Background
Hepatocellular cancer (HCC) is the most frequent type of malignancy originating from the liver with a recently rising incidence in the United States [
1]. It is the second most common cause of cancer-related death worldwide with more than 500,000 new cases per year. The incidence of the disease approximates the death rate, which reflects the aggressiveness of this tumor [
2]. HCC is one of the few types of cancer in which the various risk factors are well characterized. Specifically, infections with the hepatitis B and C virus as well as aflatoxin B1 (AFB) are responsible for almost 80 % of the cases [
3].
At the same time, the molecular mechanisms that lead to the pathogenesis of HCC are not completely understood. Up to date, there are several genes involved in the signaling pathways essential for the initiation and progression of hepatocellular carcinogenesis and these include, but are not limited to, c-myc, PTEN, e-cadherin, cyclin D1 and p53 [
4].
MicroRNAs are small non-coding RNA molecules, 18–25 nt long, that act as negative regulators of gene expression, through binding in the 3′UTR of the coding sequence of genes [
5]. Previous studies have identified different microRNAs to be deregulated in liver pre-cancerous and cancer stages [
6,
7]. Specifically, microRNAs have been identified to regulate cell cycle through regulation of cyclin G1 [
8]. In addition, miR-21 was identified to have a potent oncogenic potential in HCC by blocking directly the PTEN tumor suppressor gene [
9]. Furthermore, another study revealed a 20-microRNA metastasis signature that could significantly predict primary HCC tissues with venous metastases from metastasis-free solitary tumors with 10-fold cross-validation [
10]. Interestingly, Xu Y et al. showed that a polymorphism in the promoter region of miR-34b/c was associated with an increased risk for primary hepatocellular carcinoma [
11]. Also, serum microRNAs were found to potentially serve as biomarkers for HBV infection and diagnosis of HBV-positive HCC [
12], suggesting the potential of measuring circulating microRNA levels as biomarkers in HCC. However, it has not been extensively studied which microRNAs have both clinical and functional relevance in this type of cancer. Here, we are describing that miR-9 is potentially a novel oncogene in liver cancer, regulating the tumor initiation, growth and metastatic potential of liver cancer cells. On the other hand, inhibition of miR-9 expression blocks the tumor properties of liver cancer cells, including cell growth and migration, suggesting its therapeutic potential. Interestingly, we found that miR-9 suppressed CDH1 mRNA expression levels, directly through binding in its 3′UTR and indirectly through regulation of PPARA expression levels. Taken together, this study reveals a novel role for the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis.
Methods
RNA from HCC and liver control samples
RNA was extracted from 24 Fixed-Formalin- Paraffin-Embedded (FFPE) HCC and 14 liver control (adjacent non-tumor) tissue specimens obtained from consenting patients in the Department of Surgery at Stanford University and were approved by the Ethics Committee of the Stanford University Medical School.
MicroRNA library screen
SNU-449 liver cancer cells were plated in 96-well plates and transfected with a microRNA library consisting of 316 microRNA mimics and 2 negative control microRNAs (100 nM) (Dharmacon Inc). At 48 h post-transfection, SNU-449 cell invasiveness was evaluated in Boyden chamber invasion plates. Assays were conducted according to manufacturer’s protocol, using 2 % FBS as a chemoattractant. Invading cells were fixed and stained with 0.1 % crystal violet, 24 h post seeding. The cells that migrated through the filter were quantified by counting the entire area of each filter. MicroRNAs that affected >2-fold (50 %) SNU-449 invasiveness relative to microRNA negative control treated SNU-449 cells were considered as positive hits.
Invasion assay
We performed invasion assays in SNU-449 cells 24 h after transfection with miR-9 or anti-miR-9 and their respective controls. Invasion in matrigel has been conducted by using standardized conditions with BD BioCoat Matrigel invasion chambers (BD Biosciences). Assays were conducted according to manufacturer’s protocol, using 2 % FBS as the chemoattractant. Non-invading cells on the top side of the membrane were removed, while invading cells were fixed and stained with 0.1 % crystal violet, 24 h post-seeding. The cells that migrated through the filter were quantified by counting the entire area of each filter, using a grid and an Optech microscope at a 20× magnification.
Real-time PCR analysis
Quantitative real-time RT-PCR was performed to determine the expression levels of miR-9, miR-21 and miR-224 in 24 human HCC (stage I n = 5; stage II n = 9; stage III n = 6; stage IV n = 4) and 11 liver control tissues. RNA was isolated using Trizol, according to manufacturer’s instructions (Invitrogen). Real-time RT-PCR was assessed on a CFX384 detection system (BioRad) using the Exiqon PCR primer sets according to manufacturer’s instructions. MicroRNA expression levels were normalized to the levels of U6 small nuclear snRNA (203907, Exiqon). Normalized miRNA levels were quantified relative to the levels of a given control tissue. Real-time PCR was employed to determine the expression levels of CDH1, PPARA, vimentin and PDK4. Reverse transcription was carried out using the Retroscript Kit (AM1710, Applied Biosystems). Real-time PCR was carried out using the IQ SYBR Green Supermix (170–8882, BioRad). Gene expression levels were normalized to the levels of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin. Normalized gene expression levels were quantified to the respective control. The sequences of the primers used are the following:
CDH1-F: 5′-TGAAGGTGACAGAGCCTCTGGAT-3′
CDH1-R: 5′-TGGGTGAATTCGGGCTTGTT-3′
PPARA-F: 5′-GGCGAGGATAGTTCTGGAAGC-3′
PPARA-R: 5′-CACAGGATAAGTCACCGAGGAG -3′
Vimentin-F: 5′-CCAAACTTTTCCTCCCTGAACC -3′
Vimentin-R: 5′-GTGATGCTGAGAAGTTTCGTTGA -3′
PDK4-F: 5′-CCCCGAGAGGTGGAGCAT-3′
PDK4-R: 5′-GCATTTTCTGAACCAAAGTCCAGTA-3′
SNU-449 and HepG2 liver cancer cell lines were transfected with miR-9 or anti-miR-9 and their respective controls. Then, triplicate samples of 2×105 cells from each cell line were assayed for colony formation using the CytoSelect Cell Transformation kit (Cell Biolabs, Inc). The number of colonies were counted after 7 days.
Cell growth assay
SNU-449 and HepG2 liver cancer cell lines were transfected with miR-9 or the respective control and plated on a 96-well plate (5×103 cells/well). 48 and 72 h later, cell growth was assessed using the Cell-Titer Glo Luminescence Cell Viability Assay (Promega).
SNU-449 liver cancer cell lines were transfected with miR-9 or anti-miR-9 were plated in ultra-low attachment plates (Corning), 24 h post-transfection and were grown in DMEM F12 (Invitrogen) medium supplemented with B-27 (Gibco), bFGF and EGF in the culture medium containing 1 % methyl cellulose to prevent cell aggregation. The number of spheres was evaluated 6 days post plating.
3′UTR luciferase assay
SNU-449 cells were transfected with the reporter vectors carrying the 3′UTR of CDH1 (cat. no 25038, Addgene) or PPARA (cat. no HmiT054001-MT06, Genecopoeia). The constructs harbored the seed sequence of miR-9 (wildtype) or had a deletion of this sequence (miR-9 mutant). At 24 h, they were transfected with miR-9 or miR-control and at 48 h luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega).
Statistical analysis
All experiments were performed in triplicate unless otherwise stated. Statistical analyses were performed with the use of Origin software, version 8.6. Student’s t-test was used to examine the statistical difference in miR-9 expression between control and HCC tissues. The correlation significance was determined by means of Spearman and Pearson correlation analyses. A P-value of 0.05 or less was considered statistically significant.
Discussion
Different signaling pathways have been implicated in HCC pathogenesis [
19], however the role of non-coding RNAs has not been studied extensively until recently. Non-coding RNAs consist primarily of the microRNAs and long non-coding RNA (lincRNAs) and several studies have implicated their role in HCC initiation and progression [
6,
7,
20,
21]. Specific microRNA signatures have been identified to be deregulated in HCC patient tissues and also to correlate with different clinicopathological parameters [
10,
22]. Furthermore, microRNAs have been associated with hepatitis infection, cirrhosis and patient survival [
23].
In this study, we have screened the human microRNAome, aiming to identify microRNAs that are potent regulators of HCC invasiveness. Interestingly, we found 28 microRNAs to affect significantly (>2-fold) the invasiveness of SNU-449 liver cancer cells. Five of these microRNAs behaved as HCC invasion inducers, while 23 microRNAs as HCC invasion suppressors. This screen revealed novel microRNAs potentially involved in HCC pathogenesis and also validated findings from previous studies. Specifically, microRNAs such as miR-21, miR-29a/b, miR-26a, miR-101, miR-122a, miR-124a, miR-375 and let-7a/b have been correlated with HCC pathogenesis through regulation of essential signaling pathways [
9,
24‐
30]. More recently, we have identified that miR-24 is part of a feedback loop circuit involved in HCC pathogenesis [
7]. On the other hand the role of miR-9, miR-148b, miR-203 and miR-507 in HCC pathobiology is not well understood. Recently, high miR-9 expression levels were found to be correlated with poor prognosis in HCC patients [
31]. Furthermore, miR-148b expression was found to be decreased in HCC patients [
32], however it is not known which signaling pathways are mediators of miR-148b activity in HCC. In addition, it has been shown that miR-203 is suppressed in HCC tissues due to DNA methylation on its regulatory area [
33]. Finally, nothing is known regarding the role of miR-507 in HCC pathogenesis.
Here, we provide evidence that miR-9 affects different liver cancer cell properties, including liver tumor sphere formation. When liver cancer cells are placed in low attachment plates or in suspension, they have the ability to form liver tumor spheres, which potentially represent the cellular population harboring tumor-initiating properties [
34,
35]. Here, we evaluated for the first time the role of miR-9 to affect the growth of these liver tumor spheres and identified that miR-9 overexpression induced the formation of liver spheres derived from SNU-449 cells, suggesting its potential involvement in early stages during HCC oncogenesis. On the other hand, inhibition of miR-9 by an anti-sense microRNA-9 molecule, suppressed the growth of SNU-449-derived tumor spheres.
Bioinformatics and molecular analyses revealed that miR-9 is involved in HCC pathogenesis through direct regulation of CDH1 and PPARA genes, by binding on their 3′UTR regions. Previous studies have shown that reduced expression of CDH1 correlate with poor outcomes in HCC patients [
36]. Consistent with our findings, Tan HX et al. showed that miR-9 was significantly up-regulated in primary HCC tumors with metastases in comparison with those without metastases [
37]. In the same study, CDH1 levels were found to be up-regulated after miR-9 inhibition. Other studies have shown that high levels of CDH1 have been correlated with suppression of liver carcinogenesis [
38]. In addition, we found that miR-9 overexpression resulted in increased vimentin levels, which is a well-known mesenchymal marker correlated with CDH1 loss of expression in HCC [
39]. More importantly, the role of PPARA in HCC pathogenesis has not been previously described. PPARA is a transcription factor that has been implicated in hepatic steatosis [
40] and hepatic metabolic homeostasis through regulation of the hepatocyte nuclear factor-4 alpha (HNF4A) gene [
41]. Interestingly, we have recently found that HNF4A is a tumor suppressor gene in HCC pathogenesis [
7]. Furthermore, it has been described that there is a positive correlation between CDH1 and the PPARA signaling pathways [
17,
18]. Our analysis revealed that there is not only a positive correlation between PPARA and CDH1 mRNA levels in HCC, but also that PPARA regulates CDH1 mRNA expression levels in HCC. This observation is very interesting and novel, since miR-9 is using two discrete molecular pathways to suppress CDH1 expression in HCC. First, miR-9 directly suppresses CDH1 mRNA levels through binding on its 3′UTR and in the second indirect mechanism miR-9 suppresses PPARA mRNA levels directly, resulting in decreased CDH1 levels. Overall, these data suggest that microRNAs could use complementary mechanisms to regulate a specific downstream signaling target.
Recent studies have shown that manipulation in the expression levels of microRNAs could have therapeutic potential
in vitro and
in vivo. Specifically, administration of miR-26a or miR-124a has resulted in suppression of liver cancer tumor growth in vivo [
7,
42]. On the other hand, miR-21 inhibition suppresses HCC growth [
43]. Here, we have found that miR-9 inhibition of expression by an antisense-miR-9 suppressed the ability of liver cancer cells to form colonies in soft agar, tumor spheres and decreased their invasiveness, suggesting that targeting miR-9 could be a promising strategy to be further evaluated for the treatment of HCC.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conception and design of study: AD, DI, VG; Data acquisition, analysis and interpretation: AD, MH, CV, CP, GAP; Writing and revising the manuscript: AD, JS, VG; Study supervision: DI, MH. All authors have read and approved the final manuscript.