Background
Hepatocellular carcinoma (HCC) is one of the most common and lethal tumors worldwide due to metastases and recurrence [
1,
2]. Epithelial to mesenchymal transition (EMT) is a complex biological process that plays key role in tumor metastases, and it disrupts the intercellular junctions, polarity, order, and consistency of the cells, leading to tumor recurrence [
3]. More and more studies have elucidated the indispensable role of EMT in metastatic dissemination of HCC; and it highlighted the need for further study of EMT-related molecular mechanisms in HCC [
3,
4].
Covalently closed circular RNAs (circRNAs) are connected by the back-splicing of exons (3′ and 5′ ends) or introns of precursor mRNAs [
5]. Recent studies have shown the critical functions of circRNAs, such as microRNAs (miRNAs) or RNA-binding proteins (RBPs) sponging, proteins translation, transcription, and splicing modulation [
6,
7]. As reported, circRNAs perform better as diagnostic and therapeutic targets than linear transcripts by virtue of its structural stability, species conservation, and cell/tissue-specificity [
8,
9]. More importantly, abnormal expression of non-coding RNAs is closely related to EMT in the metastatic process of cancers [
10,
11].
In the present study, we investigated the expression profile of circRNAs in HCC patients with or without portal vein tumor thrombus (PVTT) metastasis using RNA-sequencing (RNA-seq). And we characterized a novel circRNAs, circ0003998 (circBase ID: hsa_circ_0003998) that derived from ADP ribosylation factor guanine nucleotide exchange factor 2 (ARFGEF2) and located at chr20:47570092–47,580,435. We further showed that circ0003998 plays a key role in HCC metastasis through circ0003998/miR-143-3p/FOSL2 axis and circ0003998 /PCBP1/CD44v6 axis.
Materials and methods
Patient tissues and cell lines
The study recruited 25 HCC patients with PVTT metastasis (cohort 1) and 50 HCC patients without PVTT metastasis (cohort 2) from the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). The patients with a history of preoperative chemoradiotherapy were excluded. HCC tissues, PVTT tissues, and corresponding adjacent normal liver tissues (ANL) tissues were collected from cohort 1; and another HCC tissues and corresponding ANL tissues were collected from cohort 2. The tissues were verified by two pathologists independently. The study complied with the Declaration of Helsinki and was approved by the Human Ethics Committee of the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). Moreover, all patients in this study provided written informed consent. HCC cell lines (HepG2, HuH7, Hep3B, and PLC/PRF/5) were purchased from the American Type Culture Collection (ATCC, USA). The MHCC97H cells, hepatocyte cell line L02, and HEK293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). No cell lines were contaminated by other cells, such as Hela, as shown by short tandem repeat (STR) data (Biowing Applied Biotechnology Co., Ltd., Shanghai, China) (Additional file
1). All cells were maintained in humidified incubators (37 °C) with 5% CO
2 and were cultured in Dulbecco’s modified Eagle’s medium (SIGMA, USA) with 10% fetal bovine serum (GIBCO, BRL).
Total RNA isolation, RNA-seq, and RNase R treatment
Total RNA were isolated from the cultured cells and fresh tissues by Trizol reagent (Invitrogen, USA). Total RNA was extracted by RNAprep pure Tissue Kit (TIANGEN), and rRNA was depleted by GeneRead rRNA Depletion Kit (QIAGEN). The mRNA was purified from total RNA by oligo (dT) magnetic beads, and fragmented into 200–500 bp; the cleaved RNA fragments were reverse-transcribed into cDNA, and enriched by PCR to create the final complementary DNA (cDNA) libraries. The harvested target bands were quantified by Agilent 2100 and then subjected to deep sequencing with the Illumina HiSeq 2000. HCC cells were mixed with RNase R (3 U/μg, Epicentre, Madison, WI) at 37 °C for 15 min, and then qRT-PCR was used to assess the expression stability of circ0003998 as compared to ARFGEF2 mRNA.
Reverse transcription reaction and quantitative real-time PCR
The first-strand cDNA was synthesized by the PrimeScript RT reagent kit (Takara Bio Inc., China), and the reverse transcription of miRNAs was generated by miRNAs First Strand cDNA Synthesis Poly A Tailing Kit (Sangon Bio Inc., China). SYBR Premix Ex TaqII (Takara Bio Inc., China) was used in Quantitative real-time polymerase chain reaction (qRT-PCR). β-actin and U6 were used as endogenous references for mRNA and miRNAs respectively. The comparative 2
-ΔΔCt method was used to calculate the relative fold-change of target expression. The sequences of the primers in this study were showed in Additional file
2. All primers were designed and purchased from Sangon (Shanghai, China).
Tissue microarray (TMA) and in situ hybridization (ISH)
After dewaxed in xylene and rehydrated with gradient alcohol, the TMAs were digested by protease K (20 μg/mL) at 37 °C. 3% methanol-H2O2 was used to block endogenous peroxidase. Next, TMAs were hybridized with specific digoxin-labeled circ0003998 probe (8 ng/μL, Digoxin- 5′-Digoxin-GGCCTCCTGCAACTTTAATGGCAGATG-Digoxin-3′, Service Biotechnology, Wu han, China) at 37 °C overnight. TMAs were incubated in BSA serum at room temperature for 30 min and then incubated with mouse anti-digoxin labeled peroxidase (anti-DIG-HRP) at 37 °C for 40 min. The TMAs were stained with freshly prepared diaminoaniline (DAB) solution, and the nucleus was stained with Harris hematoxylin stain.
Fluorescence in situ hybridization (FISH)
FISH assay was used to find the intracellular location of circ0003998. Probes of FISH assay for circ0003998, human U6, and human18S were synthesized by RiboBio Co., Ltd. (Guangzhou, China). Briefly, cells were rinsed with phosphate buffer saline (PBS) and fixed in 4% formaldehyde solution for 10 min at room temperature, and then incubated with 0.5% Triton X-100 solution for 5 min at 4 °C. After pre-hybridization for 30 min, the cells were hybridized with fluorescence-hybridization probes overnight in the dark at 37 °C. Then, cells were washed three times in 4X/2X/1X SSC solution in the dark at 42 °C for 5 min respectively, and laser scanning confocal microscopy was used to visualize the images at 400 × magnification.
Nuclear-cytoplasmic fractionation
Cytoplasmic and nuclear RNA isolation was detected by the PARIS™ Kit (Invitrogen, USA). Briefly, after washed in PBS, the cultured cells were resuspended in cold cell fraction buffer and then incubated on ice for 10 min. The cells samples were centrifuged at 4 °C to separate the supernatant containing cytoplasmic fractions from the pellet containing nuclear fraction. The nuclear pellet was rewashed by the cell fraction buffer and was lysed by the disruption buffer. Both the cytoplasmic fraction and the nuclear fraction were respectively mixed with 2X lysis/ binding solution, followed by 100% ethanol. After centrifugation and washing, the RNA of nuclear and cytoplasmic were respectively obtained with the eluting solution.
Establishment of stable cell lines over expressing hsa_circ_0003998
The circ0003998 expressing lentivirus vector (HBLV-circ0003998-GFP-PURO, termed as OE-circ-vector) and its control vector (HBLV-GFP-PURO, termed as NC-circ-vector) were supplied by Hanbio (Shanghai, China). HCC cells were infected with the lentivirus vectorfoloowed by selection with 2 μg/mL puromycin for 1 week.
Construction of siRNA, plasmids, miRNA mimic, and transient transfection
The small interfering RNAs (siRNAs) of circ0003998 (si-circ0003998) and the corresponding negative control (siRNA-NC) were synthesized by Hanbio (Shanghai, China). The PCBP1 expressing plasmid (OE-PCBP1), FOSL2 expressing plasmid (OE-FOSL2) and the corresponding negative plasmid vector (vector) were provided by Hanbio (Shanghai, China). The siRNAs of PCBP1 (si-PCBP1), siRNAs of FOSL2 (si-FOSL2) were synthesized by GenePharma (Shanghai, China). The microRNA-143-3p mimics (miR-143-3p) and corresponding negative control (miR-143-3p-NC) were synthesized by GenePharma (Suzhou, China). After digestion with Xho I/Xba I, circ0003998 cDNA (full-length: 304 bp) from HepG2 was amplified by qRT-PCR which cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, USA) to synthesize wild type-plasmid for the luciferase reporter assay (Luci-circ0003998-WT). The corresponding mutant type-plasmid (Luci-circ000 3998-MT) contained with mutated miRNA binding sites and was synthesized by Genscript (Nanjing, China). Lipofectamine 3000 kit (Invitrogen) was used in transient transfection according to the manufacturer’s instructions.
Wound-healing assay
The cells were seeded in six-well plates and the center of each well was straightly scratched using a 10-μL plastic pipette tip. After floating cells were gently removed with PBS, cells in the six well plates continued to be cultured for 48 h. The wound-healing process were monitored by inverted light microscope.
Cell counting Kit-8 assay
Cell proliferation was assessed by Cell Counting Kit-8 kit (CCK-8, Dojindo Chemical Laboratory, Kumamoto, Japan). HCC cells (3 × 103/well) were seeded in 96-well plates with 6 replicates. 10 μL/well of CCK8 solution and 90 μL/well of fresh medium were mixed and added to each well at 0, 24, 48, 72, and 96 h, respectively, After incubation for 1.5 h in cell incubator, the absorbance of the medium at 450 nm was measured.
Migration assay and invasion assay
24-well transwell migration champers (8 μm size, Corning-3422, USA) and pre-biocoated matrigel transwell invasion chambers (8 μm size, Corning-354,480, USA) were used for migration assay and invasion assay respectively. Briefly, a total of 5 × 104 HuH7, 8 × 104 MHCC97H, and 8 × 104 HepG2 cells were resuspended in 200 μL serum-free DMEM medium and were seeded into the inner chambers, respectively. 600 μL DMEM medium containing 10% FBS as the attractant was loaded to the bottom chambers. For migration assay, the cells were incubated for 24 h, and for invasion assay, the cells were incubated for 48 h. After incubation, the cells that migrated or invaded through the pores were fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet, and then counted at least in five random fields under 200 × microscope.
Luciferase reporter assay
HCC cells or HEK293T cells (3 × 105) were seeded in a 24-well plate overnight. 250 ng of Luci- 0003998-WT/Luci-circ0003998-MT were co-transfected into the cells with 100 nM miR-143-3p mimic/miR-143-3p mimic-NC using Lipofectamine 3000. In 48 h after transfection, firefly and Renilla luciferase fluorescence were detected by the Dual-Luciferase Reporter Assay kit (Promega, USA). The relative luciferase activities were quantified by Firefly/Renilla fluorescence.
RNA immunoprecipitation (RIP) assay
Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was used in RIP assay according to the manufacturer’s protocol. HepG2-OE cells (2 × 107) were lysed in RIP lysis buffer. 5 μg of human anti-targeted protein antibody or negative anti-IgG antibodies was incubated with magnetic beads for 2 h. Then, 100 μL RIP lysate was incubated with bead-antibody complex in 900 μL RIP Immunoprecipitation buffer (shaking at 4 °C overnight). After the beads were incubated with proteinase K buffer for 30 min at 55 °C, the immunoprecipitated RNAs were finally extracted to further detect the expression levels of circ0003998 by qRT-PCR.
RNA pull-down
HepG2 cells (2 × 107) were washed in cold PBS and then lysed in a lysis buffer. Biotin-labeled circ0003998 probe (5′-Biotin-aaaTTCCAGTTCTCTGGCCTCCTGCAACTTTAATGGCAGATG TGACTACAT-3′) and control probe (5′-Biotin-aaaATGTAGTCACATCTGCCATTAAAGTTGC AGGAGGCCAGAGAACTGGAA-3′) were synthesized by CloudSeq Biotech Inc. (Shanghai, China). Biotin-coupled probes were bounded on magnetic beads and then incubated with lysates of HepG2-OE cells overnight at 4 °C. After purification, the pull-down product was analyzed by miRNA sequencing and protein spectrum.
The animal experiments met the demands of laboratory animal welfare and ethics, and approved by the Institutional Animal Care and Use Committee of Naval Medical University (Shanghai, China). The nude male BALB/c mice (four weeks old) were purchased from Animal Center of Naval Medical University and housed under pathogen-free conditions with standard pellet diet and water. Twenty mice were randomly divided into four groups for the construction of two model. MHCC97H-OE cells (3 × 106) or MHCC97H-NC cells (3 × 106) were injected into the tail vein to establish the lung metastatic model. MHCC97H-OE cells (5 × 106) or MHCC97H-NC cells (5 × 106) were injected into the spleen to establish liver metastatic model. IVIS@ LuminaII system was used to measure the fluorescence value of GFP at excitation wave-length of 488 nm, which monitor the metastases of two types of mice models. Mice were sacrificed with CO2; their lungs and livers were stained with H&E and re-examined microscopically for the development of metastatic foci.
Western blot
HCC cells were lysed in a RIPA buffer (Beyotime Biotechnology, Nantong, China). BCA Protein Assay kit (Beyotime Biotechnology, Nantong, China) was used to detect protein concentration. The cell lysates were separated by polyacrylamide gel electrophoresis (4–20% SurePAGE Gel, Genscript, Nanjing, China), and then transferred onto to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, CA, USA). After blocking with 5% nonfat milk in Tris-buffered saline-Tween (TBST) buffer at room temperature for 90 min, the membranes were incubated with the primary antibody at 4 °C overnight. After wash with TBST buffer (10 min × 3), the membranes were incubated with the secondary antibody at room temperature for 1 h. The membranes were examined using the enhanced chemiluminescence kit (Beyotime Biotechnology, Nantong, China). Anti-E-cadherin, anti-Vimentin, anti-N-cadherin, anti-Slug and anti-Snail antibodies were purchased from Cell Signaling Technologies (CST#9782; Boston, MA, USA). Anti-β-actin (ab227387), anti-CD44v6 (ab78960) and anti-FOSL2 (ab124830) antibodies were purchased from Abcam (Cambridge, UK).
Statistical analysis
SPSS 19.0 software and Prism version 7.0 were used for statistical analyses. Data from three independent experiments were shown as mean ± standard deviation (SD), followed by Student’s t-test, one-way analysis of variance (ANOVA) and chi-square tests. The correlations were analyzed by Pearson correlation. The result with *p-value< 0.05, **p-value < 0.01, or ***p-value < 0.001 was considered as statistically significant.
Discussion
More and more circRNAs are discovered using high-throughput sequencing techniques and bioinformatics analysis. Previous study has showed that circ0003998 could promote cell proliferation and invasion by targeting miR-326 in non-small cell lung cancer [
24]. In this study, our data for the first time identified that circ0003998 could promote EMT in HCC by in vitro functional assays and in vivo mice models; and meanwhile circ0003998 was upregulated in HCC tissues and PVTT tissues, which were correlated with advanced TNM stage and high serum levels of AFP. Furthermore, our previous study has confirmed that expression of circ0003998 is also upregulated in plasma in HCC, which is significantly correlated with poor overall survival (OS) in HCC patients; and therefore, circ0003998 could be used as independently prognostic factors for poor OS in HCC [
25].
Mechanically, we identified that circ0003998 could sponge miR-143-3p, the suppressor of proliferation, invasion, and metastasis in multiple cancers [
26‐
28] as well as the inhibitor of the EMT-related genes [
29‐
31]. Furthermore, there was no significant correlation between the expression of miR-143-3p and circ0003998. Previous reports showed that ceRNAs could regulate the activity of miRNAs; however, whether ceRNAs regulate the expression levels of miRNAs might rely on the cellular context [
32,
33]. Our data further showed that circ0003998 could sponge with miR-143-3p to increase the expression of FOSL2 and thereby promoted the EMT in HCC and it has been consistently showed that FOSL2 is the target gene of miR-143-3p in osteosarcoma in previous study [
34].
The tertiary structures of circRNAs result in higher protein adsorbing capacity than those of linear RNA sequences. As such, the circRNA-interacting RBPs serves as an essential molecular action mode in genesis, translation, transcriptional regulation of target genes, and extracellular transport [
35]. PCBP1 as one of the the RBPs inhibited the tumor formation and metastasis by translation silencing, mRNA alternative splicing, or transcription of carcinogenic genes [
36]. Meaningfully, PCBP1 is reported to participate in EMT pathways in cancer, especially in the TGF-β pathway [
37‐
39]. And our data showed that circ0003998 bound to PCBP1 to increase the expression of CD44v6 and thereby promoted the EMT in HCC, which was consistent with former studies that PCBP1 regulated alternative splicing of the CD44v6 and thereby inhibited the invasion in HCC [
40].
Previous study showed that PCBP1 regulated DAB2 in the translation process [
21]. However, there were no significant changes in the protein level of DAB2 with circ0003998 regulating in our data. The sponging affinity of circRNAs with RBPs was not the same as that between circRNAs and miRNAs, and circRNAs absorbing RBPs might be more complicated than expected [
41]. Thus further research is necessary to understand whether circ0003998 inhibits the translation regulating-role of PCBP1 or whether circ0003998 induces the degradation of PCBP1.
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