Background
Hepatocellular carcinoma (HCC) ranks as the fifth most common cancer in men and the seventh in women with annual incidence rates of ~ 750,000 worldwide [
1,
2]. Despite curative improvements made in HCC therapy recent years, the 5-year survival rate of HCC subjects still remains poor due to the spread, metastases and high rate of recurrence [
3,
4]. To date, HCC has become the second most frequent cause of cancer-related death [
1]. Although many altered pathways and aberrantly expressed genes involved in hepatocarcinogenesis were identified, the precise molecular mechanisms for HCC are not entirely clear [
2,
5]. Thus, in order to improve the prognosis of patients with HCC, an urgent need for novel molecular markers that can help in early diagnosis, risk assessment and therapy appears to be imperative.
Most of the eukaryotic genome is transcribed, yielding a complex network of transcripts. And those greater than 200 nt in length with limited or no protein-coding capacity are generally defined as long non-coding RNAs (lncRNAs [
6]. LncRNAs have been shown to emerge as essential regulators in a diverse range of cellular functions, such as development, differentiation, and cell fate as well as tumorigenesis [
7,
8]. Mounting evidence has linked mutation and dysregulation of lncRNAs to cancer initiation, growth and metastasis and they may act as oncogenic factors or tumor suppressors [
8,
9]. Recently, several lncRNAs have been reported to participate in diverse biological processes involved in hepatocarcinogenesis, including cell proliferation, apoptosis, metastasis and angiogenesis [
6,
10,
11]. Examples include DANCR [
12], AFAP1-AS1 [
13], UCA1 [
14] and ZEB1-AS1 [
15]. However, investigations on the function and clinical significance of the majority of HCC-related lncRNAs still remain limited.
In this study, we identified a novel lncRNA PTTG3P, termed pituitary tumor-transforming 3, pseudogene (NCBI Accession NO.NR_002734) via microarray analysis. The expression and localization of PTTG3P were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH), respectively, using patient samples from 2 HCC cohorts. Our data showed that PTTG3P was frequently up-regulated in HCC and high levels of PTTG3P positively correlated with poor prognosis in patients with HCC. Further investigations on the role of PTTG3P and its molecular basis in HCC revealed that PTTG3P promoted cell growth, metastasis and tumorigenicity via targeting PTTG1 (pituitary tumor-transforming 1) and activating PI3K/AKT signaling pathway. These results indicated that PTTG3P harbors great potential significance as a prognostic biomarker and a therapy target for HCC.
Methods
Patient samples
Two independent cohorts involving 136 HCC patients were enrolled in this study. In cohort 1, fresh HCC samples and adjacent non-tumor tissues were obtained from 46 patients who had undergone routine surgery from 2012 to 2014 at Nanfang Hospital, Southern Medical University. Tissues were frozen and stored in liquid nitrogen until further use. In cohort 2, paraffin-embedded samples were obtained from 90 patients diagnosed as HCC between January 2007 and December 2009 at the same hospital. Medical records of all patients provided information of age, gender, and following parameters: liver cirrhosis, tumor size, tumor number, Edmonson grade and TNM stage. The patients in cohort 2 were followed up for 5 years. Written informed consent for the biological studies was obtained from each patient involved in the study, and the study was approved by the Ethics Committee of Nanfang Hospital.
Microarray analysis
Total RNA from 3 HCC tumor tissues and paired non-tumor tissues were isolated using Trizol (Invitrogen, Carlsbad, CA). Total RNA was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. For microarray analysis, Agilent Array platform was employed. Total RNA was amplified and transcribed into fluorescent cRNA using Agilent’s Quick Amp Labeling Kit. The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (8 × 60 K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.0 software package (Agilent Technologies). After quantile normalization of the raw data, lncRNAs and mRNAs that at least 2 out of 6 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed lncRNAs and mRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Pathway analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, hierarchical clustering was performed to show the distinguishable lncRNAs and mRNAs expression pattern among samples.
Cell culture
The HepG2 and Hep3Bcell lines were obtained from the Cell Bank of Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Gaithersburg, MD,USA) supplemented with 10%fetal bovine serum (FBS, Gibco)and incubated at 37°Cin an atmosphere of 5% CO2.
RNA extraction and quantitative real-time PCR analysis (qRT-PCR).
RNA extraction and qRT-PCR were performed as described previously [
16,
17]. The primers used are presented in Additional file
1: Table S1.
Western blot analysis
Western blotting was performed using a SDS-PAGE Electrophoresis System according to the previous description [
16,
17] with antibodies specific for C-myc (Cell Signaling Technology, Beverly, MA, USA), CDK4 (Cell Signaling Technology), CDK6(Cell Signaling Technology), CyclinD1(Cell Signaling Technology), Rb (Cell Signaling Technology), p-Rb (Cell Signaling Technology), Caspase3 (Immunoway, USA), Cleaved Caspase3 (Cell Signaling Technology), Snail (Proteintech, USA), Slug (Proteintech), E-cadherin (Cell Signaling Technology), N-cadherin (Cell Signaling Technology), PI3K (Abclonal Technology), p-PI3K(Cell Signaling Technology), AKT(Cell Signaling Technology), p-AKT(Cell Signaling Technology), PTTG1(Cell Signaling Technology), β-Tublin (Cell Signaling Technology) and β-actin (Proteintech).Signals were detected using enhanced chemiluminescence reagents (Millipore, Schwalbach/Ts., Germany).
Construction of stable cell lines
To obtain cell lines stably over-expressing PTTG3P, HepG2 and Hep3B cells were infected with the Lv-PTTG3P and Lv-con viruses (LAND, Guangzhou, China). To observe the knockdown effects of PTTG3P, HepG2 and Hep3B cells were transfected with the shRNA-PTTG3P (sh-PTTG3P)or control (sh-con) viruses purchased from GeneChem (Shanghai, China). The infection efficiency was confirmed by qRT–PCR.
Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, Colony formation assay and Cell cycle analysis.
CCK-8 assay, EdU incorporation assay, Colony formation assay and Cell cycle analysis were performed as described in previous study [
16].
Cell apoptosis analysis
The apoptosis assay was done with the AnnexinV-7AAD apoptosis detection kit (KeyGEN BioTECH, Nanjing, Jiangsu Province, China) according to the manufacturer’s instructions. To detect the effect of PTTG3P over-expression on 5-FU-induced cell apoptosis, Lv-PTTG3P and Lv-con cells were seeded in 6-well plates and treated on the following day with 5-FU. After incubation for 48 h, cells were harvested, stained using AnnexinV-7AAD apoptosis detection kit and then analyzed by FACS cytometry (BD Biosciences, San Jose, CA, USA).All experiments were performed in duplicate and reproducibility was checked in three independent experiments.
Cell migration and invasion assays
Cell migration and invasion assays were performed with Boyden chambers (pore size: 8 μm, 24-well; BD Biosciences) with Matrigel (for the invasion assay)or without Matrigel (for the migration assay) following the manufacturer’s protocol. For cell migration assays, cells were detached and washed with PBS, resuspended in serum-free medium, and 200 μl of cell suspensions (a total of 5 × 104 cells) was added to the upper chamber. Medium with 20% FBS was added to the bottom wells of the chambers. The cells that had not migrated were removed from the upper face of the filters using cotton swabs, and the cells that had migrated to the lower face of the filters were fixed with fixed with methanol, stained with crystal violet solution, photographed under microscope and quantified. The mean of triplicate assays for each experimental condition was used. Similar inserts coated with Matrigel were used to determine the invasive potential in the invasion assay.
Animal studies
All animal studies were approved by the Animal Experimental Committee of Nanfang Hospital. The male BALB/C nude mice (4–5 weeks old) were purchased from the Experimental Animal Center of Southern Medical University, bred and maintained in a specific pathogen-free facility. For in vivo tumor growth assays, a total of 1 × 107 HepG2 cells stably transfected with Lv-PTTG3P or Lv-con, sh-PTTG3P or sh-con in 100 μl DMEM medium was independently injected subcutaneously into the left back and right back of 10 nude mice. Tumor volumes were monitored and calculated bylength×width2 × 0.5 weekly after implantation. All mice were sacrificed four weeks later. Mean percent of body weight (±SEM) and tumor size for each group was measured. For in vivo tumor metastasis assays, 1 × 107HepG2/sh-PTTG3P and HepG2/sh-con cells were injected into nude mice through the spleen, respectively. Two months later, all mice were sacrificed to observe the tumor metastasis in liver and abdominal cavity. The metastatic tissues were photographed and analyzed by H&E staining.
In situ hybridization
The ISH probe used for detecting PTTG3P-labeled digoxin was designed and synthesized by Exiqon (Shanghai, China). ISH was performed using the ISH Kit (Boster Bio-Engineering Company, Wuhan, China) and the stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters. The score standard for the staining intensity was as follows: 0 (negative), 1 (weak), 2(medium), and 3 (strong); and 0 (0%), 1 (1–25%), 2(26–50%), 3 (51–75%), and 4 (76–100%) for the staining extent. For statistical analysis, a final staining score of 3 or higher was considered to be high expression, respectively.
Statistical analysis
SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) were used to analyze all data for statistical significance. The Chi-Square test was applied to the examination of relationship between lncRNAPTTG3P levels and clinicopathological characteristics. Survival curves were plotted by Kaplan–Meier method and log rank test. The significance of various survival-related variables was assessed by Cox regression model in the multivariate analysis. Two-tailed Student’s t-test was used for comparisons of two independent groups. Repeated measurement data analysis of variance was performed for results from CCK-8 assays and tumor growth curve determinations. Statistical significance was set at *P < 0.05.
Discussion
PTTG3P, also known as PTTG3 or rcPTTG1, was firstly identified by Leilei Chen and his colleagues in 2000 [
22]. They reported that PTTG3P is located at chromosome 8q13.1 and show high homology to hPTTG gene through Southern blot analysis, Northern blot analysis, sequencing and restriction map analysis of the genomic clones. At that time, they regarded PTTG3P as a protein coding gene. However, to date, PTTG3P has been confirmed to be a known processed pseudogene rather than a protein coding gene by automated computational analysis using gene prediction method (
http://www.pseudogene.org). Pseudogenes are structurally similar to genes that encode functional proteins, but contain “defects” that, in most cases, render them unable to encode fully functional proteins [
21]. Recently, the attribution of function to specific pseudogenes has raised them to the status of a new class of regulatory lncRNAs involved in both physiological and pathological processes [
21]. For instance, lncRNA PTENP1, a processed pseudogene of the tumor suppressor PTEN, has been demonstrated to increase PTEN abundance and then be actively involved in cancer pathogenesis [
23]. Apart from PTENP1, examples include FTH1 pseudogenes [
24], PDIA3P [
25] and BRAFP1 [
26]. However, few studies have investigated the role and mechanism of PTTG3P in HCC progression.
In this study, we demonstrated that PTTG3P is frequently up-regulated in HCC tissues relative to corresponding adjacent non-tumor tissues from 2 cohorts by qRT-PCR and ISH assays. Clinical data revealed that high levels of PTTG3P significantly correlate with the invasive and aggressive characteristics of HCC (positively correlated with tumor size and TNM stage) as well as poor survival in patients with HCC. These results implicated that over-expression of PTTG3P may be a common feature in HCC and might serve as a valuable prognostic biomarker for HCC. Consistent with our findings, a microarray analysis carried out by Jennen, D. G. et al. [
27] revealed that compared with control cells, higher levels of PTTG3P is observed in HepG2 cells exposed to aflatoxin B1 for 48 h. Aflatoxin B1 exposure is a well-known risk factor for HCC [
28,
29]. Moreover, higher level of PTTG3P has been reported to be correlated with shorter disease-free survival and overall survival in patients with gastric cancer [
30]. Thus, the meaning of PTTG3P in HCC pathophysiology is strongly suggested.
To further study the biological function of PTTG3P in HCC, we firstly performed gain-of-function and loss-of-function experiments in HepG2 and Hep3B cells. Our results showed that stably decreased expression of PTTG3P converts HepG2 and Hep3B cells into less aggressive cells, with higher capability of cell apoptosis and lower capability of cell-cycle G1/S transition, cell growth in vitro and tumorigenicity in vivo, cell migration and invasion in vitro as wells as metastasis in vivo. In contrast, elevated PTTG3P expression has opposite effects. Thus, these data indicated that PTTG3P functions as an oncogene in HCC. Similar to our results, a genome-scale RNAi profiling of cell division in human tissue culture cells showed that siRNA specially targeting PTTG3P results in G0/G1 arrest [
31]. Moreover, Kho, P. S et al. [
32] reported that 5-fluorouracil results in decreased level of PTTG3P, partly in support to our finding that over-expression of PTTG3P partially restores 5-fluorouracil-induced cell apoptosis. Consistently, PTTG3P has been demonstrated to promote gastric tumor cell proliferation and invasion [
30].
The biological roles of PTTG3P found in this study provide a mechanistic basis in HCC. It has been well-defined that unscheduled proliferation is often induced by cell cycle defects as well as dysregulation of cyclins and cyclin-dependent kinases (CDKs) complexes [
33]. Here, we showed that the molecular mechanisms of PTTG3P on promoting cell proliferation might be the acceleration of G1/S transition, elevated expression of C-myc, CyclinD1and p-Rb under enhanced expression of PTTG3P. CyclinD1, a key G1 phase-associated cell cycle regulator, is required for hyperphosphorylation of Rb and dissociation of transcription factor E2F from Rb, leading to the transcription of genes implicated in S phase progression [
34]. Moreover, a bulk of findings reported that C-myc over-expression stimulates cell cycle progression by targeting multiple genes related to cell cycle control including Cdc25A, CyclinE1 as well as Cyclin D1 [
35]. However, whether PTTG3P up-regulates Cyclin D1 via activation of C-myc need further investigation. Intriguingly, CDK4 and CDK6 are not involved in cell proliferation induced by PTTG3P. In addition to enhanced proliferation, resistance to apoptosis is also a hallmark of cancer cells [
18]. In current study, PTTG3P down-regulation activated caspase3 whereas PTTG3P over-expression partially abrogated 5-fluorouracil-induced activation of caspase3. Although PTTG3P has been linked to regulation of metastasis, the molecular mechanisms remain poorly elucidated. EMT, which is characterized as a down-regulation of epithelial markers, particularly E-cadherin, and an up-regulation of mesenchymal markers, particularly N-cadherin, snail and slug, is a crucial step for cancer invasion and metastasis in various cancer cells [
36,
37]. Here, more specifically, our data revealed that depletion of PTTG3P inactivates EMT processes by up-regulation of E-cadherin and down-regulation of snail and slug. However, N-cadherin is not implicated in EMT induced by PTTG3P.
PI3K/AKT pathway, activated in 40–50% of HCCs, plays a crucial role in the cell growth and metabolism ultimately influencing the invasion, metastasis and aggressiveness of cancer cells [
20,
38]. Once activated, PI3K/AKT signal could increase the levels of C-myc and CyclinD1, thereby promoting G1/S transition and cell proliferation [
39]. Moreover, previous study has revealed that inhibition of PI3K/AKT induces apoptotic and autophagic cell death [
40,
41]. Additionally, accumulating evidence supports a vital role of PI3K/AKT pathway on EMT and metastasis by down-regulating E-cadherin as well as up-regulating Snail and Slug [
40].Currently, studies have identified several lncRNAs to function through PI3K/AKT signaling [
41]. Here, we showed that PI3K/AKT pathway was activated since p-PI3K and p-AKT were up-regulated by PTTG3P. Therefore, the up-regulation of C-myc, CyclinD1, Snail and Slug as well as down-regulation of E-cadherin by PTTG3P probably results from enhanced PI3K/AKT pathway activity. This thus explains the induction of cell growth and metastasis by PTTG3P in HCC.
Accumulating evidence suggest that pseudogenes play critical roles in various diseases through regulating parental gene expression [
42]. Examples include PTENP1 and its parental gene PTEN, OCT4-pg4 and its parental gene OCT4 [
42].Since PTTG3P is a known processed pseudogene, we found PTTG1 to be a parental gene of PTTG3P. PTTG1 was reported to be up-regulated in various cancers [
43]. Overexpression of PTTG1 can promote cell proliferation, inhibit cell apoptosis and induce EMT [
44,
45]. Moreover, PTTG1 has been demonstrated to activate PI3K/AKT signaling [
46]. Thus, we hypothesized that PTTG3P could activate PI3K/AKT signaling and promote HCC progression by regulating PTTG1. To test our hypothesis, the regulatory relationship between PTTG3P and PTTG1 in both tumor tissues and HCC cells was further clarified. We revealed that PTTG1 was frequently up-regulated and positively correlated with PTTG3P in 46 HCC tumor tissues. Overexpression of PTTG3P resulted in up-regulation of PTTG1 whereas knockdown of PTTG1 inhibited PTTG1 expression. Our results suggest that PTTG1 could be induced by PTTG3P. Consistently, the microarray data in Oncomine database (
http://www.oncomine.org) support a positive correlation between PTTG3P and PTTG1 in ovarian serous adenocarcinoma tissues, breast carcinoma tissues and rectal adenocarcinoma tissues. However, the study carried out by Weiwei Weng et al. [
30] found PTTG3P expression is independent of PTTG1 in both gastric cancer tissues and cells. It seems that the relationship between PTTG3P and PTTG1 varies in different cancers. Nevertheless, a limitation of this current study is that we did not demonstrate a direct molecular function of PTTG3P in up-regulating PTTG1, and further investigation is needed.
Acknowledgements
We thank Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China for providing the HCC tissue samples and related anonymous clinical data.