Long noncoding RNA MRCCAT1 promotes metastasis of clear cell renal cell carcinoma via inhibiting NPR3 and activating p38-MAPK signaling

A total of 34 metastatic and 34 non-metastatic ccRCC primary tissues were collected from patients who underwent surgery for ccRCC at Changzheng Hospital, Second Military Medical University (Shanghai, China), including 6 cases of ccRCC tissue samples (3 metastatic tumor samples and 3 non-metastatic tumor samples) used in this study for lncRNA microarray analysis. These tissues were flash frozen in liquid nitrogen immediately after surgery and subsequently stored at −80 °C. All the resected nodules were identified by pathologic examination. No patients received anticancer treatments before surgery in this study. All samples were staged according to the tumor node metastasis (TNM) classification and criteria of World Health Organization (WHO), and tumor grade was assessed in accordance with the WHO criteria. The median fellow-up time of these 68 ccRCC patients is 52 month. Written informed consent was obtained from all patients. The Ethics Committee of Changzheng Hospital, Second Military Medical University approved the use of these tissues in this study.

Microarray analysis for the expression of lncRNAs and mRNAs was performed by Shanghai Gminix Biological Information Company (Shanghai, China), using Affymetrix glue grant human transcriptome array. The accession number for the microarray data reported in this paper is Gene Expression Omnibus database GEO: GSE88948.

To further elucidate correlations between lncRNAs and mRNAs, potential lncRNA-mRNA co-expression analysis was conducted according to the normalized signal intensity of specific expression genes.

Plasmid pcDNA3.1⁺-MRCCAT1 and pcDNA3.1⁺-vector were purchased from BioBoer Technologies (Wuhan, China). Short hairpin RNA (shRNA) sequences were designed by Hanbio Biotechnology Co. Ltd. (Shanghai, China) to target human MRCCAT1. After annealing, double strands of shRNA were inserted into lentiviral pHBLV-U6-Scramble-Luc-Puro vector (Hanbio, Shanghai, China), named Lv-shMRCCAT1, and negative control was named Lv-shNC. Human full-length cDNA of NPR3 was cloned into expression plasmid pLVX-MCS-3flag-IRES-ZsGreen1-PGK-Puro (Genomeditech, Shanghai, China), named pLVX-NPR3, and the empty pLVX-puro lentiviral expression vector was used as control (pLVX-vector).

Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Cells were incubated for 24 h before use in experiments. Stable cell lines were generated by using puromycin. The sequences of primers used for plasmid construction in this study were provided in Additional file 1: Table S1.

The human ccRCC cell lines 786-O and Caki-1 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and McCoy’s 5A medium (Gibco) and supplemented with 10% fetal bovine serum (Gibco).

The total RNA of the tissue samples was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was converted from total RNA by using a Reverse Transcription Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with SYBR Green (Takara, Dalian, China), and the data collection was performed on the Applied Biosystems® 7500 Real-Time PCR Systems (Thermo Fisher Scientific) according to the manufacturer’s instructions. The primers were synthesized by Biosune (Shanghai, China). The relative expression level of indicated genes was compared with that of β-actin and expression fold changes were calculated using 2^-△△Ct methods [14]. Each qRT-PCR reaction was performed in triplicate. Sequences of primers used for qRT-PCR in this study are shown in Additional file 1: Table S2.

We performed the 5′-RACE and 3′-RACE analyses to determine the transcriptional initiation and termination sites of MRCCAT1 using a SMARTer™ RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. The gene-specific primers used for the PCR of the RACE analysis were 5′-GGTCATTGGAGTCAGTGCTCTC-3′ (5′-RACE) and 5′-CGCTGTATCTTCTCCTCAGGTATG-3′ (3′-RACE).

Cell proliferation was assessed using a cell proliferation kit, Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kyushu, Japan), according to the manufacturer’s instructions. Cells were seeded into 96-well culture plates at a density of 2 × 10³ cells/well the day before transfection. Approximately 10 μl of CCK8 regent was added to each well after transfection and incubated at 37 °C for 2 h. Cell growth was analyzed at a wave length of 450 nm at 24, 48, 72, and 96 h after transfection using Spectra Max M3 (Molecular Devices). Experiments were performed in triplicate.

ccRCC cells were seeded at 5 × 10⁵ cells/well in 6-well plates and cultured until the plates were confluent. The cell monolayer was scraped in a straight line using a 10 μl pipette tip to create a scratch, washed with PBS twice and the medium was replaced with serum-free medium. Images were captured at 0, 24(18), and 48(36)h following the initial scratch to evaluate cell migration.

The invasion of ccRCC cells was assessed based on the number of cells through Matrigel-coated Transwell inserts, as previously described [15]. In brief, 3 × 10⁵ cells were seeded into 24-well plate-sized inserts (a chamber containing a pore size of 8 μm, Corning Life Sciences, NY, USA) with Matrigel (BD Biosciences, San Jose, CA, USA). Cells were plated in medium without serum, with the lower chamber containing the medium plus 10% FBS, and thus serving as a chemo-attractant. After incubation for 24 h, the cells that did not invade through the pores were carefully wiped out with cotton wool. All cells that had migrated from the upper to the lower side of the filter were fixed with 4% paraformaldehyde and stained with 1% crystal violet, and then counted and imaged (magnification, ×100). The assay was conducted three separate times.

Western blot was conducted under standard procedures [16]. Briefly, cells were lysed to obtain proteins using RIPA. Proteins were separated by 8% SDS–PAGE and then transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: NPR3 (Proteintech-25,248-1-AP), p-ERK, p-JNK, p-p38 MAPK, total ERK1/2, total JNK, total p38 MAPK (Cell Signaling Technology, Boston, MA, USA), and β-actin (Santa cruz-81,178); then the following secondary antibodies were used: rabbit anti-mouse IgG and goat anti-rabbit IgG (SAB, Bethesda, MD, USA). All antibodies were diluted in nonfat dry milk. The immunoreactive protein bands were visualized by ECL Kit (Pierce, Thermo Fisher Scientific, IL, USA). The experiment was performed three separate times.

We performed RNA immunoprecipitation (RIP) experiments using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. The EZH2 antibody used for RIP was clone AC22 (17-662; Millipore). An aliquot of lysate was removed as an input control. RNA enrichment was determined by qRT-PCR and normalized to the input control.

RNA pull-down was performed as described previously [17]. Briefly, biotin-labeled RNAs were transcribed 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 the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA). Cell nuclear proteins were extracted using the Proteo JETTM Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, St. Leon-Rot, Germany). One milligram of 786-O or Caki-1 cell nuclear extract was then mixed with 50 pmol of biotin-labeled RNAs. 60 μl of washed streptavidin agarose beads (Invitrogen) were added to each binding reaction and further incubated at room temperature for 1 h. Beads were washed briefly five times and boiled in sodium dodecyl sulfate buffer, and the retrieved protein was detected by the standard western blot technique.

Chromatin immunoprecipitation (ChIP) was performed using the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore), according to the manufacturer’s instructions. Briefly, cross-linked chromatin was sonicated into 200-1000 bp fragments. The chromatin was immunoprecipitated using anti-EZH2 (Millipore), anti-H3K27me3 (Millipore), anti-RNA Pol II antibodies. Normal mouse immunoglobulin G (IgG) was used as negative control. qRT-PCR was conducted using SYBR Green (Takara) and the gene-specific primers with the sequences 5′-GCCTGCGGGGAGTGGTGGT-3′ (forward) and 5′-CCGAGGCCGGGCTTGTGTT-3′ (reverse).

Male nude mice (BALB/c Nude; 4 weeks old) were purchased from the Shanghai Institute of Material Medical (Chinese Academy of Science, Shanghai, China) and maintained in a pathogen-free condition in accordance with relevant guidelines and regulations for the care and use of laboratory animals, with the approval of the Institutional Animal Care and Use Committee at Second Military Medical University. To establish a metastatic cancer model in vivo, 786-O cells infecting Lv-shMRCCAT1 and 786-O cells infecting Lv-shNC stably expressing luciferase were elected by puromycin (2 μg/ml). Cells (2 × 10⁶) in 200 μl PBS were injected into the tail vein of mice. Metastatic progression was monitored weekly and quantified using a noninvasive bioluminescence In-Vivo Imaging System (IVIS; Xenogen) 10 min after intraperitoneal injection of 4.0 mg of luciferin (Gold Biotech) in 50 μl of saline, as previously described [18, 19]. Mice were sacrificed at 6 weeks after inoculation, and consecutive sections of the whole lung were subjected to hematoxylin-eosin staining. In addition, lungs with metastatic renal cell carcinomas were measured and used for further analysis, such as Western blot.

To identify lncRNAs essential for ccRCC metastasis, 3 metastatic ccRCC tissues and 3 non-metastatic ccRCC tissues were subjected to lncRNA expression microarray. Cluster analysis showed a clear distinction between metastatic ccRCC tissues and non-metastatic ccRCC tissues (Fig. 1a). We identified 206 upregulated lncRNAs and 114 downregulated lncRNAs (P value <0.0001, fold change >2). Among the upregulated lncRNAs in metastatic ccRCC, TI18530REF_ELL2_009 (ENST00000505584) is found to be significantly higher in 34 metastatic ccRCC tissues than that in 34 non-metastatic ccRCC tissues (Fig. 1b). Compared with TI20445REF_ATF3_003, TI18717REF_TPM4_006 and TI18530REF_ELL2_004, the expression difference of TI18530REF_ELL2_009 is more significant (Fig. 1b-e). As a result, we named TI18530REF_ELL2_009 as metastatic renal cell carcinoma-associated transcript 1 (MRCCAT1).

Analyses of the correlation between MRCCAT1 expression and clinicopathological traits showed that MRCCAT1 expression is much higher in Fuhrman III and IV grades than Fuhrman I and II grades (P = 0.001); and in tumors >7 cm than tumors ≤7 cm (P = 0.007) (Fig. 2a and b). There is no significant correlation between MRCCAT1 expression with age (P = 0.091) or gender (P = 0.397) (Table 1). These data suggest that high MRCCAT1 expression may have an important role in ccRCC progression and metastasis.

We also compared the survival time following surgery in these 68 cases of ccRCC patients with higher MRCCAT1 expression (above the median value, n = 34) with those with lower MRCCAT1 expression (below the median value, n = 34). As shown in Fig. 2c and d, cumulative recurrence free survival and overall survival rates were significantly better in patients with lower MRCCAT1 expression than in those with higher MRCCAT1 expression. Additionally, univariate analysis showed that patients with high MRCCAT1 expression, metastases, high Fuhrman grade, and large tumor size were significantly associated with an increased risk of cancer-related death. Multivariate analysis identified high expression level of MRCCAT1 as an independent prognostic factor for ccRCC patients (Table 2). These data indicated that MRCCAT1 expression is a new prognostic factor for ccRCC patients.

Since actual sequences of lncRNAs generally differ from their predicted sequences [20‐22], we determined the transcriptional initiation and termination sites of MRCCAT1 using 5′- and 3′-RACE. As shown in Fig. 2e and Additional file 2: Figure S1a, the full-length of MRCCAT1 is 1718 bp, longer than the predicted sequences. MRCCAT1 includes two exons that are located at Chromosome 5:95,900,228-95,901,132 (Fig. 2f). ORF Finder (National Center for Biotechnology Information) failed to predict a protein of more than 62 amino acids, as shown in Additional file 2: Figure S1b. Consistently, the txCdsPredict score of MRCCAT1 is 245.0, supporting that MRCCAT1 has no protein-coding potential. We also performed a codon substitution frequency analysis using PyhloCSF [23, 24]. MRCCAT1 showed very low codon substitution frequency scores (−0.35), indicating that it is a non-coding RNA (Additional file 2: Figure S1c). Furthermore, CNCI software (The Broad Institute, Cambridge, MA,USA) showed that MRCCAT1 did not contain a valid Kozak sequence and is identified to be an lncRNA, rather than a protein-coding transcript, with a score of −0.19 [25]. Thus, we identified a novel lncRNA highly expressed in metastatic ccRCC.

To investigate the functional role of MRCCAT1 in ccRCC cells, we performed loss- or gain-of function experiments. First, three independent MRCCAT1-specific shRNAs were designed and transfected into786-O and Caki-1 cells to reduce MRCCAT1 expression (Fig. 3a). CCK-8 assay showed that MRCCAT1 knockdown inhibites the proliferation rate of 786-O and Caki-1 cells (Fig. 3b). Wound healing assay demonstrated that MRCCAT1 knockdown suppresses the migration ability of ccRCC cells compared with NC group (Fig. 3c and d). Moreover, Transwell invasion assays revealed that MRCCAT1 knockdown inhibites ccRCC cells invasion (Fig. 3e and f).

In parallel, the roles of MRCCAT1 overexpression in 786-O and Caki-1 cells were also investigated. Ectopic overexpression of MRCCAT1 was achieved by transfection of pcDNA3.1⁺-MRCCAT1 into 786-O and Caki-1 cells (Fig. 4a). CCK8 assay suggested that cell proliferation is enhanced by MRCCAT1 overexpression in 786-O and Caki-1 cells (Fig. 4b). Furthermore, MRCCAT1 overexpression promotes the migration and invasion of ccRCC cells (Fig. 4c-f). Together, these data indicated that MRCCAT1 could increase metastatic potential of ccRCC cells.

To examine the effect of MRCCAT1 on ccRCC metastasis in vivo, we established a lung metastatic mice model using MRCCAT1 stably depleted 786-O cells with luciferase expression by tail vein injection [26]. Compared with control group, bioluminescent signals were lower in the MRCCAT1 knockdown group (Fig. 5a and b). Six weeks later, less and smaller metastatic lesions were microscopically detected in the lungs of nude mice inoculated with MRCCAT1 knockdown cells compared with those inoculated with control cells (Fig. 5c and d). These results suggested that MRCCAT1 could promote pulmonary metastasis of ccRCC cells in vivo.

An lncRNAs-mRNAs co-expression network was built according to the normalized signal intensity of specific expression genes in the microarray. For each pair of genes, we calculated the Pearson correlation and chose the significant correlated pairs to construct the network (Additional file 3: Figure S2). In this lncRNAs-mRNAs co-expression network, we found that MRCCAT1 is negatively correlated with NPR3. To verify the prediction, we examined the RNA level of MRCCAT1 and NPR3 in 68 clinical ccRCC samples. Real-time PCR data showed that the expression of MRCCAT1 is negatively correlated with NPR3 (P = 0.042, r = −0.28), in line with the lncRNAs-mRNAs co-expression network (Fig. 6a).

Next, we examined whether MRCCAT1 could negatively regulate NPR3 expression in ccRCC cells. As shown in Fig. 6b, Real-time PCR data showed that NPR3 mRNA level is significantly increased in MRCCAT1 knockdown 786-O and Caki-1 cells. While, the NPR3 mRNA level is significantly decreased upon MRCCAT1 overexpression in 786-O and Caki-1 (Fig. 6c). Furthermore, western blot analysis revealed that the protein level of NPR3 is markedly increased in response to MRCCAT1 knockdown in 786-O and Caki-1 cells (Fig. 6d). Additionally, NPR3 expression is elevated in MRCCAT1 knockdown lung metastatic tumors (Fig. 6e). These data suggest that the NPR3 expression is inhibited by MRCCAT1 in ccRCC cells.

Based on these observations, we further sought to determine whether NPR3 was responsible for the pro-metastatic role of MRCCAT1. As shown in Fig. 6f and g, induction of NPR3 could abolish the role of MRCCAT1 in promoting cell proliferation. Consistently, the enhanced cell invasion by overexpression of MRCCAT1 is reversed by coexpression of NPR3 (Fig. 6h and i). Together, these data indicated that MRCCAT1 promotes ccRCC progression by suppressing NPR3.

NPR3 has previously been shown to play a pivotal role in tumor proliferation and metastasis by being negatively coupled to adenylyl cyclase and MAPK signaling pathways [11‐13]. Adenylyl cyclase can result in stimulation of mitogen-activated protein kinase (MAPK) signal pathways [27, 28]. To address whether MAPK signaling pathways were involved in the function of MRCCAT1, the phosphorylation levels of ERK, p38 and JNK were detected by Western blot. We found that phosphorylated p38 is increased in MRCCAT1 overexpressed cells and could be reversed by NPR3 induction, while the phosphorylation levels of ERK and JNK are unchanged (Fig. 7a and b). Furthermore, transwell invasion assay revealed that MRCCAT1-enhanced cell invasion could be perturbed by SB203580 (50 μmol/L) treatment, a p38-MAPK inhibitor (Fig. 7c). Taken together, these data suggested that MRCCAT1 promotes ccRCC metastasis by negatively regulating NPR3 expression and then activating p38-MAPK signaling pathway.

To investigate the mechanisms by which MRCCAT1 regulate NPR3, we first examined the subcellular localization of MRCCAT1. As shown in Fig. 8a, MRCCAT1 is predominately localized in nucleus. Recent studies have reported that lncRNAs in nucleus could recruit polycomb-group proteins to regulate gene expression [7, 17]. Twenty percent of all human lncRNAs have been shown to physically associate with Polycomb Repressive Complex 2 (PRC2) [29], which comprises of SUZ12, EED, EZH1/2 (H3K27 methyltransferase) and RbAp16/48, and represses gene transcription by inducing trimethylation of H3K27. Some lncRNAs have been shown to act in trans to alter the target specificity of PRC2 and thus inhibit a number of anti-metastatic genes [7]. Thus, we hypothesized that MRCCAT1 might repress NPR3 expression in such manner. To test this, we performed RIP assay with an antibody against EZH2 (an important subunit of the PRC2 complex), and found a significant enrichment of MRCCAT1 with EZH2, compared with IgG control (Fig. 8b). Moreover, RNA pull-down further confirmed the interaction between MRCCAT1 and EZH2 (Fig. 8c). To further address whether MRCCAT1 repressed NPR3 transcription through recruiting EZH2 to NPR3 promoters, we conducted ChIP analysis in MRCCAT1-overexpressing 786-O cells. ChIP assay demonstrated that MRCCAT1 increases the binding of EZH2 and H3K27me3 at NPR3 promoter regions (Fig. 8d), indicating that MRCCAT1 bound to EZH2 to repress NPR3 transcription.