC7 expression in HCC tissues had notably worse prognosis.
TUSC7 was also confirmed to be an independent risk factor for HCC patients. Altogether, these clinical data suggest strongly that
TUSC7 is critical for prognosis determination in HCC patients. Furthermore, we tested the action of
TUSC7 on tumor invasion and metastasis of HCC cells by taking different approaches and found that
TUSC7 inhibited cell invasion and metastasis in HCC.
EMT, a dynamic and reversible cellular process, is characterized by a loss of cell polarity and intracellular junctions and acquirement of mesenchymal features, which could result in increased HCC cell migration and invasion [
42]. Recent studies showed that lncRNAs may play critical roles in the EMT progress not only in HCC but also in other cancers [
43‐
45]. Furthermore, it has been found that some lncRNAs could promote EMT [
45,
46] while some could restrain EMT [
47,
48]. For example,
lncRNA-AOC4P has been shown to act as an HCC tumor suppressor by enhancing vimentin degradation and suppressing EMT progress [
47]. Overexpression of
lncRNA-UCA1 induced EMT and increased the migratory and invasive abilities of bladder cancer cells [
49].
lncRNA-ATB may also act on colon tumorigenesis by suppressing E-cadherin expression and promoting EMT [
50]. In this study, we analyzed EMT biomarkers of HCC tissues by using immunohistochemistry and qRT-PCR and those of HCC cells by western blot. Then, we determined the expression of an epithelial marker (E-cadherin) and mesenchymal marker (vimentin) in HCC with either low or high
TUSC7 expression. Interestingly, it was found that
TUSC7 expression was positively associated with E-cadherin expression and negatively associated with vimentin expression in HCC. We concluded that TUSC7 could suppress EMT in HCC.
Growing evidence suggests that lncRNA may act as a ceRNA to regulate miRNAs in cancer progression [
51]. As we have stated before,
TUSC7 acts as a tumor suppressor in human cancers by interacting with miRNAs, such as miR-23b [
20] and miR-211 [
38]. It has been reported that miR-10a could facilitate cell migration, invasion, and EMT by directly targeting the 3′-UTR of
EphA4 transcript to reduce its expression in HCC [
32].
EphA4 could inhibit cell migration and invasion by regulating the EMT process through the β1-integrin signaling pathway [
32]. Hence, combining our previous results and the bioinformatics analysis, we focused on miR-10a and its downstream target
EphA4. Our results showed that miR-10a was indeed a downstream target of
TUSC7. We found that acting as a sponge of miR-10a,
TUSC7 could therefore directly interact with miR-10a to restrain its function. Thus, when the expression level of
TUSC7 was reduced, its inhibition on miR-10a would be attenuated. The expression level of miR-10a would then be increased, which could lead to decreased expression of
EphA4. Therefore, we have confirmed that the downregulation of
TUSC7 could enhance miR-10a expression to reduce
EphA4 expression, thereby promoting migration, invasion, and EMT in HCC, at least in part.
In summary, our data indicate that TUSC7 may function as a tumor suppressor in HCC. Mechanistically, our experimental data demonstrate that targeting the TUSC7-miR-10a-EphA4 axis may represent a novel therapeutic application in HCC.