Background
EOC is the third most common and second most lethal gynecological malignancy worldwide [
1]. The standard treatment is maximal cytoreductive surgery followed by platinum-based chemotherapy regimen [
2]. However, the occurrence of metastasis and acquired drug resistance often lead to poor prognosis in patients with advanced ovarian cancer [
3,
4]. Therefore, there is an urgent clinical need to understand the molecular mechanisms leading to the occurrence of tumor metastasis and drug resistance in EOC. In addition, novel biomarkers which can accurately predict tumor metastasis and drug resistance are needed to provide valuable insight into the clinical management of EOC.
EMT is a biological process in which polarized epithelial cells adopt mesenchymal cell phenotype which includes enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of extracellular matrix components [
5,
6]. It has been well demonstrated that the activation of EMT program endows cancer cells with the multiple features that are required to execute most steps of the invasion-metastasis cascade [
7,
8]. In addition, activation of EMT pathway induces chemoresistance in multiple cancers such as pancreatic cancer [
9], lung cancer [
10,
11], gastric cancer [
12], liver cancer [
13] and EOC [
14].
LncRNA and miRNAs both belong to non-protein encoding RNAs which are implicated to play important roles in a variety of cellular processes such as cell proliferation, apoptosis, and migration [
15,
16]. The aberrant expression of lncRNAs in tumors can induce EMT and increase the invasion and migration of tumor cells. For example, XIST, which is a lncRNA over-expressed in colorectal cancer tissues and cells, can promote invasion, proliferation, and EMT progress through XIST/miR-200b-3p/ZEB1 pathway [
17]. Another example is PVT1, a highly expressed lncRNA in prostate cancer cells that can co-regulate Twist with miR-186 and promote tumor invasion, migration, and the progress of EMT [
18]. This lncRNA/miRNA/mRNA pathway has been proposed as a new model by which lncRNAs regulate target gene expression through miRNA response elements [
19]. Salmena et al. have termed this phenomenon as the competing endogenous RNA (ceRNA) theory [
20].
In this study, we identified a HOXD-AS1/miR-186-5p/PIK3R3 pathway in EOC as another example exhibiting the ceRNA property. The HOXD-AS1/miR-186-5p/PIK3R3 pathway plays an important role in invasion, migration and EMT of EOC.
Methods
Patients and EOC tissue samples
We collected EOC (
n = 36) and normal ovarian (
n = 14) tissue samples from patients who underwent surgery at the Hunan Cancer Hospital (Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China) from 2012 to 2014. The resected tissues were separately identified and confirmed by two pathologists and kept in liquid nitrogen immediately for subsequent use. In addition we also collected formalin-fixed paraffin-embedded (FFPE) specimens from an independent cohort of 200 patients to analyze the relationship of PFS and OS with the expression levels of HOXD-AS1. The clinicopathologic characteristics of patients are shown in Table
1. The ethics committee of Hunan Cancer Hospital approved the protocol, and all patients signed informed consents before sample collection.
Table 1
Top ten significantly up-regulated and down-regulated lncRNAs in EOC
Top ten up-regulated lncRNAs |
| lncRNA ID | FC |
p
|
1 | CTD-2116 N17.1 | 54 | 0.001 |
2 | MAL2 | 50 | 0.006 |
3 | LINC00707 | 19 | 0.005 |
4 | CXADRP3 | 18 | 0.001 |
5 | HOXD-AS1 | 18 | 0.007 |
6 | CDKN2B-AS1 | 16 | 0.004 |
7 | ALDH3B1 | 15 | 0.000 |
8 | XXbac-BPG254F23.6 | 11 | 0.004 |
9 | HCP5 | 10 | 0.004 |
10 | LINC00152 | 9 | 0.000 |
Top ten down-regulated lncRNAs |
| lncRNA ID | FC |
p
|
1 | LINC00473 | − 132 | 0.000 |
2 | PWRN1 | − 85 | 0.004 |
3 | TRHDE-AS1 | −38 | 0.000 |
4 | ADAMTS9-AS2 | −33 | 0.000 |
5 | LINC00478 | − 28 | 0.002 |
6 | CECR7 | −28 | 0.000 |
7 | CTC-454 M9.1 | −26 | 0.000 |
8 | ADAMTS9-AS1 | −26 | 0.007 |
9 | NR2F2-AS1 | − 22 | 0.006 |
10 | AL132709.5 | −22 | 0.007 |
Differentially expressed gene analysis
Differentially expressed genes and lncRNAs were identified using the threshold of fold change ≥2 and Student’s t-test p-value < 0.01 between EOC tumor versus normal tissues. The R package, Significance analysis of microarray (SAM) was used to identify differentially expressed miRNA at a threshold of fold change≥1.5, false discovery rate (FDR) q-value< 0.05. Hierarchical clustering was performed on the pheatmap package of R language and heatmap was constructed. Volcano maps were drawn using the ggplot2 packages.
Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis
DAVID (The Database for Annotation, Visualization and Integrated Discovery,
https://david.ncifcrf.gov/) was used to annotate the function of differentially expressed mRNA and enrichment of the signaling pathway. GO included cell component, biological process, and molecular function. Aberrant mRNAs were enriched into different pathways according to the KEGG database. TOP 10 terms were presented. The recommended
p-value cutoff of
p < 0.05 was used.
Cell culture
EOC cell line A2780 was cultured in Dulbecco’s modified Eagle’s medium (TransGen, Beijing, China), and EOC cell line SKOV3 and normal ovarian cell line IOSE80 were cultured in RPMI-1640 (TransGen, Beijing, China) with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (TransGen, Beijing, China). All of the cells were cultured in an incubator at 37 °C with 5% CO2.
RNA extraction and RT-qPCR
Total RNAs from tissues and cells were extracted using Trizol reagent (Invitrogen). Total RNAs from FFPE were extracted using RNeasy®FFPE Kit (Qiagen, Germany). cDNA was synthesized with the TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, Beijing, China) according to the manufacturer’s instructions. RT-qPCR was performed on the LightCycler®96 system (Roche) using a standard protocol from the TransStart® Tip Green qPCRSuperMix Kit (TransGen, Beijing, China). The results were normalized to GAPDH. The sequences of primers used for RT-qPCR assays are listed in Additional file
1.
For the detection of miRNA expression, reverse transcription and qPCR were performed using stem-loop primers and Bulge-Loop™ miRNA RT-qPCR Starter Kit from Ribobio (Guangzhou, China). U6 snoRNA was used as the endogenous control. All experiments were performed in triplicate according to the manufacturer’s manual. Expression fold changes were calculated using 2-∆∆Ct methods.
In situ hybridization (ISH)
In situ hybridization was performed to detect the expression of HOXD-AS1, miR-186-5p, and PIK3R3. The probes of HOXD-AS1, miR-186-5p, and PIK3R3 were designed by BOSTER (Wuhan, China). RNA ISH was performed using the in situ hybridization detection kit (BOSTER) and DAB chromogenic kit (ZSGB-BIO) on 3-μm FFPE tissue sections according to the manufacturer’s instructions. The staining scores were determined based on both the intensity and proportion of target gene-positive cells under 20X and 40X objective.
Small interfering RNA (siRNA) and miRNA inhibitor transfections
LncRNA HOXD-AS1, siRNA (si-HOXD-AS1), PIK3R3 siRNA (si-PIK3R3), miR-186-5p inhibitor, and negative control (si-NC, miR-NC) were all obtained from Ribobio (Guangzhou, China). RiboFECT™ CP transfection Kit (Guangzhou, China) was used for cell transfection based on manufacturer’s instructions. SKOV3 and A2780 were seeded in a six-well plate and transfection would be performed when cell’s confluence reached 50%. Cells were transfected with a final concentration of 100 nM of individual siRNA and 200 nM of miR-186-5p inhibitor. All of the steps were carried out according to the manufacturer’s specification. Experiments were performed three times.
Wound healing assay
The cell monolayer was scraped in straight lines using a 10ul pipette tip and washed with PBS two times when transfected cells were up to 80% confluence in a six-well culture dish. The cells were cultured in culture medium with 3% FBS and 1% penicillin/streptomycin (TransGen, Beijing, China). Images were captured at 0, 12, 24, 36, and 48 h following the initial scratch from five different areas for each wound. We used Image J (National Institutes of Health, Bethesda, MD, USA) software to calculate cell wound healing rate as: (the original wound areas - the actual wound areas at different times)/ (the original wound areas).
Trans-well assays
The invasion ability was measured using transwell chambers coated with 1:30 diluted Matrigel Basement Membrane Matrix (Corning Life Sciences, NY, USA). Migration assays were performed without matrigel. 1 × 105 cells were seeded into upper chambers (8 μM pore size, Corning Life Sciences, NY, USA) with 200ul serum-free media. A total of 600 ul of 20% FBS media was added into the lower chambers. After incubation for 48 h at 37 °C, the cells in the upper chamber that did not invade through the pores were wiped out with cotton wool. The cells in the lower chamber were fixed with methanol and stained with 0.5% crystal violet. Cells were counted and imaged at × 200 magnification. The experiments were repeated three separate times.
Western blotting
After transfection for 48 h, cells were lysed using RIPA buffer (Thermo, USA) with proteinase inhibitor cocktail (Roche), then incubated at 4 °C for 15 min. The lysate was centrifuged and protein concentration was measured by BCA Protein Assay Kit (Thermo, USA). The protein was separated on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.22 μm PVDF membrane (Thermo, USA). After blocking with 5% fat-free milk for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight with gentle shaking. Next, the membranes were incubated with secondary antibody at room temperature for 2 h. Finally, the immunofluorescence of protein bands was visualized using Pierce™ ECL Western Blotting Substrate (32,109, Thermo Scientific, USA) by Image J 2 software (Madison, WI, USA). Primary antibodies are as following: E-cadherin (1: 500, ab15148, Abcam company), Vimentin (1:500, #7431, Cell Signaling Technology (CST)), GAPDH (1:5000, 60,004-l-lg, Proteintech company), Rac1 (1:500, ab33186, Abcam), Twist (1500, GTX60776, Gene Tex).
Dual-luciferase reporter assay
The dual luciferase reporter plasmids (LncRNA-HOXD-AS1-WT, LncRNA-HOXD-AS1-Mut, PIK3R3-WT, PIK3R3-Mut, miR-186-5p, and negative control) were synthesized by GenePharma (Shanghai, China). The SKOV3 cells were seeded into 96-well plates and cultured to 80% confluence. The plasmid vectors or empty plasmid were transiently transfected into SKOV3 cells using lipofectamine 3000 (Invitrogen). After 48 h incubation, the luciferase activity was examined by Dual-Glo® Luciferase Assay System (Promega, USA) according to the manufacturer’s protocol.
Statistical analysis
All experiments were performed three separate times. Statistical analysis was performed with SPSS 13.0 and graphs were constructed with GraphPad Prism 5 (La Jolla, CA, USA). The data were presented as the mean ± Standard Deviation (SD). The significance of differences between groups was assessed by Student’s t-test and Chi-square test. The Kaplan-Meier method test was used for PFS analysis and OS analysis. Pearson correlation analysis was used to determine the relationship between two genes. We used the univariate and multivariate Cox proportional hazards modeling to determine the effects of variables on survival. A p-value of < 0.05 was considered statistically significant.
Discussion
Tumor metastasis is a major factor contributing to the poor prognosis of EOC patients [
21,
22]. EMT is a crucial cellular process associated with metastasis in tumor cells [
23,
24]. Our group has previously demonstrated that platinum-based drugs could induce the activation of Rac1 and initiate EMT [
25]. Understanding the crucial players in the EMT process in EOC will provide insights into the development of new therapeutic strategies to treat the disease.
LncRNAs are an class of non-coding RNAs which plays an important regulatory role in the biological processes of tumorigenesis, tumor cell proliferation, invasion, and migration [
15]. In EOC, aberrant expression of lncRNA regulates target genes through different mechanisms affecting biological behaviour of tumor cells. For example, lncRNA ABHD11-AS1 through regulating RhoC can promote A2780 and OVCAR3 proliferation and inhibit apoptosis [
26]; lncRNA PTAR through miR-101 -3p/ZEB1 pathway behave in ceRNA model promotes invasion, migration, and EMT in EOC [
27]; lncRNA PANDAR through SFRS2/p53 phosphorylation involved in the acquired Cisplatin resistance [
28]. lncRNA aberrant expression in EOC can affect tumor proliferation, migration, and drug sensitivity. It also plays an important regulatory role in cancer initiation and progression.
lncRNAs have been divided into 5 categories based on the location of the lncRNA gene in the genome: (1) sense lncRNA, (2) antisense lncRNA, (3) bidirectional lncRNA, (4) intronic lncRNA, and (5) intergenic lncRNA [
29]. In this study, we systematically investigated lncRNAs deregulation in EOC tissues versus normal ovary tissues and identified HOXD-AS1 to be significantly up-regulated in EOC tumors. HOXD-AS1 is an antisense lncRNA located on chromosome 2q31.2. Lu et al. have previously shown that HOXD-AS1 is over-expressed in metastatic hepatocellular carcinoma and promotes hepatocellular carcinoma metastasis through miR19a/ARHGAP11A signaling pathway [
30]. Furthermore HOXD-AS1 has been demonstrated to cooperate with miR-130a to regulate SOX4 and E2F8, and promote metastasis in hepatocellular carcinoma and glioma respectively [
31,
32]. In this study, we demonstrated that HOXD-AS1 acting as a cellular sponge in a ceRNA mechanism inhibits miR-186-5p and resulted in the corresponding up-regulation of PIK3R3 in EOC cells. This novel HOXD-AS1/miR-186-5p/PIK3R3 pathway plays an important role in EMT in EOC cells. In addition, high HOXD-AS1 level is significantly associated with poorer PFS and OS of EOC patients (Figs.
2 &
6), and hence demonstrating the clinical relevance of this novel pathway.
MiRNA-186-5p can bind to HOXD-AS1 and affect the expression of downstream target genes in the form of ceRNA. Abnormal expression of miRNA-186-5p in various tumors plays a regulatory role in the process of EMT. For example, high expression of miR-186-5p in colorectal cancer cells can inhibit the proliferation, metastasis, and EMT process of tumor cells; Expression changes of miR-186-5p are closely related to the proliferation and invasion ability of non-small cell lung cancer [
33]. Therefore, the study further focused on whether HOXD-AS1 can regulate the expression of miR-186-5p through a ceRNA sponge model. Data of the present study suggests that HOXD-AS1 can negatively regulate miR-186-5p.
PIK3R3 is a regulatory subunit of PI3K, whose differential expression can promote the proliferation, invasion, and migration process of NSCLC cells, cervical carcinoma cells, and pancreatic cancer cells. Our studies have suggested that PIK3R3 is the target gene regulated by HOXD-AS1/miRNA-186-5p in the mode of ceRNA. PIK3R3 was down-regulated in SKOV3 cells transfected with si-PIK3R3 accompanied with the down-regulation of Twist and Rac1, which suggests Twist and Rac1 may mediate changes in PIK3R3 expression and affecting EMT in tumor cells [
34‐
36].
In this study, we used lncRNA, microRNA, and mRNA biochips as carriers to discover a number of differentially expressed genes in ovarian cancer tissues. We utilized GEO, KEGG, DIANA and other common tumor databases for bioinformatics analysis. The results suggest that HOXD-AS1, as a ceRNA, upregulates PIK3R3 expression by sponging miR-186-5p. The higher expression of HOXD-AS1 is associated with shorter PFS and OS of EOC patients. Collectively, lncRNA-HOXD-AS1 may function as a promising biomarker and therapeutic target for human EOC treatment.
While this paper is in the process of preparation, Wang et al. reported HOXD-AS1 could regulate cell migration and invasion through miR-608 and FZD4 in ovarian cancer [
37]. Therefore, these data suggest that HOXD-AS1 may function as a master regulator of metastasis through its interaction with a number of miRNAs and their respective target genes. HOXD-AS1 functions as a cellular sponge to modulate the availability of specific miRNAs at the 3’UTR of the miRNA target genes in ceRNA theory [
20].
Conclusions
In summary, we profiled the transcriptome landscape to report significant differentially expressed mRNA, lncRNA and miRNAs in EOC tissues compared with normal ovary tissues. We delineated a novel HOXD-AS1/miR-186-5p/PIK3R3 molecular pathway that regulates cell migration, invasion, and EMT process in EOC. Suppressing HOXD-AS1 can be a possible strategy to improve the clinical outcome of EOC patients.
Acknowledgements
We would like to thank our colleagues especially Dr. Junjun Li from the department of pathology for their assistance in this project; and thank the staff of Hunan Cancer Hospital / The Affiliated Cancer Hospital of Xiangya School of Medicine; Central South University for their assistance to the study.