Mujie Ye , Meng Wei, Runnan Gao, Kuiran Dong*

Abstract Background: We have shown that downregulation of materally expressed gene 3 (MEG3) promoted autophagy and epithelialmesenchymal transition to promote neuroblastoma (NB) progression. In present study, we aim to further discover the role of MEG3 in NB. Methods: Expression levels of MEG3 in stabled transfected cells were detected by qPCR. Flow cytometry was used to examine the effects of MEG3 overexpression on apoptosis and cell cycle. Moreover, tumor stemness was detected by tumor sphere formation assay. Results: QPCR showed that MEG3 was successfully overexpressed in SK-N-BE(2)C and SK-N-AS cells. CCK8 indicated MEG3 reduced cell proliferation in two NB cells. Flow cytometry revealed that MEG3 promoted apoptosis and caused G0/G1 arrest.Using chromatin isolation by RNA purification (ChIRP) and tumor sphere formation assays, we revealed that upregulation of MEG3 decreased tumor stemness by decreasing Nestin transcription. Conclusions: Overall, present study confirmed an anti-cancer role of MEG3 in NB, demonstrated that MEG3 could be a potential therapeutic target of NB.

Keywords: Neuroblastoma; long non-coding RNA; MEG3; epigenetics; tumor stemness

Introduction

Neuroblastoma (NB), derived from sympathetic neural crest cells,commonly develops in sympathetic nerve chains and the adrenal medulla [1-3]. It is an extra-cranial and embryonic solid tumor in children, accounting for 8% to 10% of childhood malignancies and 15% of childhood cancer deaths [4-7]. Traditional surgery,radiotherapy and chemotherapy have not yet achieved satisfactory prognosis, especially in high-risk NB8-10. Although novel treatment strategies, such as chimeric antigen receptor T-cell (CART) and PD1/PDL1 immunotherapy, have shown promise, issues concerning their short- and long-term side effects remain [11,12].

Long noncoding RNAs (lncRNAs) are a kind of RNAs that are more than 200 nucleotides long and usually do not encode proteins [13,14]. However, multiple researches have shown that lncRNAs play important roles in various biological processes,such as embryonic development, tumorigenesis and osteogenic differentiation [15-17]. Maternally expressed gene 3 (MEG3), a well-known lncRNA,which is located on chromosome 14q32.3,has been reported as a cancer suppressor in various types of cancers [18,19]. For example, in gallbladder cancer,MEG3was shown to regulate LATS2, which is the downstream target gene, by facilitating the ubiquitination of EZH220. Wang et al. demonstrated thatMEG3regulated the EMT of ovarian cancer cells via acting as sponge of miR-219a-5p and modulating EGFR expression[21]. In addition, Xu et al. reported thatMEG3suppressed cell proliferation and cell migration while promoted cell autophagy in glioma cells by regulation of Sirt7 and PI3K/AKT/mTOR pathway[22]. Furthermore, silencing ofMEG3expression through aberrant methylation in its promoter region has been identified in cancers[23,24].

Previously, we unveiled thatMEG3was silenced in NB tissues compared to paratumor tissues.MEG3expression was negatively associated with NB International Neuroblastoma Staging System(INSS) stage and survival rate. Overexpression ofMEG3inhibited NB progression via FOXO1-mediated cell autophagy and mTOR pathway-mediated EMT. Moreover, we found thatMEG3downregulates EZH2 expression by promoting ubiquitin-mediated degradation, while EZH2 also negatively regulatesMEG3expression through an as yet undetermined mechanism [25]. Here, we show thatMEG3inhibits NB by inhibiting cell cycle progression, tumor stemness and promoting apoptosis.

Materials and Methods

Human NB cell lines and tissues

293T, SK-N-BE (2)C and SK-N-AS, were gifts from the lab of Professor Kai Li in Children’s Hospital of Fudan University. Among these, 293T cells and SK-N-AS cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Biological Industries, Israel). And SK-N-BE (2)C cells were cultured in DMEM/F12 (1:1) medium(Biological Industries, Israel). It’s well known that SK-N-BE (2)C is an MYCN amplification cell and SK-N-AS is an MYCN nonamplification cell line. 10% fetal bovine serum (FBS) (Biological Industries) and 1% penicillin-streptomycin solution (New Cell &Molecular Biotech, Suzhou, China) was added to naked cell culture media. All cells were cultured in a cell incubator with 5% CO2 at 37°C.

Quantitative real time-polymerase chain reaction (qPCR)

Tissues were obtained from patients at Children’s Hospital of Fudan University with informed consent. TRIzol reagent (Vazyme,Nanjing, China) was used to extract total RNA. Relative cDNA was synthesized by cDNA Synthesis kits (Yeasen, Shanghai, China).First, 42°C for 2 min was used to digest genomic DNA, followed by 25°C (5 min), 55°C (15 min) and 85°C (5 min) for reverse transcription. Hieff Universal Blue SYBR Green Mix (Yeasen,Shanghai, China) was used for qPCR assay with Roche machine.The PCR protocol was as follows: initial denaturation at 95°C(5 min), followed by 35 cycles at 95°C(30 s), 58°C (30 s), and 72°C(30 s). GAPDH was used as internal control. Primers used were as follows:MEG3-forward, 5’-CCTCACCTCCAATTTCCTCTTC-3’,MEG3-reverse, 5’-TCCAGCAGCTAACCTCATTAAC-3’;Nestin-forward, 5’-CTGCTACCCTTGAGACACCTG-3', Nestinreverse, 5’-GGGCTCTGATCTCTGCATCTAC-3'; and GAPDHforward 5'-GGAGCGAGATCCCTCCAAAAT-3', GAPDH-reverse,5'-GGCTGTTGTCATACTTCTCATGG-3'. Expression ofMEG3and Nestin was analyzed by GraphPad Prism 6 software.

Stably transfected cell lines

MEG3overexpression plasmids were purchased from Genomeditech(Shanghai, China), which was constructed in the pcDH vector. Lentivirus was packaged into 293T cells using an L-PEI transfection reagent (Sigma, USA). Briefly, 72 μL PEI-MAX(Polysciences,USA) transfection reagent was added to 6 μgMEG3plasmid and the four auxiliary plasmids (Rec/TAT/GAG/Vsvg = 1:1:1:1), followed by the addition of 1.5 mL serum-free DMEM. After 15 min, the plasmids were added to the cells in serum-free DMEM. After 6 h, 10% FBS was added to the cells. The virus was collected 48 h later. Stably transfected cells were acquired after virus infection and puromycin screening.

Cell proliferation and cell cycle assays

Proliferation was detected by Cell Counting Kit-8 (CCK-8) (New Cell & Molecular Biotech). Briefly, 1×103 cells were cultured in 96-well plates in 100 μL medium, then incubated with 10 μL reagent for 2.5 h and analyzed via a microplate reader at 450 nm absorbance.For the cell cycle experiments, cells at 80% confluence were digested and fixed with 75% ethanol overnight, then resuspended in 0.3 mL PI/RNAase staining solution. The tube was wrapped with a layer of tin foil and incubated at room temperature for 30 min.At last it was measured by flow cytometry and analyzed by Modfit software.

Cellular apoptosis assay

The cellular apoptosis assay was accomplished as previously described 6 cm dish cells were washed with PBS twice and digested with trypsin without EDTA [26]. The cell pellet was collected and resuspended with 200 μL binding buffer. Then, 5 μL Annexin V/APC and 5 μL 7-AAD was added to the tubes in dark and incubated at room temperature for 30 min, at last samples were measured by flow cytometry.

Western blot analysis

Western blot assay was carried out as previously described by Jin et al27. Cellular proteins were extracted using NP40 lysis buffer(Beyotime, China) on ice for 20 min. After measuring the protein concentration using the Bradford method (Beyotime, China), it was boiled for 5 min in 1× SDS protein loading buffer (Yeasen,Shanghai, China). Following standard electrophoresis and transfer(Rapid transfer buffer was purchased from New Cell & Molecular Biotech), the membrane (Millipore, USA) was blocked with skimmed milk (8%) for 45 min. Next, primary antibodies were added to the membrane in glass plate and placed in overnight at 4°C.Primary antibodies against Nestin (33475S, 1:1000) and CD133(64326S, 1:1000) were purchased from Cell Signaling Technology(USA), an β-actin (20536-1-AP, 1:5000) antibody was bought from Proteintech (Wuhan, China). After washing three to four times with TBST buffer, secondary antibodies (Goat Anti-Mouse IgG(CW0102S, 1:5000, CWBIO, China) and Goat Anti-Rabbit IgG(CW0103S, 1:5000, CWBIO, China) was added to the membrane respectively for 1 h at room temperature. Signals of bands were detected by Enhanced Chemiluminescent Reagent kit (New Cell &Molecular Biotech, China）.

Tumor sphere formation

The tumor sphere formation assay was executed following the article previously described by Yin et al. [28]. Ten thousand cells were seeded in 96U 3D cell culture plates (Sbio, Japan) and flat ultralow attachment plates (Corning, USA). 20 ng/mL EGF, 40 ng/mL bFGF (PeproTech, USA) and B27 (1:50, Gibco, USA) was added to the media without FBS Spheres were observed by microscopy(Invitrogen, USA) after 48 h culture in incubator.

Statistical analysis

All experiments were repeated at least three times. Results were presented as the mean ± SD. T-test was used to compare difference between groups, and p ＜ 0.05 was considered statistically significant.

Results

Overexpression of MEG3 inhibited cell proliferation

To explore the role ofMEG3in NB tumorigenesis, we first constructedMEG3-overexpressing stably transfected cell lines using two NB cells. qPCR analysis confirmed thatMEG3-overexpressing stably transfected cell lines were successfully constructed (Figure 1A, 1C). CCK-8 assay demonstrated thatMEG3significantly restrained cell proliferation (Figure 1B, D).

Over-expression of MEG3 promoted apoptosis

To explore the role ofMEG3in apoptosis, flow cytometry was performed and the results analysis revealed upregulation ofMEG3induced cellular apoptosis in SK-N-BE(2)C and SK-N-AS cells(Figure 2A-C).

Up-regulation of MEG3 caused G0 and G1 cell cycle arrest

Next, we also detected cell cycle via folw cytometry, through Modfit software analysis, we foundMEG3-overexpressing increased levels of G0 and G1 cell cycle arrest in two NB cells(Figure 3A-D).

MEG3 overexpression leads to decreased Nestin and CD133 expression and reduced tumor sphere formation

Among the genes enriched by ChIRP analysis in previous study,MEG3was found binding directly to the promoter region of Nestin(Figure 4A). In addition, western blot analysis demonstrated that overexpression ofMEG3resulted in decreased expression of markers of tumor stemness, including Nestin and CD133 (Figure 4B). qPCR analysis indicated that overexpression ofMEG3decreased Nestin expression at the transcriptional level (Figure 4C). Moreover, overexpression ofMEG3led to decreased sphere formation in vitro regardless of whether the culture plate promoted a large ball (U-shaped plate) or multiple balls (flat bottom) (Figure 4D, 4E).

Discussion

Figure 1. MEG3 suppresses cell proliferation. A) Efficiency of MEG3 upregulation in SK-N-BE (2)C; B) MEG3 restrains cell proliferation in SK-N-BE (2)C; C) Efficiency of MEG3 upregulation in SK-N-AS; D) MEG3 inhibits cell proliferation in SK-N-AS cells. **, p ＜ 0.01, ***, p ＜ 0.005.

Figure 2. MEG3 promotes cell apoptosis. A, B) Flow cytometry was performed to detect cell apoptosis; C) Proportion of apoptotic cells(Quadrant 2+ Quadrant 3) was counted.

Figure 3. Up-regulation of MEG3 leads to G0/G1 cell cycle arrest. A, C) The cell cycle distribution in two NB cells was analyzed by flow cytometry; B, D) Proportion of cells in different phases of the cell cycle in MEG3 upregulation and control groups. ** p ＜ 0.01, *** p ＜ 0.005.

As a well-known tumor suppressor lncRNA, down regulation ofMEG3has been implicated in multiple processes associated with tumor development, including proliferation, migration, invasion,apoptosis, and autophagy, in many tumor types. For example, low expression ofMEG3was observed in breast cancer, andMEG3was shown to decrease proliferation and increase apoptosis in breast cancer cells through miR-141-3p/RBMS3 axis [29]. Lin et al. reported thatMEG3induced autophagy through up-regulation of PTEN via miR-21 in nasopharyngeal carcinoma [30]. Li et al.demonstrated thatMEG3inhibited the epithelial-to-mesenchymal transition by suppressing PSAT1-dependent GSK-3β pathway in esophageal squamous cell carcinoma [31]. Accumulating evidence found thatMEG3plays multiple regulatory roles in cellular stemness, especially in cancer stem cells [32]. However, to date, the role ofMEG3in tumor stemness in NB, which is an important factor in NB recurrence and drug resistance, has not been reported [19,33].Previously, we found thatMEG3was downregulated in NB, and that upregulation ofMEG3not only significantly restrained autophagy and clonal formation of NB cells, but also reduced their migration and invasion abilities [25]. The present study has revealed a novel role forMEG3in NB tumorigenesis. Here, we demonstrateMEG3not only represses cell proliferation and cell cycle transition, but also significantly promotes NB cellular apoptosis. Thus, we show that the inhibitory effect ofMEG3overexpression in NB is consistent with its previously reported anti-tumor effect in other types of tumors.

ChIRP analysis not only confirmed thatMEG3was associated with the cell cycle, but also through sequencing found thatMEG3was enriched in the promoter region of Nestin, which is a neural differentiation marker and is often considered to be a marker of tumor stemness34. The stem cell characteristics of tumors are closely related to tumor recurrence and chemotherapeutic drug resistance, which leads to high mortality rates in high-risk NB patients35. Indeed, we found thatMEG3could negatively regulate Nestin expression via inhibiting its transcription. Furthermore,we demonstrated that overexpression ofMEG3inhibited tumor sphere formation in vitro. Since Nestin plays an important role in maintaining tumor stemness, the suppression of tumor stemness byMEG3may be regulated by Nestin36. Therefore, increasingMEG3expression to reduce the proportion of NB stem-like cells may help reduce the risk of recurrence in children with high-risk NB and improve their prognosis.

Cell differentiation and cell cycle are closely related biological processes, with cell cycle arrest being conducive to cell differentiation [37,38]. The degree of differentiation of tumor cells predicts the malignancy of the tumor and the prognosis of NB39.Poorly differentiated NB is representative of a higher degree of malignancy and poorer prognosis [40]. Retinoic acid (RA) and its receptors have the ability to control cell differentiation, proliferation and apoptosis in NB. Studies have shown that RA can treat NB and reverse NB cells from an undifferentiated state to a differentiated state [41]. Here, we show thatMEG3overexpression leads to cell cycle arrest of NB cells and inhibits tumor stemness. This may promote differentiation of NB, thereby inhibiting tumor proliferation.In addition, tumor stemness and the stage of differentiation are highly correlated with clinical drug resistance [42,43]. Thus, our findings may help clarify the drug resistance mechanism of NB.

In summary, the current study extends our previous findings and demonstrates thatMEG3plays an important role in NB tumorigenesis. We showed that overexpression ofMEG3can inhibit NB development by promoting cell cycle arrest, inhibiting tumor stemness and increasing apoptosis. Due to low level ofMEG3in NB, the effects ofMEG3depletion on NB progression is difficult to detecte. Although more studies are required to further elucidate the specific mechanisms ofMEG3, the present findings indicate thatMEG3could be a potential biomarker or therapeutic target for NB.

Figure 4. MEG3 inhibits tumor stemness via transcriptional regulation of Nestin. A) MEG3 binds to the promoter region of Nestin;B) Overexpression of MEG3 decreases Nestin and CD133 expression levels; C) Overexpression of MEG3 decreases Nestin expression at the RNA level; D) Overexpression of MEG3 decreases sphere size in cells cultured in a U-shaped bottomed, ultra-low adhesion culture plate; E)Overexpression of MEG3 decreases sphere size in cells cultured in a flat bottomed, ultra-low adhesion culture plate. *, p ＜ 0.05.