Knockdown of NEAT1 restrained the malignant progression of glioma stem cells by activating microRNA let-7e

Nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA, promotes oncogenesis in various tumors, including human gliomas. Herein, we studied the expression and function of NEAT1 in glioma stem cells (GSCs). Quantitative real-time PCR demonstrated that NEAT1 was upregulated in GSCs. NEAT1 knockdown inhibited GSC cell proliferation, migration and invasion and promoted GSC apoptosis. A potential binding region between NEAT1 and microRNA let-7e was confirmed by dual-luciferase assays. Upregulation of NEAT1 reduced the expression of let-7e, and there was reciprocal repression between NEAT1 and let-7e in an Argonaute 2-dependent manner. Let-7e expression was lower expression in glioblastoma tissues and GSCs than in normal brain tissues and cells. Restoration of let-7e suppressed tumor function by inhibiting proliferation, migration and invasion while promoting apoptosis in GSCs. NEAT1 knockdown and let-7e overexpression both reduced NRAS protein expression. NRAS was identified as a direct target of let-7e and promoted oncogenesis in GSCs. As NEAT1 promoted oncogenesis by downregulating let-7e expression, both of these genes could be considered for application in glioma therapy.


INTRODUCTION
Glioma is one of the most prevalent and aggressive primary brain tumors in adults.Despite treatment with advanced therapeutic strategies, patients with this disease only have a median survival of 15 months [1].Glioma stem cells (GSCs) are a subpopulation of glioma cells, and are characterized by self-renewal, promotion of angiogenesis, and multi-differentiation [2].GSCs are important contributors to the malignant progression of glioma, from its development to therapeutic resistance and recurrence [3].Therefore, it is urgent to discover the molecular mechanisms by which GSCs are maintained, as this would provide a new focus for the development of glioma treatments.
Long non-coding RNAs (lncRNAs, ~200nt) are a class of RNAs that do not encode proteins [4].LncRNAs are widely expressed in various human somatic tissues, and are involved in diverse cellular events, including epigenetic regulation, gene transcription, mRNA processing and gene translation [5].LncRNAs are ubiquitously dysregulated in tumor cells and have crucial regulatory roles in the malignant progression of tumor cells, such as promotes or suppresses proliferation, migration and invasion, and apoptosis [6].Nuclear paraspeckle assembly transcript 1 (NEAT1) is a 4-kb lncRNA, and has been reported to localize to the nucleus, where it serves as a core component of the paraspeckle sub-organelles [7][8][9].NEAT1 is upregulated and has important functions in a variety of cancers including glioma, such as favors cell proliferation, migration and invasion and impaired apoptosis [10][11][12][13].However, whether NEAT1 is associated with the malignant progression of GSCs remains unclear.
MicroRNAs (miRNAs, ~22nt) are a group of small non-coding RNAs with aberrant expression in various Research Paper tumors.MiRNAs are involved in diverse biological processes, such as cell growth, migration, apoptosis and differentiation, by binding to the 3′-UTRs of mRNAs [14,15].The let-7 family of miRNAs comprises 13 members, one of which is let-7e [16].The let-7 family is characterized by regulating diverse biological processes in cancer cells, including inhibited proliferation and promoted apoptosis [17].The downregulation of let-7e is known to be a prognostic marker of squamous-cell lung carcinoma [18].Further, let-7e is repressed in medulloblastoma [19].Let-7e expression is reduced in deep vein thrombosis and prevents cell endothelial progenitor function by binding to FASLG [20].Remarkably, let-7e can bind to MMP9 and induce adipose-derived stem cell differentiation [21].However, the expression and function of let-7e in GSCs remain unclear.
The RAS gene is recognized to encode three isoforms -HRAS, KRAS and NRAS [22] -and is involved in diverse cellular events and signaling pathways.NRAS is approximately 4.3 kb in length and is aberrantly expressed in many tumors, including colorectal cancer and cutaneous melanoma [23][24][25].However, little has been studied about the function of NRAS in GSCs.
In the current study, we sought to determine the expression and function of NEAT1, let-7e and NRAS in glioma tissues and GSCs.We also investigated the interactions among them in the regulation of GSC malignant behavior and the potential molecular pathways involved.

Isolation and identification of GSCs
Cells isolated from U87 and U251 cell lines were cultured in serum-free medium and allowed to form cell spheres (Figure 1A (a, c)).In an effort to verify the selfrenewing abilities of the cells, we harvested the spheres and performed a second round of the sphere-forming assay.As expected, spheres were generated again from single cells (Figure 1A (b, d)).The positive staining of Nestin and CD133 confirmed that most cells within the spheres expressed these neural stem cell lineage markers on their membranes (Figure 1B).Moreover, the cell spheres stained positively for glial fibrillary acidic protein (GFAP) and beta-tubulin-III (lineage markers), suggesting that they were undergoing typical morphological differentiation towards astrocytic and neuronal lineages (Figure 1C).Further, GSCs-U87 and GSCs-U251 induced larger xenografted tumors in mice than non-GSCs, indicating their stronger tumorigenicity (Figure 1D).

NEAT1 was upregulated while let-7e was downregulated in GSCs
As previously reported, NEAT1 was upregulated in glioblastoma (GBM) tissues [10].Also, we found NEAT1 was upregulated in GBM U87 and U251 cell lines (Figure 2A and 2B).Quantitative real-time PCR (qRT-PCR) was conducted to determine the expression of NEAT1 in two additional glioma cell lines and GSCs.As shown in Figure 2A and 2B, NEAT1 expression was significantly upregulated in T98, A172, GSC-U87 and GSC-U251 cells.On the contrary, let-7e expression was significantly lower in GBM tissues and GBM cell lines than in normal brain tissues and normal human astrocytes, and correlated negatively with the glioma pathological grade (Figure 2C-2E).These results suggested that NEAT1 promotes oncogenesis in GSCs, while let-7e functions as a tumor suppressor.

Knockdown of NEAT1 impaired the malignant progression of GSCs
To determine the effect of NEAT1 on GSCs, we divided cells into three groups: the control group, sh-NC group (transfected with the sh-NC plasmid), sh-NEAT1 group (transfected with the sh-NEAT1 plasmid) and the sh-NC group (transfected with the sh-NC plasmid).The Cell Counting Kit-8 (CCK-8) assay indicated that GSC proliferation was lower in the NEAT1-knockdown group than in the sh-NC group (Figure 3A).The migration and invasion of GSCs were also significantly lower in the sh-NEAT1 group than in the sh-NC group (Figure 3B).Moreover, GSCs treated with sh-NEAT1 exhibited weaker migration abilities than control cells in 3D Spheroid-based tumor migration assays (Figure 3C).Flow cytometry analysis revealed that the inhibition of NEAT1 significantly increased GSC apoptosis (Figure 3D).These results indicated that NEAT1 might act as an oncogene in GSCs.

Let-7e functioned as a tumor suppressor
Similarly, to determine the effect of let-7e on GSCs, we divided cells into five groups: the control group, pre-NC group (transfected with let-7e agomir NC), pre-let-7e group (transfected with the let-7e agomir), anti-NC group (transfected with let-7e antagomir NC) and anti-let-7e (transfected with the let-7e antagomir).A CCK-8 assay revealed that GSC proliferation was lower in the let-7e overexpression group (pre-let-7e) than in the pre-NC group (Figure 4A).Transwell assays were conducted to assess the effects of let-7e overexpression on the invasiveness and migratory abilities of GSCs.The migration and invasion of GSCs were lower in the pre-let-7e group than in the pre-NC group (Figure 4B).Similarly, GSC spheroid migration was attenuated (Figure 4C) and apoptosis was enhanced in the let-7e overexpression group relative to the anti-let-7e group.Thus, it is conceivable that let-7e, in contrast to NEAT1, functions as a tumor suppressor in GSCs (Figure 4D).

NEAT1 is a direct target of let-7e
There is increasing evidence that lncRNAs could be competing endogenous RNAs (ceRNAs) or molecular www.impactjournals.com/oncotargetsponges in down-regulating the expression and biological functions of miRNAs, Using a bioinformatics database (Starbase), we determined that NEAT1 harbors two putative binding sites for let-7e (Figure 5B).To validate our hypothesis that let-7e could directly bind to NEAT1, we first measured the expression of let-7e in sh-NEAT1 GSCs by qRT-PCR.Let-7e expression was elevated in the sh-NEAT1 group but not in the sh-NEAT1-Mut group.In contrast, NEAT1 expression was reduced in the pre-let-7e group (Figure 5A).Further, dual-luciferase gene reporter assays were used to assess the binding sites of NEAT1 and let-7e.The luciferase activity was significantly lower in the NEAT1-Wt1+prelet-7e group than in the control group (Figure 5C), while the luciferase activity did not differ between the NEAT1-Mut1+pre-let-7e group and the control group, suggesting that binding site 1 between NEAT1 and let-7e was functional.
An RNA-binding protein immunoprecipitation (RIP) assay was conducted to determine whether NEAT1 and let-7e were in the expected RNA-induced silencing complex (RISC).We explored RNA levels by qRT-PCR, and found NEAT1 and let-7e immunoprecipitated with Argonaute 2 were repressed than those in the control group, respectively (Figure 5D).Thus, NEAT1 inhibition was confirmed to restore let-7e expression in a RISCdependent manner, and there was a reciprocal repression feedback loop between NEAT1 and let-7e.

NEAT1 inhibition hindered the malignant progression of GSCs by upregulating let-7e
Further, to explore the mechanism whereby NEAT1 promoted malignant behavior in GSCs by attenuating let-7e expression, we divided cells into five groups: the control group, the sh-NC+pre-NC group (cells stably expressing sh-NC, co-transfected with pre-NC), sh-NEAT1+pre-let-7e group (cells stably expressing sh-NEAT1, co-transfected with pre-let-7e), sh-NC+anti-NC group (cells stably expressing sh-NC, co-transfected with anti-NC) and sh-NEAT1+anti-let-7e group (cells stably expressing sh-NEAT1, co-transfected with anti-let-7e).GSC proliferation was markedly lower in the sh-NEAT1+pre-let-7e group than in the sh-NC+pre-NC group (Figure 5E).Moreover, the numbers of migrating and invading GSCs were significantly lower in the sh-NEAT1+pre-let-7e group than in the control group (Figure 5F).In addition, GSCs treated with sh-NEAT1+pre-let-7e exhibited weaker migration abilities than controls in 3D Spheroid-based tumor migration assays (Figure 5G).The apoptotic ratio of GSCs with knockdown of NEAT1 and overexpression of let-7e was robustly elevated compared with that in the sh-NC+pre-NC group (Figure 5H).These results indicated that knockdown of NEAT1 hindered malignant behavior in GSCs by upregulating let-7e.

NRAS was upregulated in glioma tissues and GSCs, and facilitated GSC proliferation, migration and invasion and reduced GSC apoptosis
NRAS was previously identified as an oncogene in several cancers.NRAS protein levels in normal brain tissues, glioma tissues and GSCs were investigated by immunohistochemistry and Western blot.As shown in Figure 6A, NRAS localized to the cytoplasm and was upregulated in glioma tissues compared with normal brain tissues.Similarly, the Western blot revealed greater protein levels of NRAS in glioma tissues and GSCs than in normal brain tissues and non-GSCs (Figure 6B and 6C).Thus, we inferred that NRAS promotes the malignant progression of   GSCs.NRAS has been reported to act as an oncogene in various tumor cells.To determine the effect of NRAS on GSCs, we divided cells into five groups: the control group, NRAS (+)-NC group (transfected with the empty plasmid), NRAS (+) group (transfected with the NRAS full-length plasmid), NRAS (-)-NC group (transfected with the empty plasmid) and NRAS (-) group, (transfected with the shorthairpin NRAS plasmid).CCK-8 assays revealed that GSC proliferation was enhanced in the NRAS overexpression group compared to the NRAS (+)-NC group (Figure 6D).Transwell assays demonstrated that GSC migration and invasion were greater in the NRAS overexpression group than in the NRAS (+)-NC group (Figure 6E).Further, overexpression of NRAS induced the migration of GSCs in a 3D Spheroid-based tumor migration assay (Figure 6F).NRAS overexpression also significantly reduced GSC apoptosis (Figure 6G).

NRAS was involved in the NEAT1/let-7edependent malignant progression of GSCs
Bioinformatics databases (Targetscan, Starbase and miRanda) predicted that several genes would be downstream targets of let-7e, including NRAS.We first determined the effect of let-7e on the protein levels of NRAS by Western blot, and found that NRAS expression was significantly downregulated, among several downstream molecules of let-7e predicted by the bioinformatics databases.Next, the mRNA and protein levels of NRAS were detected in GSCs treated with sh-NEAT1, pre-let-7e or anti-let-7e by qRT-PCR and Western blot.NRAS mRNA and protein levels were lower in the sh-NEAT1 group than in the sh-NC group (Figure 7A  and 7E).On the contrary, GSCs treated with anti-let-7e exhibited higher mRNA and protein levels of NRAS than those treated with anti-NC (Figure 7B and 7F).NRAS expression was significantly lower in the sh-NEAT1+prelet-7e group than in the sh-NC+pre-NC group (Figure 7G).
Having confirmed that both NEAT1 and let-7e influence the malignant behavior of GSCs, we performed a dual-luciferase reporter assay to verify the putative binding site (Figure 6C) between let-7e and NRAS.Luciferase activity was lower in the NRAS-Wt+let-7e group than in the NRAS-Wt+let-7e-NC group, but did not differ significantly between the NRAS-Mut+let-7e and NRAS-Mut+let-7e-NC groups (Figure 7D).
To further confirm the binding sites between let-7e and NRAS, we mutated the expected binding sequence in let-7e.As shown in Figure 7H, NRAS protein levels did not change in the pre-let-7e-Mut group than in the pre-NC group.

DISCUSSION
In this study, we demonstrated that NEAT1 was upregulated in GSCs.NEAT1 inhibition impaired the malignant behavior of GSCs and attenuated NRAS expression.On the contrary, let-7e expression was downregulated in glioma tissues and GSCs.Restoration of let-7e suppressed proliferation, migration and invasion, promoted apoptosis, and reduced NRAS expression in GSCs.Moreover, let-7e was found to bind to NEAT1 in a sequence-specific manner, and there was reciprocal repression between let-7e and NEAT1, possibly induced by the RISC.NRAS was identified as a direct target of let-7e and was involved in the NEAT1-induced malignant progression of GSCs.Further, NRAS was confirmed to promote oncogenesis in GSCs by stimulating cell proliferation, migration and invasion while inhibiting apoptosis.
There is much evidence that lncRNAs are aberrantly expressed in various tumors.Due to their involvement in tumorigenesis and cancer progression, lncRNAs could be used as diagnostic or prognostic biomarkers and potential therapeutic targets.NEAT1 is an lncRNA confirmed to be upregulated in gliomas, and promotes cell proliferation, migration and invasion while suppressing apoptosis in glioma U87 and U251 cells [10].
GSCs are a subgroup of glioma cells characterized by self-renewal, promotion of angiogenesis and multidifferentiation. Conventional therapies against glioma may be limited mainly due to the existence of GSCs, which facilitate the recurrence, drug-resistance, rapid growth, invasion and metastasis of glioma [2].Therefore, therapy against GSCs has become an urgent and promising field.In the current study, we found that NEAT1 was upregulated in GSCs, suggesting that it might promote GSC progression.Indeed, inhibition of NEAT1 reduced the malignant progression of GSCs.Consistent with our results, NEAT1 was reported to be upregulated in non-small cell lung cancers and to promote lung cancer cell growth [26].Likewise, the overexpression of NEAT1 contributes to the malignant behavior of laryngeal squamous cell cancer by downregulating the miR-107/ CDK6 pathway [27].We further examined the expression of NRAS, which promotes oncogenesis in various cancers, and found that NEAT1 knockdown significantly reduced NRAS expression in GSCs.However, whether NRAS is also involved in the NEAT1-induced enhancement of GSC progression needs to be further investigated.
We also found that let-7e, a conserved miRNA, was downregulated in glioma tissues and GSCs, which suggested that let-7e might be associated with GSC progression.As reported earlier, low expression of let-7e may be a biomarker of non-small cell lung cancer [28].Also, let-7e expression is significantly reduced in retinoblastoma [29], and low let-7e expression contributes to cisplatin resistance in epithelial ovarian cancer [30].To explore the function of diminished let-7e expression in GSCs, we studied the effects of restoring or inhibiting of let-7e expression in GSCs.Our results indicated that overexpression of let-7e suppressed the proliferation, migration and invasion and promoted the apoptosis of GSCs, whereas inhibition of let-7e facilitated the malignant progression of GSCs.In addition, NRAS expression was reduced in GSCs that overexpressed let-7e.
LncRNAs have been confirmed to function as endogenous miRNA sponges by binding to miRNAs and restraining their function [31].We used bioinformatics software (Starbase) to determine whether NEAT1 harbors a let-7e binding site, and identified two putative binding sites (Figure 5B).Additional experiments revealed that let-7e expression was significantly rescued when NEAT1 was inhibited.Let-7e overexpression reduced the expression of NEAT1, while let-7e inhibition increased NEAT1 expression.This suggested that NEAT1 and let-7e participate in a reciprocal repression feedback loop.Dual-luciferase reporter assays verified that let-7e binds to NEAT1 in a sequencespecific manner.Further, an RIP experiment confirmed the involvement of the RISC complex in the reciprocal repression between NEAT1 and let-7e.This finding was consistent with a previous report that XIST functions as tumor suppressor by upregulating miR-152 in a RISC-dependent manner, and harbors a miR-152 binding site that allows XIST function as a ceRNA of miR-152 in GBM stem cells [32].HOTAIR is confirmed to promote the malignancy of renal carcinoma cells, and to stimulate oncogenesis by suppressing miR-141 in the RISC complex [33].
To validate our hypothesis that NEAT1 promoted the malignant progression of GSCs by suppressing let-7e, we established GSCs in which NEAT1 was stably inhibited and let-7e was overexpressed or inhibited.The inhibition of let-7e in these cells largely reversed the suppression of GSC progression resulting from the knockdown of NEAT1.Furthermore, in NEAT1-knockdown GSCs, the inhibition of let-7e restored NRAS expression.These results indicated that NEAT1 promotes oncogenesis by downregulating let-7e in GSCs.
MiRNAs regulate cellular events by binding to the 3′-UTRs of downstream genes.For instance, miR-186 inhibits GSC growth by binding to the 3′-UTRs of XIAP and PAK7 [34].As our bioinformatics analysis and dualluciferase assay demonstrated, NRAS is a direct target of let-7e and promotes malignant behavior in GSCs.For over four decades, scientists have searched for a safe and effective method of hindering the aberrant activity of RAS in human cancers [22].NRAS belongs to the RAS gene family, and its encoded protein is essential for controlling the activity of several cellular signaling pathways, including PI3K/ AKT/mTOR and MEK/ERK [35][36][37].It is well known that the PI3K/AKT/mTOR and MEK/ERK pathways can directly and effectively promote cancer cell proliferation, migration and invasion, while inhibiting the apoptosis of tumor cells [38][39][40][41].NRAS has been confirmed to activate these two important pathways, suggesting that therapies targeting NRAS may be promising anticancer treatment methods.Moreover, high-frequency NRAS mutations occur in various tumors, such as melanoma, juvenile myelomonocytic leukemia and colorectal cancer [42,43].NRAS mutations contribute to the response to cetuximab treatment in colorectal cancer cells by reducing patients survival [43].Localized immunotherapies exhibit enhanced activity in high-grade melanoma and may be especially effective in those with NRAS mutations [44].Further, NRAS protein levels are dysregulated in several tumor cells.NRAS is overexpressed in lung cancer cells and can be directly downregulated by miR-515-5p [45].MiR-421 can inhibit prostate cancer progression by attenuating NRAS protein expression [46].Dual-luciferase reporter assays confirmed our hypothesis that let-7e could bind to NRAS in a sequence-specific manner, and NRAS expression was inversely affected by the overexpression or downregulation of let-7e in GSCs.Importantly, the NRAS 3′-UTR reversed the let-7e-induced impairment of malignant behavior in GSCs.Whether NRAS protein is a viable clinical therapy target for patients with glioma remains to be investigated.
In conclusion, we have shown that the knockdown of NEAT1 impaired malignant behavior in GSCs by upregulating let-7e expression.Moreover, let-7e suppressed GSC malignant behavior by binding to NRAS.The significance of the correlation among NEAT1,let-7e and NRAS expression was highlighted for the first time.Thus, therapies targeting the NEAT1/let-7e/NRAS axis may be promising options for the treatment of human glioma.

Clinical tissues
All glioma tissues and normal brain tissues (NBTs) were collected from surgical resections of human glioma in the Department of Neurosurgery, Shengjing Hospital of China Medical of University, from January 2013 to September 2015.Informed consent was gathered from all patients, and the research method was approved by the Ethics Committee of Shengjing Hospital of China Medical University.Grades of glioma were assigned by neuropathologists according to World Health Organization (WHO) classification.These tissues were divided into four groups: grade I (n=30), grade II (n=30), grade III (n=30) and grade IV (n=30).NBTs were used as the negative control group (n=30), and were obtained from patients who had been in traffic accidents that required immediate partial resections of brain tissue to reduce intracranial pressure, but unfortunately died after surgery.The tissues we obtained were far from the trauma tissue.All informed consent forms were obtained from relatives of the trauma victims.The specimens were immediately frozen and preserved in liquid nitrogen until use in this research.

Tumor xenografts
Four-week-old male nude mice were purchased from the National Laboratory Animal Center (Beijing, China).All mice were given free access to autoclaved food and water during the experiment.All experiments with nude mice were performed strictly in accordance with a protocol approved by the Administrative Panel on Laboratory Animal Care of the China Medical University.The mice were subcutaneously injected with 5 × 10 4 GSC-U87 (or GSC-U251) and non-GSC-U87 (or non-GSC-U251) cells (n = 6 in each group).The subcutaneous tumor-bearing mice were sacrificed six weeks after injection.The tumor volume was calculated by the formula: volume (mm 3 ) = length × width 2 /2.

RNA extraction and qRT-PCR
Total RNA was extracted from clinical specimens and cells with the Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA).The primers for NEAT1 and GAPDH were synthesized by Takara Bio (Japan).A One-Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Takara Bio) was used for qRT-PCR.The primers for NEAT1: forward 5′-ATGCCACAACGCAGATTGAT-3′, reverse 5′-CGAGAAACGCACAAGAAGG-3′; GAPDH: forward 5′-TGCACCACCAACTGCTTAGC-3′, reverse 5′-GGCATGCACTGTGGTCATGAG-3′.cDNA was generated from miRNA with a TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA).TaqMan Universal Master Mix II was used to perform TaqMan microRNA assays for let-7e and U6 (Applied Biosystems, Foster City, CA, USA) on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems).GAPDH and U6 were used as endogenous controls for gene and miRNA expression, respectively.Gene and miRNA expression were normalized to those of the respective endogenous controls, and the fold-change in gene expression was calculated as 2 −ΔΔCt .

Cell proliferation assay
After transfection efficacy was confirmed, GSCs were dissociated with Accutase (Life Technologies Corporation, Carlsbad, CA, USA), resuspended, and seeded in 96-well plates at 3000 cells per well.The CCK-8 assay (Dojin, Japan) was used to measure GSC proliferation.CCK-8 solution (10 μL) was added to each well and the plate was incubated for 2 h at 37°C.The absorbance was recorded at 450 nm on a SpectraMax M5 microplate reader (Molecular Devices, USA).

Cell migration and invasion assay
A 24-well insert with an 8-mm pore size (Corning, USA) was employed for the GSC migration and invasion assays.GSCs were dissociated with Accutase, resuspended in 100 μL serum-free medium and placed in the upper chamber (without or pre-coated with 500 ng/mL Matrigel solution (BD, Franklin Lakes, NJ, USA) for the migration and invasion assays, respectively), while 600 μL of 10% FBS medium was placed in the lower chamber.After incubation at 37°C for 48 h, the cells on the upper membrane surface were scraped off.The cells on the lower side of the member were fixed and then stained with 10% Giemsa.Cells were counted from five random vision fields under a microscope for statistics.

3D Spheroid-based tumor migration assays
For further analysis of the invasion abilities of GSCs, 3D Spheroid-based tumor migration assays were performed.Briefly, a Matrigel matrix (BD, State of New Jersey) was added to each well of a 96-well black, clearbottom spheroid microplate (Corning, New York).The plate was then transferred to a 37°C/5% CO 2 incubator for 1 h to initiate gel formation.GSCs were diluted to a concentration of 3×10 4 /mL in medium, and 100 μL of this dilution was plated to each of the appropriate wells.After 48 h, cells were monitored from five random vision fields under a microscope for statistics.

Apoptosis detection
Apoptosis was measured with Annexin V-PE/7AAD staining (Southern Biotech, Birmingham, AL, USA) according to the manufacturer's instructions.After being washed twice with 37°C PBS and stained with Annexin V-PE/7AAD, cells were analyzed by flow cytometry (FACScan, BD Biosciences), and apoptotic fractions were recorded with CELL Quest 3.0 software.

RNA immunoprecipitation
To investigate whether NEAT1 was associated with the RISC, we performed RNA immunoprecipitation.GSCs were lysed in complete RNA lysis buffer containing protease inhibitor and RNase inhibitor from an EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol.Whole cell lysates from the control groups and let-7e groups were incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute 2 antibody (Millipore) and the negative control (normal mouse IgG; Millipore).Samples were incubated with Proteinase K buffer, and then the immunoprecipitated RNA was isolated.The RNA concentration was measured with a NanoDrop (Thermo Scientific) and the RNA quality was assessed with a bioanalyser (Agilent, Santa Clara, CA, USA).Purified RNA was obtained, and qRT-PCR was performed with the primers mentioned above to demonstrate the presence of the binding targets.

Immunohistochemistry assays
Slides of specimens (4 μm thick) were dewaxed, rehydrated, and incubated in 0.3% H 2 O 2 for 10 minutes to inhibit endogenous peroxidase activity.Slides were then blocked with 10% normal goat serum (MXB, Fuzhou, China) for 30 minutes and incubated overnight at 4°C with a rabbit polyclonal antibody against NRAS (1:50, Abcam, UK) and XIAP (1:50, Abcam, UK).Slides were washed with PBS three times and then incubated with biotinylated rabbit anti-rabbit IgG for 1 h at room temperature.After incubation with an avidin-biotinperoxidase complex for 10 minutes, samples were stained with 3,3'-diaminobenzidine.Slides were imaged under a light microscope (Olympus, Japan) at 100× and 200× magnification.

Western blot
Total proteins were extracted from cells with RIPA buffer containing protease inhibitors (Beyotime Institute of Biotechnology) on ice, and these proteins were then subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes.After non-specific binding was blocked with 5% nonfat milk at room temperature for 2 h, membranes were incubated with primary antibodies as follows: NRAS (1:2000, Abcam, UK) and GAPDH (1:1000, Santa Cruz Biotechnology).Then, the membranes were incubated with HRP-conjugated secondary antibodies (1:5000 goat anti-rabbit or goat anti-mouse, respectively; Santa Cruz Biotechnology) at room temperature for 2 h.Immunoblots were visualized by enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology) and recorded with ChemImager 5500 V2.03 software.The relative integrated density values were calculated with GAPDH as an internal control.

Statistical analysis
Data are presented as the mean ± standard deviation (SD).All experimental results were statistically analyzed with Student's t-test or one-way analysis of variance (ANOVA).All statistical analyses were performed with SPSS 18.0 statistical software, with P<0.05 considered as statistically significant.

Figure 3 :
Figure 3: Knockdown of NEAT1 restrained cell proliferation, migration and invasion and promoted apoptosis of GSCs. A. ACCK-8 assay was used to determine the effect of NEAT1 on GSC proliferation.B. Quantification of the migration and invasion of NEAT1-knockdown GSCs.Representative images and accompanying statistical plots are presented.C. 3D Spheroid-based tumor migration assays of the effect of NEAT1 expression on GSC migration.Scale bars, 60 μm.D. Flow cytometry analysis of the effect of NEAT1 knockdown on GSCs.Data are presented as the mean ± SD (n=5, each group).* P<0.05 vs. sh-NC group.Scale bars, 20 μm.

Figure 4 :
Figure 4: Restoration of let-7e inhibited cell proliferation, migration and invasion and facilitated apoptosis of GSCs. A. The CCK-8 assay was employed to determine the effect of let-7e on GSC proliferation.B. Quantification of GSC migration and invasion in groups according to let-7e expression.Representative images and accompanying statistical plots are presented.C. 3D Spheroid-based tumor migration assays of the effect let-7e expression on GSC migration.Scale bars, 60 μm.D. Flow cytometry analysis of GSCs in groups according to let-7e expression.Data are presented as the mean ± SD (n=5, each group).* P<0.05 vs. pre-NC group; # P<0.05 vs. anti-NC group.Scale bars, 20 μm.

Figure 6 :
Figure 6: NRAS was upregulated in glioma tissues, glioma cell lines and GSCs.And NRAS promoted cell proliferation, migration and invasion and inhibited apoptosis of GSCs. A. Immunohistochemistry of NRAS protein levels in non-tumorous brain, Grade I-II glioma, and Grade III-IV glioma tissues.Original magnification: 100× (above), 200× (below).Scale bar, 50 μm.B. NRAS protein expression in non-tumorous brain tissues and glioma tissues, with GAPDH as an endogenous control.Representative protein expression and integrated light density values of NRAS in non-tumorous brain tissues, low-grade glioma tissues (WHO I-II), and high-grade glioma tissues (WHO III-IV) are shown.Data are presented as the mean ± SD (n=15, each group).* P<0.05 vs. non-tumorous brain tissue group.C. Western blot of NRAS expression in non-GSCs and GSCs, with GAPDH as an endogenous control.* P<0.05 vs. non-GSC group.D. The CCK-8 assay was employed to determine the effect of NRAS on GSC proliferation.E. Quantification of GSC migration and invasion upon NRAS inhibition.Representative images and accompanying statistical plots are presented.F. 3D Spheroid-based tumor migration assays of the effect of NRAS expression on GSC migration.Scale bars, 60 μm.G. Flow cytometry analysis of the effects of NRAS overexpression or downregulation on GSCs.Data are presented as the mean ± SD (n=5, each group).* P<0.05 vs. NRAS (+)-NC group; # P<0.05 vs. NRAS (-)-NC group.Scale bars, 20 μm.

Figure 7 :
Figure 7: NRAS was a target gene of Let-7e.And the expression of NRAS was regulated by both NEAT1 and let-7e.A. qRT-PCR analysis of the effect of NEAT1 on NRAS mRNA expression in GSCs.* P<0.05 vs. sh-NC group.B. qRT-PCR analysis of the effect of let-7e on NRAS mRNA expression in GSCs.* P<0.05 vs. pre-NC group; # P<0.05 vs. anti-NC group.C. NRAS harbored one putative let-7e binding site, the designed mutant sequence is indicated.D. Dual-luciferase reporter assay of HEK 293T cells co-transfected with NRAS-Wt and let-7e-NC; NRAS-Wt and let-7e; NRAS-Mut and let-7e-NC; or NRAS-Mut and let-7e.* P<0.05 vs. NRAS-Wt+let-7e-NC group.E. Western blot analysis for NRAS in NEAT1-knockdown GSCs, with GAPDH as an endogenous control.* P<0.05 vs. sh-NC group.F. Western blot analysis for NRAS in let-7e-overexpression and let-7e-inhibition GSCs, with GAPDH as an endogenous control.* P<0.05 vs. pre-NC group; # P<0.05 vs. anti-NC group.G.Western blot analysis for NRAS in GSCs co-transfected with sh-NEAT1 and pre-let-7e or anti-let-7e, with GAPDH as an endogenous control.* P<0.05 vs. sh-NC+pre-NC group.H.Western blot analysis for NRAS in let-7e-Mut-overexpression or let-7e-Mut-inhibition GSCs, with GAPDH as an endogenous control.For G-K, data are presented as the mean ± SD (n=5, each group).

Figure 8 :
Figure 8: Let-7e inhibited GSC malignant progression by binding to the NRAS 3′-UTR.A. The CCK-8 assay was used to evaluate the effects of let-7e and NRAS (+) on U87 and U251 cell proliferation.B. Quantification of cell migration and invasion in groups according to let-7e and NRAS (+)-NC expression.Representative images and accompanying statistical plots are presented.C. Migration abilities of GSCs in groups according to let-7e and NRAS (+) expression.Scale bars, 60 μm.D. Flow cytometry analysis of GSCs in groups according to let-7e and NRAS (+) expression.Data are presented as the mean ± SD (n=5, each group).* P<0.05 vs. let-7e+NRAS group.Scale bars, 40 μm.