Background
Hepatocellular carcinoma (HCC) is the fifth most common malignancies worldwide and the second leading cause of cancer-related death which contribute to increasing morbidity and mortality in China according to world health organization (WHO) [
1,
2]. Despite great advancement in diagnosis and therapeutic strategy, including novel chemotherapeutic interventions and liver transplantation, the long-term survival remains dismal because of high rate of intrahepatic and distal metastasis [
3,
4]. Therefore, it is urgent to elucidate the molecular mechanisms underlying HCC progression and develop promising biomarkers for cancer treatment.
MicroRNAs (miRNAs), a family of small, single-stranded and non-coding evolutionarily conserved RNAs of approximately 21–25 nucleotides in sequence length, act as post-transcriptional modulator of gene expression in cancer progression by interacting with complementary sequences within the 3′-untranslated region (UTR) of target mRNA to induce mRNA degradation or translational repression [
5‐
7]. Increasing evidence confirm that dysregulated miRNAs are involved in various biological processes in HCC [
8], including cell proliferation, cell cycle, apoptosis and metastasis [
9,
10]. Therefore, miRNAs have been recognized as promising therapeutic and prognostic biomarkers in HCC diagnosis and treatment. MiR-1468, a novel cancer related microRNA, was dysregulated and could predict patients’ survival in diverse cancers [
11]. Jiang et al. confirmed that miR-1468 inhibited cell proliferation and induced cell cycle arrest by targeting ribonucleotide reductase large subunit M1 (RRM1) in glioma [
12]. In papillary renal cell carcinoma (pRCC), miR-1468 was significantly associated with patient survival and identified by multivariate Cox regression analyses as potential independent prognostic factors in pRCC [
13]. In lung adenocarcinoma, miR-1468 expression was significantly correlated with recurrence-free survival [
14]. Moreover, miR-1468 was decreased in blood-based microRNA biomarker in early diagnosis of pancreatic cancer [
15]. Previous study reported that miR-1468 showed an up-regulated tendency and might be a potential prognostic biomarker in HCC samples derived from The Cancer Genome Atlas (TCGA) database [
16]. Nevertheless, the function of miR-1468 and its underlying molecular mechanisms in HCC remain unknown.
Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-activated transcription factor, which belongs to the nuclear receptor superfamily and exerts its function on tumor promotion, cellular differentiation, cell cycle and apoptosis [
17,
18]. In HCC, PPAR-γ has been identified as a tumor suppressor gene and mediated apoptosis of HCC cells depends on modulation of phosphatidylinositol 3-kinase (PI3K) pathway [
19,
20]. Moreover, recent findings using PPAR-γ knockout mice suggest that PPAR-γ reduces HCC carcinogenesis and metastasis and acts as a tumor-suppressor gene in the liver [
21]. PPAR-γ agonist induces apoptosis by triggering the intrinsic apoptosis pathway and inhibiting PI3K/AKT survival pathway in human cervical cancer cells [
22]. Our previous study confirms that miR-130b promotes cell aggressiveness by inhibiting PPAR-γ in human HCC [
23]. These findings indicate that PPAR-γ regulates HCC tumorigenesis and progression.
In present research, we demonstrated that miR-1468 overexpression was associated with poor prognostic features and reduced survival of HCC patients. MiR-1468 promoted the growth of HCC cells in vitro and in vivo. Furthermore, we confirmed that miR-1468 inhibited PPAR-γ/AKT signaling activity through directly suppression of Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 (CITED2) and Up-frameshift protein 1 (UPF1). Therefore, our results confirm that miR-1468 exerts a critical role in HCC progression and represents a potential target for HCC diagnosis and treatment.
Methods
Patients’ tissues and cell culture
Patients’ tissues and paired adjacent non-tumor tissues were obtained from 99 patients in our hospital after the informed consent were obtained from all patients. All patients didn’t receive any therapy including radiotherapy, chemotherapy or radiofrequency ablation before surgery. The clinicopathological and demographic information of the patients was described in Table
1. The normal immortalized human hepatocyte LO2 and a panel of HCC cells (Hep3B, HepG2, Huh7, MHCC-97 L and SMMC-7721) (Chinese Academy of Sciences, Shanghai, China) were maintained in DMEM (Invitrogen, Carlsbad, USA) containing 10% FBS (Gibco, GrandIsland, USA) in 37 °C with 5% CO2.
Table 1
Clinical correlation of miR-1468 expression in hepatocellular carcinoma (n = 99)
Age (years) |
< 50 | 32 | 18 | 14 | 0.429 |
≥ 50 | 67 | 32 | 35 |
Gender |
Male | 73 | 36 | 37 | 0.692 |
Female | 26 | 14 | 12 |
Tumor size (cm) |
< 5 | 75 | 32 | 43 | 0.006*
|
≥ 5 | 24 | 18 | 6 |
Tumor number |
Solitary | 85 | 53 | 42 | 0.684 |
Multiple | 14 | 7 | 7 |
Edmondson-Steiner grading |
I + II | 77 | 34 | 43 | 0.018*
|
III + IV | 22 | 16 | 6 |
TNM stage |
I + II | 79 | 35 | 44 | 0.014*
|
III + IV | 20 | 15 | 5 |
Venous infiltration |
Present | 15 | 8 | 7 | 0.812 |
Absent | 84 | 42 | 42 |
AFP (ng/ml) |
< 400 | 27 | 15 | 12 | 0.538 |
≥ 400 | 72 | 35 | 37 |
HBsAg |
Positive | 90 | 44 | 46 | 0.487 |
Negative | 9 | 6 | 3 |
Quantitative real-time polymerase chain reaction (qRT-PCR)
qRT-PCR was conducted as reported previously [
10,
24,
25]. All RNA was extracted based on the protocol of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). qPCR primer against miR-1468 (HmiRQP0193), U6 (HmiRQP9001), CITED2 (HQP062677), UPF1 (HQP071077) and GAPDH (HQP006940) were ordered from Genecopoeia (Guangzhou, China).
Immunohistochemical staining (IHC)
The sections were dewaxed, dehydrated, and rehydrated. CITED2, UPF1 (1:100, Cell Signaling, Danvers, MA, USA) were added to the sections and incubating at 4 °C overnight. Then applying the biotinylated secondary antibodies (Goldenbridge, Zhongshan, China) according to SP-IHC assays. Specific experiment was conducted similar to previously reported [
24,
26].
Immunofluorescence (IF)
We used 4% paraformaldehyde to fix transfected cells and used 0.3% Triton X-100 to permeabilize. The primary antibody CITED2 (Novous Biologicals, Inc. Littleton, CO, USA), UPF1 (Cell Signaling Technology, Danvers, USA) was used. Then the Alexa Fluor-conjugated secondary antibody was performed the next experiment. Lastly, the images were taken by Microscope (Zeiss, Germany).
Western blot analysis
We separated proteins by SDS–PAGE and transferred proteins to PVDF membranes. Detailed experiment was performed similar to previously reported [
24,
26].
RNA interference transfection
The CITED2, UPF1 and a negative control siRNA were synthesized by GenePharm (Shanghai, China). Hep3B and MHCC-97 L cells (2 × 105 per well) were transfected in a concentration of 100 nM siRNA.
Cell proliferation, cell cycle and apoptosis detection
Cell Counting Kit-8 (CCK8) reagents (Dojindo, Kumamoto, Japan), EdU, cell cycle, colony formation and apoptosis were carried as described previously [
10,
27].
Luciferase reporter assay
The 3’-UTR sequence of CITED2 and UPF1, 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). The assays were carried out as described previously [
10,
28].
In vivo experiments
Four-to-six-week-old male BALB/c nude mice (Centre of Laboratory Animals, The Medical College of Xi’an Jiaotong University, Xi’an, China) were used to establish the nude mouse xenograft model. Hep3B (5 × 106) cells that were transfected with miR-1468 or miR-control vectors or MHCC-97 L cells with anti-miR-1468 were mixed in 150 μl of Matrigel and were inoculated subcutaneously into the flank of nude mice. The tumor volume for each mouse was determined by measuring two of its dimensions and then calculated as tumor volume = length × width × width/2. After 3 weeks, the mice were sacrificed by cervical dislocation under anesthesia with ether and the xenograft tumor tissue was explanted for examination. Animal protocols were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University.
Statistical analysis
Results are managed as the mean ± SD and analyzed by SPSS software, 16.0 (SPSS, Chicago, USA). The statistical approaches mainly included a two-tailed Student’s t test, a Kaplan–Meier plot, Pearson chi-squared testand so on. Difference with P < 0.05 was regard to be significant. Graphs were mainly made by GraphPad Prism 6 (GraphPad, San Diego, USA).
Discussion
Increasing evidence confirm that dysregulation of miRNAs plays a critical role in cancer initiation, development and progression, including HCC [
32]. Recently, miRNAs have been recognized as diagnostic, therapeutic and prognostic markers [
33,
34]. In present study, we confirmed that miR-1468 was up-regulated in HCC tissues and cell lines, which was consistent with TCGA data. Moreover, increased miR-1468 level was obviously correlated with malignant clinicopathological features, including large tumor size, high histological grade and advanced tumor stage. Moreover, miR-1468 was an independent prognostic factor in predicting survival of HCC patients. These data suggest that miR-1468 is involved in HCC progression and represents as a promising biomarker for HCC therapy.
Previous study has confirmed that blood-based circulating miR-1468 is evaluated as a diagnostic marker panel for early diagnosis of pancreatic cancer [
15]. In this study, we demonstrated that miR-1468 promoted cell proliferation, cell cycle progression, colony formation and inhibited apoptosis by gain- and loss-of function experiment in vitro and in vivo. Moreover, we provided the first evidence that the target genes of miR-1468 were CITED2 and UPF1. First, miR-1468 negatively modulated CITED2 and UPF1 expression in HCC cells. Second, miR-1468 affected the luciferase activity of wt 3’UTR but not mt 3’UTR of CITED2 and UPF1. Third, miR-1468 was inversely correlated with the expression of CITED2 and UPF1 in HCC tissues. Furthermore, CITED2 and UPF1 were down-regulated in HCC tissues, and modulating their expression could reverse the biological function of miR-1468 in HCC cells. CITED2 is a transcriptional co-regulator that directly interacts with host of transcription factors and co-activators, and influences the activation of gene transcription [
35,
36]. CITED2 is a potent prognostic predictor and associated with proliferation, migration and chemoresistance in breast cancer [
37]. Moreover, CITED2 serves as a coactivator of hepatocyte nuclear factor 4α (HNF4α) in liver development [
38]. CITED2 is a novel direct effector of PPAR-γ in suppressing HCC cell growth [
29]. UPF1 is essential for accomplishing DNA replication during S phase of cell cycle [
39]. The human RNA surveillance factor UPF1 inhibits hepatic cancer progression and EMT progress by targeting MRP2/ABC2 [
40]. Moreover, UPF1 regulates HCC tumorigenesis by up-regulation of SMAD family member 7 (Smad7) and affecting transforming growth factor β (TGF-β) pathway [
41]. In this research, we also confirmed that CITED2 and UPF1 were down-regulated in HCC tissues compared to adjacent non-tumor tissues. These data provide more evidence for establishing therapeutic strategies in miRNA-modulating networks.
Previous study reveals that the target genes of miR-1468 are enriched in PPAR signaling pathway [
16]. CITED2 signals through PPAR-γ to regulate death of cortical neurons after DNA damage [
30]. Moreover, CITED2 is a novel effector of PPAR-γ in inhibiting HCC cell growth [
29]. In HCC, PPAR-γ inhibits cell invasion by up-regulating plasminogen activator inhibitor-1. The PPAR-γ agonist has been shown to inhibit HCC development. In this study, alteration of PPAR-γ activation could abolish the effects of miR-1468 on HCC growth. Moreover, PPAR-γ exerted its biological function through AKT signaling. Here, AKT phosphorylation was critical for the biological effects of miR-1468 in HCC. Taken together, we confirm that miR-1468 promotes HCC cell growth by modulation of PPAR-γ/AKT signaling (Additional file
3: Figure S3).
In summary, we demonstrate that miR-1468 overexpression acts as an independent biomarker for indicating poor prognosis of HCC patients. Furthermore, we confirm that miR-1468 promotes cell proliferation, colony formation, cell cycle progression and inhibits apoptosis by directly targeting CITED2 and UPF1 mediated PPAR-γ/AKT pathway. Our data reveal a novel role for miR-1468 in HCC development and progression, and suggest miR-1468 as a potential target for HCC diagnosis and treatment.
Conclusions
To conclude, our data offer the promising evidence that miR-1468 overexpression acts as an independent risk factor for indicating poor prognosis of HCC patients. miR-1468 facilitates HCC cell proliferation and cell cycle progression, and induces apoptosis in vitro and in vivo. More significantly, CITED2 and UPF1-mediated PPAR-γ/AKT pathway may be directly implicated in the oncogenic function of miR-1468 in HCC. Our study considers miR-1468 as a potential diagnostic marker and therapeutic target for HCC.