Introduction
Colorectal cancer (CRC), also known as large bowel cancer, is a major public health problem worldwide [
1]. Epidemiological data have revealed that the 5-year survival rate of CRC patients ranges from 90% for patients with stage I disease to 10% for those with metastatic disease [
2]. Although numerous studies have revealed that alterations in oncogenes and tumour suppressor genes contribute to tumorigenesis and the development of CRC [
3], the precise molecular mechanisms underlying CRC pathogenesis, particularly for metastasis, remain to be fully elucidated.
Long noncoding RNAs (lncRNAs), which are more than 200 nt in length and have limited or no protein-coding capacity, play both oncogenic and tumour suppressor roles in tumorigenesis and progression [
4,
5]. LncRNAs can regulate gene expression via multiple mechanisms, including chromatin remodelling, modulation of the activity of transcriptional regulators, and posttranscriptional modifications [
5]. Dysregulated lncRNA expression has been reported to modulate the progression of various types of cancers, such as bladder, prostate, lung, breast, gastric and colorectal cancers [
6,
7]. Increasing evidence suggests that lncRNAs can trigger metastatic progression, increase chromosomal instability, and promote CRC tumorigenesis [
8‐
10]. Therefore, further identification of CRC-related lncRNAs and investigations of their functions in CRC are imperative.
Metastasis is the major cause of CRC related death [
11]. The epithelial mesenchymal transition (EMT), a process by which epithelial cells gain a migratory and invasive mesenchymal phenotype [
12], is considered as the first and most important step for cancer cell metastasis. During EMT, epithelial cells can acquire mesenchymal components and motility features, lose epithelial components and cell adhesion, and infiltrate into the tumour vasculature [
13]. Increasing evidences indicate that EMT is a pivotal step for tumour infiltration and distant metastasis in a variety of carcinomas [
14]. EMT-transcription factors (EMT-TFs), including Twist, Snail, and Zeb1, have been implicated in the control of EMT [
15]. The important role of Zeb1 in EMT regulation has been described for many cancer types [
16,
17]. LncRNAs have been reported to regulate EMT-TFs and subsequently trigger the EMT of cancer cells [
18]. We are interested in determining whether any lncRNAs exist that can regulate EMT-TFs to trigger the EMT and dissemination of CRC cells.
In this study, a CRC-associated lncRNA (RP11, RP11-138 J23.1) that displayed a remarkable trend towards increasing expression levels from normal colorectal to CRC tissues was identified and selected for further validation and functional analysis in terms of CRC progression. We demonstrated that post-translational upregulation of Zeb1 is required for the lncRNA RP11-induced EMT and dissemination of CRC cells.
Materials and methods
Microarray and computational analysis
Fresh paired normal and histologically confirmed CRC tumour tissues were obtained from 3 stage I CRC cases and 3 stage IV cases with distant metastasis before any treatment during surgery from the Sixth Affiliated Hospital of Sun Yat-sen University from February to October 2014. Total RNA from the samples (3 stage I CRC tissues, 3 stage IV CRC tissues, and their corresponding paired nontumour tissues) was extracted, amplified and transcribed into fluorescent cRNA using the Quick Amp Labeling kit (Agilent Technologies, Palo Alto, CA, USA). The labelled cRNA was then hybridized onto the Human LncRNA Array v2.0 (8 × 60 K, ArrayStar, Rockville, MD, USA), and after the washing steps, the arrays were scanned with the Agilent Scanner G2505B. Agilent Feature Extraction software (version 10.7.3.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). The differentially expressed lncRNAs with statistical significance were identified using Volcano Plot Filtering. The threshold used to screen upregulated or downregulated lncRNAs was a fold change ≥2.0 and p < 0.05.
Database (DB) search
The expression of lncRNA RP11 in CRC and other cancers was analyzed using the GEPIA (Gene Expression Profiling Interactive Analysis) online database (
http://gepia.cancer-pku.cn). The expression of RP11 between tumour and normal tissues or among different stages of CRC was also analyzed with GEPIA. GEPIA can deliver fast and customizable functionalities based on data from The Cancer Genome Atlas (TCGA) and provide key interactive and customizable functions, including differential expression analysis, correlation analysis and patient survival analysis [
19]. We used the Kaplan-Meier plotter to assess the prognostic value of RP11, Zeb1, and their normalization to Siah1 or Fbxo45 expression in CRC patients based on the data from the GEPIA online database. The high expression was defined as greater than the median of the values of transcripts, while the low expression was defined as less than the median of the values of transcripts.
Data about the expression of Zeb1 in CRC and normal tissues were further obtained from the Oncomine database (
www.oncomine.org) as follows: Hong Colorectal [
20] and Skrzypczak colorectal 2 [
21]. The sample information and expression data are available in the Gene Expression Omnibus (GEO) database [Accession nos. GSE2091 (Skrzypczak colorectal 2) and GSE9348 (Hong Colorectal) at
www.ncbi.nlm.nih.gov/geo].
The expression profiles of Zeb1, Fbxo45, METTL3 and Siah1 among the N stages of CRC in patients were downloaded from LinkedOmics (
http://www.linkedomics.org), which is a publicly available portal that includes multi-omics data from all 32 cancer types from TCGA. The LinkedOmics website allowed a flexible exploration of associations between a molecular or clinical attribute of interest and all other attributes, providing the opportunity to analyse and visualize associations between billions of attribute pairs for each cancer cohort [
22].
Animal studies
All animal experiments were complied with the Zhongshan School of Medicine Policy on the Care and Use of Laboratory Animals. To evaluate the potential roles of RP11 in the growth of CRC, ten female BALB/c nude mice (4 weeks old) purchased from Sun Yat-sen University (Guangzhou, China) Animal Center were raised under pathogen-free conditions and randomly divided into two groups. HCT-15 RP11 stable overexpression or control cells (2 × 106 per mouse) diluted in 100 μl normal medium + 100 μl Matrigel (BD Biosciences) were subcutaneously injected into immunodeficient mice to investigate tumour growth. When the tumours of all mice grew into visible tumours, the tumour volumes were measured every 3 d using manual callipers and calculated using the formula V = 1/2 × larger diameter × (smaller diameter) 2. At the end of the experiment, mice were sacrificed, and tumours were removed and weighed for use in histological and other analyses.
For the in vivo liver metastasis model, HCT-15 RP11 stable overexpression or control cells (1 × 106 per mouse) were injected into both male and female BALB/c nude mice (n = 7 for each group) via the tail vein to analyze distant metastasis. Eight weeks after injection, the experiment was terminated, and livers were analyzed for the presence of metastatic tumours.
Protein stability
To measure protein stability, cells were treated with cycloheximide (CHX, final concentration 100 μg/ml) for the indicated time periods. Zeb1 expression was measured by western blot analysis.
RNA immunoprecipitation
RNA immunoprecipitation (RIP) experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to previously described procedures [
23]. Antibodies for RIP assays of IgG, Zeb1, Siah1, Fbxo45, hnRNPA2B1, and m
6A were diluted 1: 1000. After RIP, RNA concentrations were measured using the Qubit® RNA High-sensitivity (HS) Assay Kit and Qubit 2.0. The co-precipitated RNAs were detected by reverse transcription (RT)-PCR. The gene-specific primers used for detecting RP11 were presented in Additional file
2 :Table S2. RNA expression was normalized to the total amount of RNA used for reverse transcription.
RNA pull-down/mass spectroscopy analysis
LncRNA-RP11 and its antisense RNA were transcribed in vitro from the pGEM-T-RP11 vector, biotin-labelled with the Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA) and T7/SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). One milligram of protein from the extracts of HCT-15 cells stably transfected with pcDNA3.1-RP11 was then mixed with 50 pmoles of biotinylated RNA, incubated with streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA), and washed. The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver-stained, and the specific bands were excised. In-gel proteolysis was performed using trypsin (89,871, Pierce, Rockford, IL, USA). Mass spectroscopy (MS) analysis was then performed on a MALDI-TOF instrument (Bruker Daltonics) as described elsewhere [
24].
mRNA stability
To measure RNA stability in HCT-15 RP11 stable overexpression or control cells, 5 μg/ml actinomycin D (Act-D, Catalogue #A9415, Sigma, St. Louis, MO, USA) was added to cells. After incubation at the indicated times, cells were collected, and RNA was isolated for qRT-PCR. The mRNA half-life (t1/2) of ZEB1, Siah1 or Fbxo45 was calculated using ln2/slope, and GAPDH was used for normalization.
Statistical analysis
Statistical analysis was performed using SPSS software (SPSS, Chicago, Illinois, USA). The expression levels of lncRNA RP11 in CRC patients were compared with the paired-sample t test. Survival curves were generated using the Kaplan-Meier method, and the differences were analysed with the log-rank test. The χ2 test, Fisher’s exact probability, and Student’s t-test were used for comparisons between groups. Data were expressed as the mean ± standard deviation (SD) from at least three independent experiments. All P values were two-sided and obtained using SPSS v. 16.0 software (Chicago, IL, USA). p < 0.05 was considered statistically significant.
Discussion
The application of next-generation sequencing has revealed that thousands of lncRNAs are involved in the progression of human disease. Several lncRNAs have been reported to play key roles in cancer developmental processes, including proliferation, survival, migration or genomic stability [
25]. Among the few lncRNAs that have been functionally characterized, several have been linked to cancer cell invasion and metastases [
38,
39]. Regarding CRC progression, lncRNAs have been reported to regulate cell survival [
40], tumorigenicity [
10], and asymmetric stem cell division [
41]. By using microarray analysis and functional screening, we show that lncRNA RP11, which is upregulated by m
6A methylation, can trigger the migration, invasion and EMT of CRC cells via post-translational upregulation of the EMT-TF Zeb1.
Our study highlights the function and mechanisms of RP11 in regulating CRC metastasis. Among the 8 simultaneously upregulated lncRNAs between stage I and normal tissues, stage IV and normal tissues, and stage IV and stage I tissues, RP11 expression in CRC tissues was not only greater than that in adjacent normal tissues but also higher than that in other cancers, suggesting that RP11 might be a specific target for CRC diagnosis and therapy. By screening for its potential roles in cell proliferation, colony formation, cell cycle progression, apoptosis, drug sensitivity/accumulation, and ROS generation via gain- and loss-of-function assessments, we found that RP11 can trigger the migration, invasion and EMT of CRC cells both in vitro and in vivo. This was evidenced by the observed upregulation of FN and vim and downregulation of E-Cad. Together with published reports of cancer metastasis-related lncRNAs, such as lncRNA-ATB [
38], SChLAP1 [
39], NKILA [
30], and PNUTS [
42], our study confirms the regulatory roles of lncRNAs in EMT and cancer metastasis. High RP11 expression correlates with positive lymph node metastasis and advanced TNM stage, suggesting that RP11 can be a strong predictor of CRC metastasis and prognosis.
We find that the post-translational regulation of Zeb1 plays an essential role in the RP11-triggered dissemination of CRC. Zeb1 is a well-known and powerful EMT-TF that promotes EMT, metastasis, and the generation of cancer stem cells in many types of malignancies, including CRC [
28,
43]. We findd that RP11 has no effect on mRNA expression but increases the protein expression of Zeb1 in CRC cells by increasing Zeb1 protein stability and decreasing Zeb1 ubiquitination. By screening for factors responsible for the stability of Zeb1 in cancer cells, we confirm that the downregulation of Siah1 and Fbxo45 mediates the RP11-induced stabilization of Zeb1 in CRC cells. As ubiquitin E3 ligases, Siah1 and Fbxo45 can induce Zeb1 degradation through the ubiquitin-proteasome pathway [
44,
45].
lncRNAs can modulate the stability and nuclear turnover of specific mRNAs via RBPs and miRNAs [
5]. In this work, the RP11-hnRNPA2B1-mRNA complex downregulates the mRNA stability of Siah1 and Fbxo45 in CRC cells. RP11 can be detected in both the cytoplasm and nucleus in CRC cells. The actions of RP11 towards decreasing mRNA stability through hnRNPA2B1 can be attributed to the cytoplasmic localization RP11. This is supported by the observation that RP11 increases the cytoplasmic accumulation of hnRNPA2B1, while hnRNPA2B1 overexpression decreases the expression of Siah1 and Fbxo45. Several existing studies have demonstrated that lncRNAs form complexes with RBPs and then trigger mRNA decay [
32,
46]. HnRNPA2B1 is known to form complexes with lncRNAs and is emerging as an important mediator of lncRNA-induced transcriptional repression [
47]. Recently, lncRNA
lncHC-binding hnRNPA2B1 has been reported to directly bind to the
Cyp7a1 and
Abca1 mRNAs and reduce their expression levels in hepatocytes [
32]. In addition, hnRNPA2B1 interaction with lncRNA RMST may indicate the participation of the lncRNA in alternative splicing, mRNA trafficking, and neuronal cell survival [
48]. Although our findings link RP11 and hnRNPA2B1 to suppression of mRNA stability, the detailed molecular mechanism is not currently understood in depth. This might be because hnRNPA2B1 can recruit factors involved in the mRNA degradation pathway (such as P bodies) to accelerate mRNA degradation.
Finally, we explore whether m
6A methylation, but not DNA methylation or histone acetylation, is involved in the upregulation of RP11 in CRC cells. m
6A methylation involvement is evidenced by the observation that RP11 is significantly enriched with m
6A-RIP and that Mettl3 significantly increases RP11 expression in CRC cells. As one of the most common RNA modifications, m
6A can be found on almost all types of RNAs; can modulate all stages of the RNA life cycle, such as RNA processing, nuclear export and translation [
33,
34]; and can therefore regulate cancer progression processes, such as cell proliferation [
49] and tumorigenesis [
50]. However, investigations of the functions of m
6A in lncRNAs are few. One recent study first revealed an m
6A-dependent model of the lincRNA/miRNA interaction in which the m
6A modification of
linc1281 was required for the direct binding of let-7 to
linc1281 in embryonic stem cells (ESCs) [
51]. We reveal that m
6A could increase RP11 accumulation in the nucleus and on chromatin. We find that Mettl3 overexpression could increase binding between hnRNPA2B1 and RP11 in CRC cells, which might be due to m
6A-induced alterations in the local RNA structure and enhancements in the RNA binding of hnRNPs [
52]. Considering that knowledge of the mechanism of RNA methylation is still in its infancy, additional discoveries of regulatory patterns mediated by m
6A on the biogenesis and functions of lncRNA are worth verifying in the future.
In conclusion, our findings demonstrate the pro-metastatic role of lncRNA RP11 in the dissemination of CRC cells. We have discovered that RP11 post-translationally stimulates Zeb1 expression via downregulation of the mRNA expression of Siah1 and Fbxo45 by binding to hnRNPA2B1. Furthermore, m6A modification may increase RP11 expression and function in CRC cells and tissues. Considering the high and specific levels of RP11 in CRC tissues, our present study provides a potent target that may serve as a predictive marker of metastasis and as an effective target for anti-metastatic therapies for CRC patients.
Acknowledgements
We thank Prof Chuan He at the University of Chicago for helpful discussions and data analysis.