LncRNA MSC-AS1 aggravates nasopharyngeal carcinoma progression by targeting miR-524-5p/nuclear receptor subfamily 4 group A member 2 (NR4A2)

Between June 2013 and April 2018, amount to 34 pairs of NPC tissues and pair-matched adjacent normal tissues were collected from patients who underwent surgical resection at The Second Affiliated Hospital of Harbin Medical University. Before surgery, patients who received radiotherapy and chemotherapy were excluded. After resection, the coupled tissues were immediately preserved at -80 °C. Ethical approval was obtained from the ethics committee of The Second Affiliated Hospital of Harbin Medical University. All patients for participation provided informed consent.

Two nasopharyngeal epithelial cells (NP69 and NP460) and four nasopharyngeal carcinoma (NPC) cells (SUNE1, CNE-2, 5-8F, CNE-1) were obtained from the Chinese Academy of Sciences (Shanghai, China). All cells were incubated in RPMI-1640 medium (Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS; Gibco). Cells were incubated under the standard environment (37 °C, 5% CO₂).

At 1day prior to transfection, 5-8F or CNE-1 cells were incubated in six-well plates. Then, cells were transfected via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Short hairpin RNA (shRNA) targeting MSC-AS1 (sh-MSC-AS1#1/2) and the negative control (sh-NC), miR-23b-3p mimic, miR-524-5p mimic/inhibitor, NC mimic/inhibitor, along with the overexpression of NR4A2, MSC-AS1 and pcDNA3.1 empty vector were constructed from RiboBio (Guangzhou, China). Transfection took place after 48 h, and cells were all harvested. The sequences were shown in Additional file 1: Table S1.

Total RNA was extracted using TRIzol reagent (Invitrogen), and then the PrimeScript reverse transcriptase reagent kit (Takara, Kusatsu, Japan) was applied to synthesize cDNA. Real-time PCR amplification was then performed using an SYBR Green Real-Time PCR Kit (Applied Biosystems, Foster City, CA, USA) on the Bio-Rad CFX96 System (Applied Biosystems). As the endogenous control, U6/GAPDH was used. The 2^-ΔΔCT method was selected for transcript quantification. The primers were listed in Additional file 1: Table S1.

5-8F or CNE-1 cells were seeded in 96-well plates and incubated at different periods. Each well in fresh medium was added by 10 µl of CCK-8 solution and incubated for 4 h continually. Finally, the absorbance at OD value (450 nm) was assessed through a microplate reader (Bio-Rad, Hercules, CA, USA).

Transfected 5-8F or CNE-1 cells were placed into six-well plates and maintained in medium added with 10% FBS (Gibco). Rinsed through ice-cold PBS (Sigma-Aldrich, St. Louis, MO, USA), colonies were subsequently fixed through methanol (Sigma-Aldrich), dyed using crystal violet (Sigma-Aldrich) and then counted manually.

5 × 10⁴ transfected cells of 5-8F or CNE-1 were plated into the each well of 96-well plates, and then 100 μL of EdU medium was added for 3 h. The proliferative cells were detected using EdU assay kit (Ribobio) as per instruction. Cells were fixed by 4% paraformaldehyde, permeabilized by 0.5% Troxin X-100 and incubated in 1 × Apollo^® 488 staining solution. After DAPI staining (0.3 mM; Sigma-Aldrich) for nuclear detection, cell samples were assayed using fluorescence microscope (Leica, Mannheim, Germany).

Using Beyotime C1115 Caspase 3 Activity Assay Kit (Beyotime, Nantong, China), the activity of caspase-3 was determined. Isolated proteins from transfected 5-8F or CNE-1 cells were added into 96-well plates with the reaction buffer (Invitrogen). Finally, caspase-3 activity was examined through a microplate reader (Thermo Fisher Scientific) at 405 nm.

Transfected 5-8F or CNE-1 cells were incubated with fluorescein-labelled dUTP (Thermo Fisher Scientific). After that, the nucleus was stained by 0.3 mM of DAPI from Sigma-Aldrich. Through the fluorescence microscope (Leica, Mannheim, Germany), apoptotic cells were eventually analyzed.

In serum-free medium, transfected 5-8F or CNE-1 cells were planted in the top transwell chambers (Corning Incorporated, Corning, NY, USA), which was coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The bottom chambers (Corning Incorporated) were plated with medium containing 10% FBS (Gibco). Thereafter, invaded cells were fixed, stained with 1% crystal violet and eventually evaluated through an inverted microscope (magnification: 200 × ; Nikon, Tokyo, Japan).

Cell samples of 5-8F and CNE-1 on culture slides were incubated for 24 h until adhered to the slides. After that, cells were washed in PBS, fixed by ice-cold methanol for 10 min and blocked in 5% BSA for 10 min. The primary antibodies against E-cadherin and N-cadherin, as well as the secondary antibodies were utilized for the incubation with cells. Following DAPI staining (0.3 mM), cells were observed using fluorescence microscope (Leica).

Total protein samples from 5-8F or CNE-1 cells were extracted in RIPA lysis buffer, and then separated by electrophoresis on 12% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA). Protein samples were then transferred onto PVDF membranes (Bio-Rad Laboratories) and incubated in 5% nonfat milk for blocking membranes. The diluted primary antibodies (1: 2000; Abcam, Cambridge, MA), including mouse monoclonal anti-E-cadherin antibody (ab76055), rabbit monoclonal anti-N-cadherin antibody (ab202030), mouse monoclonal anti-NR4A2 antibody (ab41917) and mouse monoclonal anti-GAPDH antibody (ab8245) as loading control, were used to probe membranes all night at 4 °C. Following washing in Tris-buffered saline containing 0.1% Tween-20 (TBST), the horse-radish peroxidase (HRP)-tagged rabbit monoclonal secondary antibodies (1: 5000; Abcam) against IgG were added for 2 h at room temperature. At length, the protein blots were observed using ECL detection system (Santa Cruz, CA, USA) in the dark, and then placed into gel imager, followed by analysis of Bio-Rad image analysis system (Bio-Rad). The gray values of the target proteins and the internal control were compared employing the Image J software (NIH, Bethesda, MD, USA).

Expression profile of MSC-AS1 in HNSC samples and its association with overall survival of HNSC patients were downloaded from GEPIA (http://​gepia.​cancer-pku.​cn/​) [31]. The binding sequences between RNAs were predicted from starBase v2.0 (http://​starbase.​sysu.​edu.​cn/​) [32].

Using the nuclear/cytoplasmic Isolation Kit (Biovision, San Francisco, CA, USA), subcellular fractionation in 5-8F or CNE-1 cells was carried out as instructed. Cells were initially lysed in the cell fractionation buffer, and centrifuged for the separation of cytosolic and nuclear fractions. The supernatant was transferred into a fresh RNase-free tube. Thereafter, the remaining lysates were rinsed in cell fractionation buffer and centrifuged. The cell disruption buffer was added for lysing cell nuclei. Lysates and the supernatant were mixed in 2 × lysis/binding solution, equal volume of ethanol was added. After washing, TRIzol reagent was added to isolate RNAs. Finally, the expression ratios of MSC-AS1 were determined by RT-qPCR, with U6 and GAPDH used as the control for nuclear RNA and cytoplasmic RNA.

The specific RNA FISH probe to MSC-AS1 was synthesized by Ribobio Company. Cells of CNE-1 and 5-8F were fixed by 4% formalin, treated with protease K at 37 °C, and then rinsed in PBS. Cells were dehydrated in ethanol. 20 μl of hybridization solution containing 2 μl of probe and 18 μl of pre–hybridization solution was prepared to incubate with cells all night at 42 °C. Afterwards, cells were rinsed twice in 25% formamide/2 × saline sodium citrate (SSC). DAPI solution (0.3 mM) was then added for nuclear counter-staining. Samples were observed under fluorescence microscope.

54 miRNAs potentially bound to MSC-AS1 were obtained from starBase. The expressions of 54 miRNAs were detected by PCR in three pairs of adjacent normal tissues and NPC tissues. Differentially expressed miRNAs (P < 0.05 and fold change > 2.0) were picked for follow-up analyses, among which miR-524-5p was most significantly down-regulated.

Using a Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA), RIP assay was carried out. After being harvested, transfected 5-8F or CNE-1 cells were lysed in RIP lysis buffer (Solarbio, Beijing, China). Subsequently, magnetic beads (Invitrogen) were added to the RIP lysis buffer (Solarbio) and then conjugated overnight with anti-Ago2 (Abcam) or anti-IgG (Abcam) at 4 °C. The immunoprecipitated RNA was acquired after digestion with proteinase K (Absin, Shanghai, China) and quantified by RT-qPCR.

The wild-type or mutant binding sequence of miR-524-5p in MSC-AS1 or NR4A2 3′-UTR was synthesized and sub-cloned into pmirGLO dual-luciferase vector (Promega, Madison, WI, USA). MSC-AS1-Wt/Mut vector or NR4A2-Wt/Mut vector was co-transfected with NC mimic or miR-524-5p mimic into 5-8F, CNE-1 or 293T cells. 48 h later, the Dual Luciferase Report Assay System (Promega) was employed to monitor luciferase activity.

MiR-524-5p no-biotin probe and miR-524-5p biotin probe were synthesized by Thermo Fisher Scientific. The biotinylated miRNA was incubated with cell lysates (Invitrogen) overnight, followed by adding streptavidin magnetic beads (Invitrogen). Finally, RT-qPCR was used to detect the expression levels.

Through GEPIA, MSC-AS1 was identified as a highly-expressed lncRNA in HNSC samples (Fig. 1a). Besides, MSC-AS1 presented an association with the overall survival rate in HNSC, which included NPC (Fig. 1b). Thus, we further researched the association of MSC-AS1 with NPC. Firstly, the expression profile of MSC-AS1 was determined in NPC samples. We found that MSC-AS1 level was elevated in NPC samples compared with the paired adjacent non-tumor samples (Fig. 1c). Based on the cut-off value (median value) of MSC-AS1 expression in 34 patient samples, the overall survival of patients with high or low MSC-AS1 level was analyzed. As indicated in Fig. 1d, high level of MSC-AS1 was associated with the low overall survival of NPC patients. In addition, MSC-AS1 expression was higher in NPC cells than in nasopharyngeal epithelial cells (Fig. 1e). Then, the function of MSC-AS1 in NPC was determined through loss-of-function assays. Since CNE-1 and 5-8F cells were validated to present the higher MSC-AS1 level, we silenced MSC-AS1 level in these two cells by sh-MSC-AS1#1/2, and the transfection efficiency was confirmed by RT-qPCR (Fig. 1f). Thereafter, we performed CCK-8, colony formation and EdU assays to explore the function of MSC-AS1 knockdown on NPC cell proliferation. As a result, silenced MSC-AS1 significantly inhibited the proliferation of two NPC cells (Fig. 1g–i). Besides, the apoptosis of NPC cells was detected by caspase-3 activity and TUNEL staining. Results showed that knockdown of MSC-AS1 induced caspase-3 activity and increased the TUNEL staining ratio in NPC cells (Fig. 1j–k), indicating that MSC-AS1 depletion prompted apoptosis in NPC cells. Above results confirmed that MSC-AS1 was upregulated in NPC tissues and cells, promoted cell proliferation and refrained cell apoptosis.

Later, we detected the effect of MSC-AS1 on invasive capacity of NPC cells. Transwell assay depicted that the number of invaded NPC cells was decreased upon the depletion of MSC-AS1 (Fig. 2a). Moreover, EMT progression was evaluated by detecting the levels of epithelial and mesenchymal markers. As shown, E-cadherin protein level was boosted, whereas N-cadherin protein level was reduced responding to MSC-AS1 knockdown in NPC cells (Fig. 2b). Accordingly, the same results were observed in IF assay (Fig. 2c). Moreover, morphology of cells was observed after MSC-AS1 was silenced. It was found that silencing of MSC-AS1 inhibited the EMT phenotype in two NPC cells (Fig. 2d). Hence, it was suggested that knockdown of MSC-AS1 hindered cell invasion and EMT in NPC. To further validate the oncogenic role of MSC-AS1 in NPC, gain-of-function assays were carried out in SUNE1 cells which presented a relative lowest level of MSC-AS1. At first, MSC-AS1 was overexpressed in SUNE1 cells (Fig. 2e). Colony formation assay and EdU assay indicated that cell proliferation was promoted after overexpression of MSC-AS1 (Fig. 2f–g). Additionally, enhanced level of MSC-AS1 aggravated cell invasion (Fig. 2h) and EMT process (Fig. 2i–k). Altogether, it was indicated that MSC-AS1 functioned as an oncogene in NPC.

The mechanisms of lncRNAs are diverse [33]; it has been reported that lncRNAs could regulate gene expression at transcriptional level in the nucleus [34]. More studies indicated that lncRNAs could function as ceRNAs at post-transcriptional level in the cytoplasm [35]. Through subcellular fractionation assay and FISH assay, we found that MSC-AS1 was mainly expressed in cytoplasm of NPC cells (Fig. 3a), indicating the post-transcriptional regulation of MSC-AS1 in NPC. Thus, the molecular regulatory mechanism that MSC-AS1 acted as a ceRNA in NPC was investigated. We found that 54 candidate miRNAs could bind to MSC-AS1 through browsing starBase v2.0 (http://​starbase.​sysu.​edu.​cn/​). To detect their implication in NPC, we analyzed the expressions of these miRNAs in NPC tissues. Consequently, miR-524-5p was found as the most significantly downregulated miRNA in NPC tissues compared with matched adjacent non-cancerous tissues (Fig. 3b). Former studies have showed that miR-524-5p was lowly expressed and served as an anti-tumor gene in multiple cancers [19‐22]. Therefore, we chose miR-524-5p for further exploration. The downregulation of miR-524-5p in NPC cells was validated as well (Fig. 3c). RIP assay showed that miR-524-5p could be co-immunoprecipitated with MSC-AS1 by Ago2 antibody in NPC cells (Fig. 3d). Besides, we obtained the binding sequence between MSC-AS1 and miR-524-5p, and mutated the sequence on MSC-AS1 for luciferase activity assay (Fig. 3e). Results showed that overexpression of miR-524-5p decreased the luciferase activity of MSC-AS1 WT rather than MSC-AS1 Mut (Fig. 3f). According to the results of heatmap, miR-23b-3p also presented significant downregulation in NPC tissues. Thus, the binding sequence between MSC-AS1 and miR-23b-3p was predicted (Additional file 2: Figure S1a). However, luciferase reporter assay indicated that the luciferase activity of both MSC-AS1 WT and MSC-AS1 Mut was not changed by miR-23b-3p mimic (Additional file 2: Figure S1b). This data suggested that only miR-524-5p was sponged by MSC-AS1 in NPC. Then, we overexpressed miR-524-5p in NPC cells by miR-524-5p mimic (Fig. 3g). RT-qPCR showed that MSC-AS1 level was downregulated in NPC cells transfected with miR-524-5p mimic (Fig. 3h). Afterwards, we found that MSC-AS1 knockdown induced the expression of miR-524-5p in NPC cells (Fig. 3i). The negative expression correlation between MSC-AS1 and miR-524-5p levels in NPC tissues was confirmed as well (Fig. 3j). Next, we also detected the function of miR-524-5p in NPC cellular processes. In response to the upregulation of miR-524-5p, cell growth was suppressed (Additional file 2: Figure: S1c–e) and EMT process was inhibited (Additional file 2: Figure S1f), indicating the tumor-suppressive role of miR-524-5p in NPC. These data indicated that miR-524-5p was sponged by MSC-AS1 and acted as a tumor-suppressor in NPC.

Subsequently, we tried to find the target genes of miR-524-5p. As presented in Fig. 4a, the intersection of 4 bioinformatics tools (PITA, RNA22, miRmap, and microT) showed that PKN2, NR4A2, NEDD4, and SIK2 were potential targets of miR-524-5p. To investigate the interaction of miR-524-5p with these 4 mRNAs, RNA pull down assay was carried out. Results showed that only NR4A2 was enriched in miR-524-5p biotin probe while other mRNAs couldn’t be pulled down (Fig. 4b), indicating that NR4A2 interacted with miR-524-5p in NPC cells. NR4A2 was known as a member of NR4A family [23]. Multiple studies have revealed that NR4A2 functioned as a carcinogene in several cancers, and indicated poor prognosis in NPC patients [28‐30]. Hence, we deduced that MSC-AS1 might regulate NR4A2 in NPC cells through miR-524-5p. We then confirmed that NR4A2 was upregulated in NPC samples versus the adjacent non-cancerous tissues (Fig. 4c). Also, NR4A2 expression was confirmed to be positively correlated with MSC-AS1 expression and negatively correlated with miR-524-5p expression in NPC samples (Fig. 4d). Moreover, NR4A2 level was higher in NPC cells than nasopharyngeal epithelial cells (Fig. 4e). Thereafter, we detected the interaction between miR-524-5p and NR4A2. The miR-524-5p binding sequence on NR4A2 and the mutated sequence were presented. Furthermore, overexpression of miR-524-5p led to the reduced luciferase activity of NR4A2 WT but not NR4A2 Mut in 293T cells (Fig. 4f). Besides, the enrichments of NR4A2, miR-524-5p, and MSC-AS1 in the precipitates of Ago2 were confirmed by RT-qPCR following the RIP analysis (Fig. 4g). Then, the regulation of MSC-AS1/miR-524-5p axis on NR4A2 level was examined. NR4A2 mRNA and protein levels were reduced by miR-524-5p mimic in NPC cells (Fig. 4h). In addition, the inhibition of miR-524-5p restored the expressions of NR4A2 mRNA and protein reduced in MSC-AS1-silenced NPC cells (Fig. 4i). Jointly, these data implied that MSC-AS1 induced NR4A2 level by sponging miR-524-5p in NPC cells.

To further reveal the function of NR4A2 in NPC, we conducted loss-of-function assays in two NPC cells. At first, NR4A2 expression was silenced in CNE-1 and 5-8F cells (Fig. 5a). Cell proliferation was monitored through several proliferation assays. Unsurprisingly, silencing of NR4A2 observably inhibited the proliferative ability of two NPC cells (Fig. 5b–d). Additionally, cell apoptosis was observed in NR4A2-downregulated NPC cells. As indicated in Fig. 5e–f, cell apoptosis was accelerated by the knockdown of NR4A2. Through western blot analysis, we concluded that EMT process was impaired by the silencing of NR4A2 (Fig. 5g). These findings reflected the same function of NR4A2 as MSC-AS1 in NPC cells.

To figure out whether MSC-AS1 promoted NPC progression by regulating NR4A2, rescue experiments were implemented in CNE-1 cells. We first confirmed that NR4A2 mRNA and protein levels reduced by MSC-AS1 silence were recovered under the co-transfection of pcDNA3.1/NR4A2 in CNE-1 cells (Fig. 6a). Besides, the prohibitive effect of MSC-AS1 silence on CNE-1 cell proliferation was abrogated by NR4A2 overexpression (Fig. 6b–d). Later, we observed that MSC-AS1 knockdown increased the apoptosis of CNE-1 cells, but such acceleratory effect was reversed by overexpression of NR4A2 (Fig. 6e–f). In addition, the invasive ability of CNE-1 cells retarded by MSC-AS1 silence was recovered by NR4A2 overexpression (Fig. 6g). Subsequently, NR4A2 overexpression reversed the induced level of E-cadherin and reduced level of N-cadherin in NPC cells with MSC-AS1 depletion (Fig. 6h). In conclusion, it was suggested that MSC-AS1 hastened NPC cell proliferation, restrained apoptosis, facilitated invasion and EMT through regulating NR4A2.