Background
Hepatocellular carcinoma (HCC) is the fifth most common malignancies worldwide and the second frequent cause of cancer-related death according to world health organization (WHO) data [
1,
2]. Despite remarkable advances in diagnosis and improvement in therapeutic modalities, including novel chemotherapeutic interventions and target therapy, the long-term survival of HCC patients remains unsatisfactory due to the high rates of intrahepatic and distal metastasis [
3,
4]. Therefore, it is critical to identify the potential molecular mechanisms underlying the progression and metastasis in HCC and thus provide novel therapeutic targets for cancer treatment.
MicroRNAs (miRNAs), a group of endogenous evolutionarily conserved non-coding small RNAs, act as post-transcriptional regulator of gene expression in cancer initiation, development and progression by binding to complementary sequences within the 3′-untranslated region (UTR) of target mRNA and subsequently inducing mRNA degradation or translational repression [
5,
6]. Numerous evidences confirm that aberrantly expressed miRNAs play critical roles in multiple biological progresses in HCC [
7‐
10], including cell proliferation, apoptosis, drug-resistance, metastasis and stem cell renewal, and have been identified as promising therapeutic and prognostic biomarkers in HCC diagnosis and treatment.
MiR-1296, a novel cancer-related miRNA, has been found to be dysregulated in cancers [
11‐
13]. Zhu et al. demonstrated that miR-1296 was involved in the regulation of cell migration and invasion in human gastric cancer via targeting ERBB2/Rac1 signaling pathway [
14]. MiR-1296 was significantly decreased in triple-negative breast cancer, and promoted cell cycle arrest and cisplatin sensitiveness of breast cancer cells [
15]. Moreover, miR-1296 increased resistance to chemotherapeutic treatment and could be used as a new potential biomarker for breast cancer stem cell diagnosis [
16]. These data suggest that miR-1296 plays a tumor suppressive role in malignancies. In addition, miR-1296 upregulation could serve as a predictive marker for the colon cancer cases with subsequent relapse [
17], which indicates that miR-1296 serves as an oncogene in colon cancer. Taken together, the expression level and biological function of miR-1296 is cancer-specific. Nevertheless, the function of miR-1296 and its underlying molecular mechanisms in HCC remain unknown.
Epithelial-to-mesenchymal transition (EMT), is implicated in the invasion and metastasis of various cancers through transformation of adherent and polarized epithelial cells into an invasive mesenchymal cell phenotype [
18,
19]. Moreover, the typical EMT process usually is characterized by decrease of the cell adhesion molecule E-cadherin and increase of Vimentin and N-cadherin expression. E-cadherin is an important determinant of epithelial cell-cell adhesion, while Vimentin and N-cadheirn are the mesenchymal markers [
20,
21]. Increasing studies reveal that EMT is a main cause for HCC invasion and metastasis [
22,
23]. However, the association between miR-1296 and EMT in HCC is poorly investigated.
Our results showed that underexpression of miR-1296 was associated with poor prognostic features of HCC patients. MiR-1296 inhibited migration, invasion and EMT progression of HCC cells in vitro and in vivo. Notably, serine-arginine protein kinase 1 (SRPK1) was identified as a direct target of miR-1296 and mediated the function of miR-1296 in HCC cells. In addition, miR-1296, SRPK1 and their combination were valuable predictors for the prognosis of HCC patients.
Methods
Clinical tissues
One hundred and twenty-six HCC tissues and matched adjacent non-tumor tissues were collected from Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi’an Jiaotong University during January 2009 to December 2011. Another cohort of ninety-eight HCC specimens were obtained from Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital. Pathological diagnosis was performed according to the WHO criteria. The tissues were stored at −80 °C or embedded in paraffin. None of the patients received chemotherapy or radiotherapy before surgery. Written informed consent were obtained from all patients.
The human HCC cell lines including MHCC-97 L, HCCLM3, MHCC-97H, Huh7, Hep3B and the normal human immortalized normal hepatic cell line LO2 were purchased from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China) and were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, USA) containing 10% FBS (Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA) in a humidified atmosphere at 37 °C with 5% CO2.
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
Total RNA from HCC tissues and cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. cDNA was reverse-transcribed from 2 μg total RNA using a Reverse Transcription Kit (Takara, Biochemical, Tokyo, Japan). cDNA was then amplified with a SYBR® Premix Ex Taq™ II (Perfect Real-Time) kit (Takara). The gene expression levels were calculated using the delta-delta Ct method with U6 or GAPDH as an internal control. Hsa-miR-1296 primer (HmiRQP0143), snRNA U6 qPCR Primer (HmiRQP9001), SRPK1 (HQP017724) and GAPDH (HQP006940) were purchased from Genecopoeia (Guangzhou, China).
Cell transfection
MiRNA vectors, including precursor miR-1296 clones (HmiR0471), precursor miR-1296 scrambled control clones (miR-control; CmiR0001), miR-1296 inhibitors (anti-miR-1296; HmiR-AN0143) and miR-1296 inhibitor control clones (anti-miR-NC; CmiR-AN0001) were obtained from Genecopoeia (Guangzhou, China). The SRPK1 overexpression plasmid and specific siRNA against SRPK1 and a scramble siRNA were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Cells were transfected with above vectors using Lipofectamine 2000 Reagent (Invitrogen Life Technologies) in accordance with the manufacturer’s protocol.
Western blot analysis
The whole proteins were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche) and the concentrations were quantified with BCA Protein Assay Kit (Tiangen, Beijing, China), and an equal amount of 40 μg protein was separated by 10% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk in TBST for 2 h at room temperature and incubated overnight with specific primary antibodies at 4 °C. Then the membranes were washed three times by TBST and incubated with HRP-conjugated secondary antibody for 2 h at room temperature (ZSGB-BIO, China). Detection was performed by enhanced chemiluminescence kit (Amersham, Little Chalfont, UK). GAPDH (G8140; US Biological, Swampscott, MA, USA) was used as protein loading control. The SRPK1 primary antibody was obtained from Abcam (Cambridge, MA, USA). The antibodies against E-cadherin, N-cadherin, Vimentin, AKT, p-AKT, ZO-1, ZEB1, Slug, Snail, Twist, ERBB2, CCND1 and MCM2 were purchased from Cell Signaling Technology (Beverly, MA, USA).
Immunofluorescence (IF)
HCC cells that transfected with corresponding miRNA vectors were seeded on chamber slides and were fixed with 4% paraformaldehyde for 10 min at room temperature. Then, cells were incubated with antibodies against E-cadherin (Cell Signaling Technology) or Vimentin (Cell Signaling Technology) at 4 °C overnight. Then, the slides were incubated with matched secondary antibodies (Invitrogen) at room temperature for 1 h. The nuclear of EC cells were stained with DAPI (Sigma) at room temperature for 10 min. Fluorescence confocal images were captured using a LSM 5 Pascal Laser Scanning Microscope (Zeiss Germany, Oberkochen, Germany).
Cell migration and invasion analyses
Matrigel-uncoated and -coated transwell inserts (8 μm pore size; Millipore) were used to evaluate cell migration and invasion. Briefly, 2 × 104 transfected cells were suspended in 150 μL serum free DMEM medium into the upper chamber, and 700 μL DMEM medium containing 20% FBS was placed in the lower chamber. After 24 h incubation, cells were fixed in 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet dye for 15 min. The cells on the inner layer were softly removed with a cotton swab and counted at five randomly selected views, and the average cell number per view was calculated.
Immunohistochemistry (IHC) analysis
Briefly, 4 μm sections were deparaffinized in xylene, rehydrated through graded ethanols, followed by blocking of endogenous peroxidase activity in 3% hydrogen peroxide for 10 min at room temperature. The corresponding antibody (1:300, Cell Signaling Technology, Inc.) was applied as the primary antibody by a streptavidin peroxidase-conjugated (SP-IHC) method. The staining results were semi-quantitatively evaluated by the multiply of staining intensity and the percentage of positive staining cells. The percentage of positive cells was given into four grades: 0 for <5%; 1 for 6%–25%; 2 for 26%–50%; 3 for 51%–75% and 4 for >75%. Staining intensity was assessed by four degrees: 0, negative; 1, weak; 2, moderate; and 3, strong. Each section was assayed for ten independent high magnifications (×400) fields to get the average scores.
Luciferase reporter assay
The 3′-UTR sequence of SRPK1 predicted to interact with miR-1296, together with a corresponding mutated sequence within the predicted target sites, were synthesized and inserted into the pmiR-GLO dual-luciferase miRNA target expression vector (Promega, Madison, WI, USA) called wt-SRPK1 3′-UTR and mt-SRPK1 3′-UTR. Subsequently, HCCLM3 or Hep3B cells that were plated into 24-well plate and were transfected with corresponding vectors. Cells were co-transfected with the wild-type or mutant 3′-UTR of SRPK1 vector using the Lipofectamine 2000 reagent (Invitrogen, USA). After 48 h, cells were harvested and measured according to the manufacturer’s instructions (Dual-Luciferase Assay System; Promega). pRL-TK expressing Renilla luciferase was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies.
In vivo experiments
4–6 week-old female BALB/c nude mice (Centre of Laboratory Animals, The Medical College of Xi’an Jiaotong University, Xi’an, China) were randomized into two groups (n = 5), and either HCCLM3-miR-1296 or HCCLM3-miR-control cells (1 × 106); Hep3B-anti-miR-1296 or Hep3B-anti-miR-NC were injected into the tail veins for the establishments of pulmonary metastatic model. Mice were sacrificed 10 weeks’ post injection and examined microscopically by hematoxylin and eosin (H&E) staining for the development of lung metastatic foci. Animals were housed in cages under standard conditions. The protocols for these animal experiments were approved by the Ethics Review Committee of Xi’an Jiaotong University.
Statistical analysis
Data are presented as the mean ± SD and performed at least three independent replicates. SPSS software, 16.0 (SPSS, Inc., Chicago, IL, USA) and Graphpad Prism 6.0 (CA, USA) were used for a two-tailed Student t-test, Pearson’s correlation analysis, Kaplan-Meier method and the log-rank test to evaluate the statistical significance. Differences were defined as P < 0.05.
Discussion
Numerous studies demonstrated that aberrantly expressed miRNAs were involved in the cancer initiation, development and progression, including HCC [
30]. MiRNAs have been identified as novel prognostic biomarkers and effective therapeutic targets of HCC [
31]. In this research, we found that miR-1296 was significantly down-regulated in HCC tissues and cell lines for the first time. Moreover, the aggressive and recurrent phenotype of HCC showed a lower expression of miR-1296. These data indicate that miR-1296 plays a tumor suppressive role in HCC.
Local and systemic metastasis is a major cause leading to a dismal prognosis of HCC. Increasing evidences confirm that miRNAs are recognized as key regulators in metastasis of cancers, including HCC [
32,
33]. In this study, gain- and loss-of-function experiment confirmed that miR-1296 overexpression inhibited the migration and invasion of HCC cells while miR-1296 knockdown increased these metastatic behaviors in vitro and in vivo. EMT is a critical process in the invasion and metastasis of HCC [
34]. In present study, we demonstrated that miR-1296 suppressed EMT events of HCC cells. Moreover, we also found that the miR-1296 high expressing HCC tissues showed increased expression of E-cadherin and decreased expression of Vimentin. These results suggest that miR-1296 inhibits HCC metastasis by suppressing EMT phenotype.
SRPK1, a highly conserved protein in precursor mRNA translation and splicing, chromatin reconstruction, is dysregulated in different cancers [
26,
35]. Moreover, SRPK1 play a critical role in EMT process of human glioblastoma [
36]. Here, we confirmed that SRPK1 was a direct downstream target of miR-1296 and mediated the biological function of miR-1296 in HCC. First, miR-1296 negatively regulated SRPK1 abundance in HCC cells. Second, the complementary sequences of miR-1296 were identified in the 3’UTR of SRPK1 mRNA. MiR-1296 overexpression or knockdown accordingly altered the luciferase activity of wt 3’UTR but not mt 3’UTR of SRPK1. Third, miR-1296 was inversely correlated with the expressions of SRPK1 in HCC tissues. Next, we demonstrated that SRPK1 mediated miR-1296-modulated migration, invasion and EMT process of HCC cells. Previous study shows that SRPK1 functions as an oncogene via promoting activation of PI3K/AKT signaling [
26]. Activation of PI3K/AKT signaling pathway is involved in the development and progression of HCC and regulates the malignant biological function of cancer cells [
37,
38]. Moreover, PI3K/AKT plays a crucial role in EMT process of HCC [
39]. Herein, we discovered that miR-1296 restrained the activation of PI3K/AKT signaling. The activator of PI3K/AKT pathway abrogated the inhibitory effect of miR-1296, while AKT inhibitor reversed the promoting effects of miR-1296 knockdown on migration, invasion and EMT process of HCC cells. Therefore, SRPK1/AKT pathway may be involved in the role of miR-1296 in HCC cells.
It’s necessary to confirm whether miR-1296 and SRPK1 could serve as valuable biomarkers for diagnosis and prognostic prediction. Here, we found that both low expression of miR-1296 and high level of SRPK1 were significantly associated with adverse clinical features of HCC patients. In addition, we confirmed that miR-1296 underexpression and SRPK1 overexpression as well as their combination were obviously correlated with poor prognosis of HCC patients. These results suggest that miR-1296 and SRPK1 may be promising predictors for the prognosis of HCC patients.
Previous study confirms that serine-arginine (SR) protein phosphorylation is increased in hypoxia condition [
28]. SRPK1 dissimilarly impacts the growth, metastasis, chemosensitivity and angiogenesis of glioma in hypoxic conditions [
29]. Moreover, hypoxia environment is a critical cause for HCC metastasis and leads to abnormal expression of miRNAs [
40,
41]. Therefore, we tried to explore the relationship between hypoxia and miR-1296 in HCC. Our data showed that miR-1296 expression was significantly decreased in hypoxia. Moreover, miR-1296 restoration abolished the promoting effects of hypoxia on migration, invasion and EMT process of HCC cells. These results suggest that hypoxia-induced miR-1296 loss promotes the metastasis and EMT of HCC.
In conclusion, we demonstrate for the first time that miR-1296 is underexpressed in HCC tissues and cell lines, and its reduced expression is correlated with malignant clinicopathological features. Furthermore, we confirm that miR-1296 inhibits migration, invasion and EMT process of HCC cells probably by directly targeting SRPK1-mediated PI3K/AKT pathway. Notably, miR-1296 underexpression, SRPK1 overexpression and their combination are potential prognostic predictors for the survival of HCC patients. Moreover, hypoxia is a key cause for miR-1296 underexpression in HCC cells. In summary, the deregulation of miR-1296 may play an important role in tumor metastasis and may be a novel prognostic factor and potential therapeutic target for HCC.
Acknowledgements
Not applicable.