Long noncoding RNA NEAT1 regulates glioma cells proliferation and apoptosis by competitively binding with miR-324-5p and upregulating KCTD20 expression

Recent studies have pointed out that long non-coding RNAs (lncRNAs) play a key role in tumorigenesis, including glioma. Nuclear paraspeckle assembly transcript 1 (NEAT1), a lncRNA, has been reported to be participated in the development and progression of many types of tumors and promotes cancer cell proliferation, migration, invasion, and drug resistance. The exact role and regulatory mechanism of NEAT1 in gliomas still need to be further explored. and KCTD20.

NEAT1 inhibited the abilities of cell proliferation and induced G0/G1 arrest and apoptosis in vitro, suppressed tumorigenesis in vivo via sponging miR-324-5p and then upregulated KCTD20 expression. In addition, NEAT1 reversed the effects of miR-324-5p on the proliferation and apoptosis of glioma cells, and involved the inhibition of potassium channel tetramerization protein domain containing 20 (KCTD20) expression.

Conclusion
Collectively, our ndings demonstrate that NEAT1 epigenetically up-regulates KCTD20 expression through competitively binding miR-324-5p, and also provides a potential therapeutic target for human glioma.

Background
Glioma, the most prevalent and aggressive tumor in the central nervous system, is accounting for 70% of highly malignant primary brain tumors (1). Despite the constantly improving multiple diagnosis and treatment strategies in past decades including surgical resection followed by radiotherapy and systemic Temozolomide (TMZ) chemotherapy(2), the prognosis for patients with glioma remains dismal, and the overall survival is only 12-14 months after diagnosis (3,4). In order to treat malignant gliomas more e ciently, approaches have been performed to nd molecular targets related with glioma cell growth, apoptosis, differentiation, invasion, and migration. However, developing effective targeted therapies to improve the cure rate for this complex disease remains a challenge. Therefore, it is urgent need to unveil the underlying mechanisms of gliomas, especially focusing on tumorigenesis and in order to develop more effective treatment strategies.
Long noncoding RNAs (lncRNAs), a subgroup of noncdoing RNAs, de ned as ncRNAs with transcripts longer than 200 nucleotides and with little protein-coding potential (5,6). The underlying regulatory mechanisms of lncRNAs function in cancer are very complex and not fully understood. Many studies have shown that lncRNA exerts a crucial role in role in the development process, genomic imprinting, cellular homeostasis, and pluripotency of embryonic stem cells (7)(8)(9). Generally, the abnormally expressed lncRNAs participate in many biological mechanisms at the epigenetic, transcriptional, and posttranscriptional levels (10)(11)(12), such as cell proliferation, metabolism, apoptosis, migration and differentiation (13)(14)(15). Recently, a new regulatory mechanism indicates that the potential effect of lncRNAs is to inhibit the expression and biological functions of miRNAs(microRNA) as competing endogenous RNAs (ceRNA) (16,17). Emerging evidence has con rmed that ceRNA is a very important pathway in regulation of cancer progress, also known as sponge, which associate with miRNAs and regulates the miRNA downstream target genes. For instance, MT1JP regulated gastric cancer by functioning as competing endogenous RNA to regulate miR-92a-3p/FBXW7 expression (18). lncRNA TUSC8 served as a ceRNA of MYLIP through competitively binding with miR-190b-5p and inhibited breast cancer metastasis (19). Hence, there is no doubt that lncRNAs are the core factors in tumorigenesis and development, but the overall pathophysiological mechanism of lncRNAs in glioma remains to be determined.
The nuclear-enriched abundant transcript 1 (NEAT1), located on chromosome 11, has been reported to be a transcriptional regulator for numerous genes (20). Previous studies suggest that abnormal expression of NEAT1 promotes tumorigenesis in a variety of human cancers (21,22). Furthermore, the lncRNA NEAT1 is involved in many tumors by acting as an endogenous competing RNA (ceRNA) for many tumor suppressor miRNAs (23). In non-small cell lung cancer, NEAT1 can promote the progression of NSCLC under hypoxic condition and regulate the Wnt/β-Catenin signaling pathway (24). Moreover, the overexpression of NEAT1 in pancreatic cancer was shown to facilitates cancer progression by negative modulation of miR-506-3p (25). Although there have been some articles on the regulation of NEAT1 in gliomas, few investigations involve intracranial tumor formation experiment. Therefore, we added the orthotopic xenograft studies experiment to this study, and selected NEAT1 to further study its regulation mode of proliferation and apoptosis in glioma.
In the current study, we determined that NEAT1 was upregulated in glioma tissues and cell lines and is closely correlated with the glioma progression and poor overall survival. Further mechanistic investigation has revealed that NEAT1 may function as ceRNA to compete with miR-324-5p via directly binding to KCTD20, which improved the expression levels of KCTD20. The inhibition of NEAT1 and KCTD20 signi cantly inhibited the proliferation of glioma cells, which was consistent with the results of miR-324-5p overexpression. Our present work provides a novel understanding of the function of NEAT1/miR-324-5p/KCTD20 in the diagnosis and treatment of glioma patients.

Human Tissue Samples
All of the 43 paired human glioma tissues as well as the paired adjacent noncancerous tissues were obtained from patients who underwent surgical resection at from the Department of Neurosurgery, Jiangsu Province Hospital, the First A liated Hospital of Nanjing Medical University. Histologic grade was staged was classi ed by pathologists using WHO criteria. No surgical patients had received preoperative radiotherapy or chemotherapy. All tissue samples were collected during surgery, frozen immediately in liquid nitrogen, and stored for total RNA or protein extraction. The clinical-pathological characteristics of the patients was summarized in Table 1. This study was approved by the Institutional Review Board and the ethics committee of Nanjing Medical University and Fourth Military Medical University, and informed consent was obtained from all patients.

Cell culture
Six human GBM cell lines U87, LN229, H4, U251, U118 and A172 and human embryonic kidney (HEK) 293T cells were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). And all cells were sustained in Dulbecco's modi ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin/mL and 100 ng of streptomycin/mL at humidi ed air at 37℃ with 5% CO 2 . NHA cells were purchased from Lonza (Walkersville, MD) and cultured in the provided astrocyte growth media supplemented with rhEGF, insulin, ascorbic acid, GA-1000, L-glutamine and, 5% FBS.

Cell transfection
The NEAT1 (siNEAT1) and KCTD20 (siKCTD20), pcDNA3.1 plasmids, pcDNA-NEAT1 and empty vector, and the expression of miR-324-5p was achieved by transfection of miR-324-5p mimics or miR-324-5p inhibitor were synthesized by Genechem (Shanghai, China). For stable transfection, the lentivirus carrying short hairpin RNA designed against NEAT1 (shNEAT1), shKCTD20 and its negative control was packaged in GBM cells using the lentiviral packaging kit (Genechem Shanghai, China). All the plasmids were transfected into cells grown in DMEM culture media using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Cell proliferation assay
At 48 hours after transfection, cells were seeded at 2000 per well in 96-well plates and cultured. Firstly, cell proliferation rate was detected at the indicated time points (24,48, 72 and 96h) using a Cell Counting Kit-8 (CCK8) assay (Dojindo, Japan) following the manufacturer's instructions. After a 1 h incubation with CCK-8 at 37 °C, absorbance (OD value) at a wave length of 450 nm was detected and used for calculating cell viability.
The colony formation assay was performed following the method described in our earlier study (26).
Brie y, cells were harvested 24h after transfection and then seeded onto 6-well plate (200 cells/well). After cultured for approximately 2 weeks until colony formation was observed, visible colonies were xed with 100% methanol and stained with 0.1% crystal violet for 15 minutes. Colony-forming e ciency was calculated as the number of colonies/plated cells ×100%.
For the 5-ethynyl-2-deoxyuridine (EdU) proliferation assay, the Cell-Light EdU labeling detection kit was purchased from Life Technologies (MA, USA), in accordance with the manufacturer's protocol. U251 and LN229 cells were transfected with plasmid DNA or siRNA for 48 hours. were incubated with 10 μM EdU for 24 h, xed, permeabilized, and stained with both the Alexa-Fluor 594 reaction cocktail for the EdU, and DAPI was used to label cell nuclei. The EdU-positive cells were visualized using a uorescence microscope.

TUNEL assay
The transfected GBM cell lines were xed in 4% paraformaldehyde for 15 minutes and stained with In Situ Cell Death Detection Kit, POD (Roche, Switzerland) according to the manufacturer's instructions.
Firstly, cells were incubated in terminal dexynucleotidyl transferase (TdT) reaction cocktail for 45 minutes at 37°C, then treated with Click-iT reaction cocktail. The nucleus was stained with hematoxylin or methyl green. Finally, the percentage of TUNEL-positive cells were observed with Nikon ECLIPSE E800 uorescence microscope.

Flow cytometric analysis
Cell cycle analysis was performed as described previously (27). For cell cycle assays, U251 and LN229 cells that were transfected with indicated lentivirus and/or plasmids for 48 hours, and then collected by centrifugation for ve minutes at 2000 r/minute. Next, cells were washed twice with PBS and xed with 75% ice-cold ethanol for 24h. The collected cells were re-suspended in PBS containing 25 mg/ mL propidium iodide, 0.1% Triton, and 10 mg/mL RNase (Multi Sciences, Hangzhou, China) and incubated for 30 minutes in the dark before being analyzed by a ow cytometry.

Western blotting assay and antibodies
Western blot analysis was done as described previously (28). Brie y, cells in culture were lysed using the RIPA buffer (KenGEN, China). Next, total protein extraction was evaluated by BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific). Equal amounts of the protein extracts were loaded, subjected to 10% SDS-PAGE, transferred onto PVDF membrane (Millipore, Billerica, MA, USA) for western blotting analysis. After blocking membranes with 5% non-fat for 2h, they were probed by antibodies against CDK4 (Abcam), Cyclin D1 (Abcam), Bcl-2 (Abcam), Bax(Abcam), KCTD20 (Abcam), and GAPDH (Santa Cruz) at 4 °C overnight. They were then incubated with corresponding secondary antibodies, and processed using enhanced chemiluminescence reagents. An enhanced chemiluminescence (ECL) detection system (Thermo Scienti c, USA) was applied to visualize the protein. GAPDH was used as an internal control.

Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis
The lncRNA NEAT1 expression level in glioma tissues and normal samples was examined by uorescence in situ hybridization (FISH). Probes against lncRNA NEAT1 were synthesized by GoodBio (Wuhan, China). FISH was performed according to the BioSense manufacturer's protocol. Frozen human samples were xed with 4% paraformaldehyde for 30 minutes and then washed 3 times with PBS. Treated sections were digested with Proteinase K for 3 minutes at 37 °C and then sequentially dehydrated for 5 min in 70, 85, and 100% ethanol. Next, probes were denatured at 78 °C for 5 minutes and then hybridized with sections overnight in a humidi cation chamber at 42 °C. Tissue sections were washed with preheated 2 × sodium citrate (SSC), 1 × SSC, and 0.5 × SSC in sequence at 37 ° C for 10 minutes.
Then sections washed 3 times with PBS and stained with 4′,6-diamidino-2-phe-nylindole (Sigma) for 15 minutes. They were then examined with a Zeiss LSM 700 confocal microscope (Oberkochen, Germany). IHC was performed on mice xenogeneic tumor tissue and human tissue as described previously (29).
RNA immunoprecipitation RIP (RNA immunoprecipitation) assay was performed with the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) following the manufacturer's protocol. Brie y, U251 and LN229 cells lysates were prepared and incubated with RIP buffer containing magnetic beads conjugated with human anti-Argonaute2 (anti-Ago2) antibody, while normal mouse IgG functioned as a negative control. Immuno-precipitated RNA was tested by qRT-PCR analysis to verify the presence of NEAT1 and miR-324-5p expressions detection.

Luciferase reporter assay
The complementary DNA fragment containing the wild type (WT) or mutant (MUT) sequences of NEAT1 or KCTD20 were subcloned downstream of the luciferase gene within the pGL3 vectors. Brie y, cells were seeded in a 24-well plate and co-transfected with either empty vector or miR-125a, miR-324-5p, miR-495-3p and miR-504, re y luciferase reporter comprising wild-type or mutant NEAT1 and 3'UTR of KCTD20 fragment using Lipofectamine 2000 (Invitrogen) according to the protocol. At 48 h after transfection, the relative luciferase activities were measured using a dual luciferase assay kit (Promega, Madison, WI, USA). The relative luciferase activity was normalized against to the Renilla luciferase activity. All experiments were performed in triplicate.

Statistical analysis
All statistical analyses were performed using Prism 7 software (La Jolla, CA, USA). All experiments were performed in triplicate with means and standard error of the mean or standard deviation subjected to the Student's t-test for pairwise comparison or ANOVA for multivariate analysis. Analysis of patient survival was performed using Kaplan-Meier analysis, and signi cance was determined by the log-rank test. Correlations between NEAT1, miR-324-5p, KCTD20 were analyzed by Spearman's rank correlation. A signi cance level set at P<0.05 was considered signi cant for all the tests.

Results
NEAT1 is identi ed as a highly overexpressed lncRNA and correlated with a poor prognosis in glioma cancer.
To evaluate the expression of glioma cancer-related lncRNAs that may be involved in tumorigenesis, we rst analyzed data from the TCGA database. The results found that NEAT1 expression was signi cantly upregulated in high grade gliomas(HGG) compared to low grade gliomas (LGG) (Fig. 1A). In order to explore the role of NEAT1 in glioma, we rst detected the relative expression levels of NEAT1 in 43 paired glioma tissues/adjacent noncancerous tissue specimens by quantitative reverse transcription PCR (qRT-PCR). As shown in Fig. 1B, an increase in NEAT1 expression was observed in glioma tissues compared to the corresponding adjacent normal tissues. In six glioma cell lines (U87, LN229, H4, U251, U118, A172), the expression of NEAT1 obviously elevated compared with normal cell line NHA (Fig. 1C), especially in U251 and LN229. In addition, using uorescent in situ hybridization (FISH), we found that the glioma tissues exhibited signi cantly higher NEAT1 staining intensity compared with adjacent normal brain tissues (Fig. 1D). Immunohistochemistry (IHC) assays indicated that proliferation marker Ki-67 expression was lower in normal samples than the gliomas (Fig. 1D). We furtherly conducted Kaplan-Meier survival analysis using the data from the TCGA database, and found that patients with high-NEAT1 levels frequently had shorter overall survival time than those with low-NEAT1 (Fig. 1E). As presented in Table 1, a higher NEAT1 expression was observed more frequently in patients with larger tumor size (P = 0.047) and advanced WHO stage (P = 0.024). Taken together, these data suggest that NEAT1 expression is signi cantly upregulated in gliomas and could serve as a prognostic marker for glioma patients.

NEAT1 promotes the proliferation of glioma cells and inhibits apoptosis in vitro.
To explore the potential biological processes of NEAT1 involvement in glioma, we performed gain-and loss-of-function studies on the function cell proliferation and apoptosis through glioma cells. Based on the above analysis of NEAT1 expression in glioma cell lines (Fig. 1C), we selected U251 and LN229 cell lines for subsequent experiments. We silenced NEAT1 expression in U251 and LN229 cells by transfecting siNEAT1. At the same time, we transfected these two cell lines with pcDNA 3.1-NEAT1 expression vector to induce the ectopic overexpression of NEAT1. The qRT-PCR analysis was performed 48 h after transfection to detected its e ciency. The results indicated that knocking down of NEAT1 showed higher interference e ciency in both U251 and LN229 cell lines ( Fig. 2A). And comparing with control groups, NEAT1 expression was remarkably higher in the pcDNA-NEAT1 groups than in the empty vector groups (Fig. 2A). Then the cell proliferation of U251 and LN229 cells were monitored using CCK-8,EdU and colony formation assays. Firstly, we conducted the CCK-8 assay to evaluate the effect of NEAT1 on cell viability. As shown in Fig. 2B, cell proliferation curve showed that downregulation of NEAT1 signi cantly inhibited cell growth compared with the controls. In contrast, overexpression of NEAT1 promoted cell proliferation in both U251 and LN229 cells (Fig. 2C). Consistently, the colony formation assays showed the similar results ( Fig. 2D-E). EdU incorporation experiments was carried out to further testify the proliferative ability of NEAT1. The data showed that silencing NEAT1 expression by siNEAT1 signi cantly suppressed the EdU-positive rate of in both U251 and LN229 cells, while ectopic expression of NEAT1 increased EdU-positive cells (Fig. 2F-G). As well, TUNEL assay showed that cell apoptosis rate was increased in siNEAT1 group compared with that in siCtrl group, while it is decreased in pcDNA-NEAT1 group compared with that in corresponding control groups (Fig. 2H-I). Collectively, these results suggested that NEAT1 may serve as an oncogene for glioma cells during cancer progression.
Knockdown of NEAT1 induces apoptosis and G1 arrest of glioma cells in vitro and glioma tumorigenesis in vivo.
Increased cell cycle arrest and apoptosis are two factors that may help clarify the research mechanism of cancer cell proliferation. Therefore, we examined differences in cell-cycle distributions following NEAT1 overexpression or silencing by ow cytometry analysis. Downregulation of NEAT1 resulted in a notable accumulation of cells in G0/G1 phase and a decrease in S-phase cells (Fig. 3A), whereas cell-cycle progression beyond the G1-S transition was observed in NEAT1-overexpression of U251 and LN229 cells (Fig. 3B). The results indicated that the proportion of apoptotic cells treated with siNEAT1 increased signi cantly, but decreased in the pcDNA-NEAT1 groups. (Fig. 3C-D). In addition, western blotting analysis showed that the expression levels of cell cycle-related proteins (CDK-4 and cyclin D1) and apoptosisrelated proteins (Bcl-2, Bax) are highly consistent with their respective rates of apoptotic and percentage of cell cycle progression (Fig. 3E).
To examine the potential role of lncRNA NEAT1 in vivo, U251 cells transfected with shCtrl or shNEAT1 was intracranially injected into immunocompromised nude mice. At 7, 14, 21, and 28 days after implantation, compared with the blank control group, silencing of NEAT1 signi cantly inhibited the intracranial tumor growth (Fig. 3F) Meanwhile,these ndings were further con rmed by the survival curves (Fig. 3G). Representative H&E staining shown in Fig. 3H, tumor volumes were signi cantly different between two groups. Compared to shCtrl tumors, immunohistochemical staining of Ki-67 showed reduced expression in shNEAT1 group (Fig. 3H). These data indicate that the inhibitory effect of NEAT1 silencing on glioma cell proliferation may be attributed to increased apoptosis and cell cycle arrest, and con rm the carcinogenic activity of NEAT1 on gliomas in vivo.
A potential mechanism for competitive RNA (ceRNA) has been proposed, and recent studies have shown that many lncRNAs function as ceRNA for speci c miRNAs (30,31), which increases the level of posttranscriptional regulation. Emerging evidence have shown that NEAT1 acts as ceRNA in various cancers, including glioma(32), but lncRNAs may regulate multiple targets by functioning as ceRNA to adsorb more than one miRNA. To gure out the potential candidate miRNAs of NEAT1, we used online target prediction tools StarbaseV2.0 and lncRNASNP2, and thirteen miRNAs were chosen (Fig. 4A). Based on the glioma expression levels in the TCGA database, we found that four of the thirteen miRNAs selected had signi cantly lower expression in tumor compared to normal tissues (Fig. 4B, Fig. S1A). The CCGA database also showed that miR-324-5p expression was signi cantly reduced in high grade glioma (Fig. 4C). We further tested four miRNAs (miR-125a, miR-324-5p, miR-495-3p and miR-504) through dual luciferase reporter assays to con rm the interaction between NEAT1 and candidate miRNAs. The results show that miR-125a and miR-324-5p can inhibit luciferase activity, and miR-324-5p has a higher inhibitory e ciency (Fig. 4D). Therefore, we nally selected miR-324-5p for further research. To verify the direct binding relationship between NEAT1 and miR-324-5p (Fig. 4E), a wild type NEAT1 luciferase reporter vector (WT-NEAT1), and a mutant NEAT1 luciferase reporter vector (MUT-NEAT1) on predicted miR-324-5p binding sites of NEAT1 were constructed. As presented in Fig. 4F, we observed that miR-324-5p mimics reduced the luciferase activities of WT-NEAT1 transfection, compared with control group. To further validate the association relationship between NEAT1 and miR-324-5p, RNA immunoprecipitation (RIP) experiments was performed on U251 and LN229 cells transiently overexpressing miR-324-5p. The analysis showed that NEAT1 was highly enriched in cells transfected with miR-324-5p mimics compared to cells transfected with controls (Fig. 4G). The qRT-PCR data revealed that miR-324-5p levels was increased upon NEAT1 knockdown in both U251 and LN229 cells (Fig. 4H). However, ectopic expression of miR-324-5p had no effect on NEAT1 expression (Fig. S1B). Additionally, we evaluated the miR-324-5p expression in human glioma tissues and U251 and LN229 cell lines, as expected, miR-324-5p was signi cantly downregulated in both of them ( Fig. 4I-J). Meanwhile, Spearman's correlation analysis demonstrated that there is a negative correlation between the expression of lncRNA NEAT1 and miR-324-5p in glioma samples (Fig. 4K). Taken together, these data demonstrated that NEAT1 associated with the miR-324-5p by functioning as a ceRNA.
Critical roles of miR-324-5p on glioma cell proliferation in vitro.
To examine whether miR-324-5p inhibits cell proliferation in glioma, we transfected U251 and LN229 cells with miR-324-5p inhibitor or miR-324-5p mimics (Fig. 5A, Fig. S1C). The CCK-8, colony formation, and EdU assays were performed and found that miR-324-5p inhibitor dramatically increased glioma cell proliferation and colony formation ability, while overexpressing of miR-324-5p could inhibit cells viability in both U251 and LN229 (Fig. 5B-D, Fig. S1D-E). Consistent with our expected results, TUNEL analysis showed that downregulated expression of miR-324-5p suppressed glioma cell apoptosis (Fig. 5E), while overexpression of miR-324-5p shows opposite results (Fig. S1F). Furthermore, data from the ow cytometry analysis showed that silencing of miR-324-5p decreased cell apoptosis and cell cycle arrest at G0/G1 phase (Fig. 5F-G); Inversely, more apoptotic cells and percentage of cells in G0/G1 phase were observed after cells were transfected with miR-324-5p mimics (Fig. S1G-H). Parallel western blotting experiments were performed in LN229/U251 cells, downregulation of miR-324-5p led to a signi cant decrease in Bax, and an increase in Bcl-2 and cell cycle-related proteins CDK4 and Cyclin D1 (Fig. 5H). As shown in Fig. S1I, the corresponding protein expression results also appeared in the mimics group.
Together, these results indicate a possible inhibitory role of miR-324-5p in the regulation of glioma progression.
Silencing of miR-324-5p reversed the suppressive effects on glioma cells induced by downregulation of NEAT1.
According to the above results, we noticed that the reduced proliferative capacity of NEAT1 knockdown was similar to those of miR-324-5p overexpression, suggesting that downregulation of NEAT1 might be a mechanism to reduce the tumorigenesis of glioma cells by regulating miR-324-5p expression. To con rm whether miR-324-5p is involved the NEAT1-regulated tumorigenesis, the combinations of transfection was performed before assessing glioma cell proliferation and apoptosis assays. As shown in Fig. 6A-C, these results showed that the inhibitory effects of siNEAT1-mediated could partially be rescued when U251 and LN229 cells were co-transfected with miR-324-5p inhibitor. The results of ow cytometry analysis showed that compared with the co-transfection groups, higher rate of cell cycle arrest and apoptosis were observed in siNEAT1 group while the miR-324-5p inhibitor group showed lower rates ( Fig. 6D-F). Analysis of the above co-transfected cells in Western blot experiments also showed the same results (Fig. 6G). Our results showed that siNEAT1 inhibited the proliferation of glioma cells and induce cell cycle arrest and apoptosis, while miR-324-5p downregulation could partially eliminate these regulatory effects.
To further clarify the ceRNA regulation mechanism by which NEAT1/miR-324-5p regulates glioma cell proliferation, we used online informatics tools TargetScan, miRDB and miRWalk to predict the downstream target genes that involved in NEAT1 competitive binding to miR-324-5p. As illustrated in Fig. 7A, there are 52 target genes with potential binding sites for miR-324-5p. Among them, we found that six genes in the TCGA database had signi cantly higher expression in glioma tissues compared to normal tissues (P < 0.05) (Fig. S2A-B). To select the most effective target gene associated with miR-324-5p, U251 and LN229 cells were transfected with a construct containing a suppressor or negative control for miR-324-5p. Immunoblotting and qRT-PCR results revealed that compared with the expression levels of the other ve genes, the expression levels of KCTD20 was signi cantly increased at the protein level and mRNA level when miR-324-5p was downregulated (Fig. 7B). We further detected the expression of KCTD20 in glioma tissues and showed that it was consistent with TCGA, CGGA and GSE10611 database results (Fig. 2C, Fig. S2B). As presented in Fig. 7D, the miRNA: mRNA alignment analysis showed that the 3 ′ UTR of KCTD20 contains one putative binding site for miR-324-5p. A signi cant repression of luciferase activity upon miR-324-5p transfection was observed in both U251 and LN229 cells when wildtype KCTD20 plasmids was present (Fig. 7E), indicating that miR-324-5p binds to KCTD20 in a sequencespeci c manner. Therefore, we further analyzed the regulatory relationship between KCTD20 and miR-324-5p in glioma cells. The protein and mRNA expression levels of KCTD20 in U251 and LN229 cells was evaluated or decreased after being transfected with miR-324-5p inhibitor or miR-324-5p mimics (Fig. 7F). We have preliminary demonstrated that miR-324-5p downregulation can partially reverse the inhibitory effects of silencing NEAT1, and miR-324-5p can regulate the expression of KCTD20. Therefore, we continue to study the association between NEAT1 and KCTD20. As exhibited in Fig. 7G, KCTD20 levels was dramatically reduced after NEAT1 knockdown in the 2 glioma cell lines. However, silencing of miR-324-5p could markedly abolished the inhibitory effects of siNEAT1 both in KCTD20 mRNA and protein levels (Fig. 7H). As expected, IHC experiment showed lower KCTD20 expression in shNEAT1 mouse xenograft tumors than control groups (Fig. S2C). Furtherly, we analyzed the correlation between NEAT1 and KCTD20 expression levels in glioma cancer tissues. As presented in Fig. 7I, the result displayed that KCTD20 was positively correlated with NEAT1 in glioma tissues, consistent with the potential regulatory axis of NEAT1/miR-324-5p/KCTD20 pathway. These data, taken together, suggested that lncRNA NEAT1 may regulates the expression of KCTD20 through post-transcriptional modulation of miR-324-5p.
KCTD20 is involved in NEAT1-induced tumor proliferative capability in vitro and in vivo.
In order to elucidate the carcinogenic effect of KCTD20 in gliomas, we rst evaluated its expression in gliomas and normal tissues. Analysis of KCTD20 expression using the TCGA, CGGA and GSE10611 database revealed that KCTD20 was signi cantly upregulated in tumor samples compared to normal samples (Fig. S2B). Similarly, IHC staining of glioma samples showed that NEAT1 protein abundance is increased in glioma tissues (Fig. S2C). In order to verify the role of KCTD20 in the proliferation of glioma cells, U251 and LN229 cells were transfected with siKCTD20 or negative control plasmids for western blot and qRT-PCR experiments (Fig. 8A). CCK-8 and colony formation assays showed that silencing of KCTD20 inhibited the proliferative viability of human glioma cells in vitro, and EdU incorporation analysis also showed the similar result (Fig. 8B-D). Next, we performed the ow cytometry analysis. The data suggested that cells transfected with siKCTD20 increased cell cycle arrest and had higher apoptotic rate in comparison with controls ( Fig. S3A-B). In cells that suppressed KCTD20 expression, we found that CyclinD1, CDK4, and Bcl-2 protein levels were reduced, while Bax protein levels were increased (Fig. S3C).
To examine the role of KCTD20 on glioma cell proliferation, U251 cells were transfected with lentiviral constructs containing shRNA against KCTD20 (shKCTD20) or the negative control (shNC) and injected into mouse brains. We found that KCTD20 silencing could notably inhibit glioma cell tumor growth in vivo (Fig. S3D-F). In addition, cell proliferation and apoptosis assays presented that knockdown of the miR-324-5p stimulated the proliferation and inhibited apoptosis of U251 and LN229 cells, and this cancer-promoting effect was partially reversed after the downregulation of KCTD20 by siKCTD20 (Fig. 8E-G, Fig. S2H). Moreover, as shown in Fig. 8H-K, concomitant suppression of miR-324-5p and KCTD20 abrogated the effect of KCTD20 knockdown on the cell apoptosis and reversed the siKCTD20-induced G1 arrest of U251 and LN229 cells compared with cells transfected with siKCTD20 and the control groups.
And the transfection of miR-324-5p inhibitor increased the KCTD20 expression at both mRNA and protein levels, while the simultaneous upregulation of KCTD20 restored to a relatively normal level after cotransfection with siKCTD20 (Fig. 8L, S3G). Furthermore, the reduced CDK4, Cyclin D1 and Bcl-2 expression in siKCTD20 group was rescued by inhibition of miR-324-5p, and Bax expression is consistent with the above results (Fig. 8L). A statistically signi cant inverse correlation was observed between miR-324-5p and KCTD20 expression levels in 43 glioma specimens (r= − 0.4582, P = 0.002, Fig. S3I). Taken together, these data provide substantial evidence that NEAT1 could promote glioma cell proliferation and exert oncogenic functions by modulating miR-324-5p/KCTD20.

Discussion
Over the past decades, tremendous efforts have been made to identify cancer-related lncRNAs and elucidate their biological role in development and progression of numerous human diseases, including cancers (33)(34)(35). Currently, lncRNAs has been accepted as important regulatory factors in cancer progression and different biological processes such as chromatin remodeling, transcriptional and posttranscriptional regulation (36). Although a large number of lncRNAs have been annotated (37), functional interpretation has only just begun. Studies show that 18% of these lncRNAs are related to human tumors, while human protein-coding genes account for only 9% (38). Therefore, more lncRNAs related to gliomas and a more systematic study of the molecular mechanisms of glioma progression need to be explored. Eventually it will become an important part of the early diagnosis and treatment of glioma, and it will help clinicians to evaluate the prognosis of glioma patients.
LncRNA NEAT1 has been reported to be upregulated in numerous tumor samples and identi ed as a potential therapeutic target for many cancers (39). According to Shin et al.'study, NEAT1 is regarded as an oncogenic factor in breast cancer, and downregulation of NEAT1 suppresses the proliferation of breast cancer cell (40). Herein, we found that NEAT1 was overexpressed in glioma specimens and cell lines and mediated miR-324-5p/KCTD20. In addition, silencing NEAT1 could signi cantly inhibit cell viability, further affect cell propagation and induce cell apoptosis capacities in glioma. Furthermore, knockdown of miR-324-5p in glioma cells demonstrated the opposite results. All these data indicated our conclusion that ectopic expression lncRNA NEAT1 may represent a poor prognostic indicator in glioma and may become a potential therapeutic strategy for the treatment of patients with gliomas.
Recently, a growing number of studies have reported that a new regulatory mechanism involving ceRNAs between lncRNAs and miRNAs, where lncRNAs could modulate related RNAs by binding and titrating them off their binding sites on protein-coding messengers (41,42). LncRNAs might function as a ceRNA or a molecular sponge in modulating miRNAs, by which lncRNAs can sponge miRNAs to block the tumor suppressor role of speci c miRNAs and release the suppression effects of oncogenes caused by miRNAs to facilitate tumor initiation. For example, lncRNA PSMA3-AS1 has been found to act as a ceRNA to promote esophageal cancer cell progression and the expression of proto-oncogene EZH2 through negative modulation of miR-101 (43). LncRNA UCA1 functioned as an endogenous sponge ofmiR-182-5p to positively regulate the expression of Delta-like ligand 4(DLL4) in renal cancer cells (44). In addition, some studies have con rmed that NEAT1 can play a regulatory role through competitively binding different miRNAs in a variety of cancers (45,46). These studies have uncovered a new way to elucidate regulatory mechanisms between NEAT1 and KCTD20. Therefore, we performed the bioinformatics analysis and found thirteen miRNAs that could bind to complementary sequences in NEAT1. Next, we con rmed that miR-324-5p can bind to NEAT1 by luciferase reporter assays and RIP analysis. Combined with the online bioinformatics prediction and the predicted expression level from TCGA database, miR-324-5p was nally selected as a candidate gene that NEAT1 can bind. By applying the methods of lossand gain-of-function, we demonstrated that NEAT1 plays a role in cell proliferation, cell cycle processes and cell apoptosis in glioma. These results revealed that NEAT1 exerts physiological functions via directly binding to miR-324-5p. Interestingly, recent research reported that lncRNA can function as ceRNA and interfere with miRNAs, and ultimately exert its regulatory effect on mRNAs at the post-transcriptional level (47,48). In this study, we further veri ed that lncRNAs can regulate mRNAs post-transcription in glioma cells by acting as ceRNA in combination with miRNAs. Our data revealed that NEAT1 reverses the effects of miR-324-5p on glioma cell proliferation, cell cycle and apoptosis through post-transcriptional regulation of KCTD20 expression levels.
KCTD20 (potassium channel tetramerization protein domain containing 20), located in chromosome 6, is a newly identi ed protein which is an isoform of BTBD10 (BTB domain-containing protein 10) and harbors a C-terminal amino-acid sequences similar to BTBD10(49). Some reports have identi ed that KCTD family proteins may be involved in regulating cell proliferation(50). Studies have shown that KCTD20 is an oncogene that can promote the development of small cell lung cancer by activating the Fak/AKT pathway(51). But the role of KCTD20 in tumorigenesis and development is still unclear, especially in glioma. To investigate whether NEAT1-induced miR-324-5p inhibition led to the overexpression of its target mRNA and promote the occurrence and progression of glioma, we further studied the miR-324-5p target gene KCTD20. We explored the association between miR-324-5p and KCTD20 through bioinformatics analysis, and found that the protein expression level of KCTD20 can be suppressed by miR-324-5p. The luciferase reporter vector assays were analyzed, suggesting that miR-324-5p inhibits KCTD20 levels by directly binding to its 3'-UTR. Furthermore, we investigated whether KCTD20 can promote tumor growth via loss-of-function assays. Our data rstly showed that knockdown of KCTD20 inhibited glioma cell proliferation, as well as promoted the apoptosis and induced G0/G1 cell cycle arrest, indicating the tumorigenic activity of KCTD20 in glioma. In addition, Spearman's correlation analysis indicated that miR-324-5p were inversely correlated with NEAT1 and KCTD20 expression, while there has a positive correlation between NEAT1 and KCTD20. These ndings are consistent with our results, which also reveal new mechanisms for regulating KCTD20 expression.
In conclusion, our current experimental results provide mechanistic insights, highlighting that lncRNA NEAT1 could act as a molecular sponge of miR-324-5p and signi cantly contributed to the occurrence and progression of glioma by activating KCTD20 protein expression. Shedding new light on the potential understanding of the molecular network of glioma cancer progression. Taken together, our ndings suggest that lncRNA NEAT1 may be used as a prognostic factor for glioma patients, and it can also be considered as a potential biomarker and target for glioma cancer treatment. The expression levels of Ki-67 in normal and glioma tissues were assessed by IHC. scale bar=100μm E.
Kaplan-Meier analysis was performed on glioma patients with high (n=314) or low (n=314) NEAT1 expression using the TCGA database (P<0.001). Values are shown as the mean ± standard errors of the mean based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 2
Page 22/34 The role of lncRNA NEAT1 in glioma cell proliferation and apoptosis in vitro. A. siCtrl/siNEAT1 or Empty Vecotr/ pcDNA-NEAT1 was transfected into U251 and LN229 cells. The expression of NEAT1 was veri ed using qRT-PCR assays. B-C. Cell proliferation rates was determined in response to NEAT1 knockdown or overexpression using CCK-8 assays. D-E. Colony formation assays were performed to detect the proliferation of siNEAT1-transfected and pcDNA-NEAT1-transfected in both U251 and LN229 cells.
Colonies were counted and captured. scale bar=100μm. F-G. EdU staining assays were used to evaluate the growth in U251 and LN229 cells transfected with siNEAT1/pcDNA-NEAT1. scale bar=100μm. H-I. The TUNEL assay was used to detect the apoptosis of U251 and LN229 cells by silencing or overexpressing NEAT1. scale bar=100μm. Values are shown as the mean ± standard errors of the mean based on three independent experiments. *P < 0.05, **P < 0.01 Values are shown as the mean ± standard errors of the mean based on three independent experiments.
*P < 0.05, **P < 0.01 PCR analysis. C. The expression level of KCTD20 was analyzed by qRT-PCR in glioma tissues and corresponding adjacent non-tumor tissues (n=43). D. Predicted interactions between miR-324-5p and the 3′-UTRs of KCTD20 from Targetscan bioinformatics algorithm. E. The relative luciferase activity in cells co-transfected with wild type 3′ UTR (KCTD20-WT) or mutant type 3′ UTR (KCTD20-MUT) and indicated constructs miR-NC or miR-324-5p mimics, Renilla luciferase vector was used as an internal control. F. The protein and mRNA expression of KCTD20 in response to miR-324-5p inhibition group or miR-324 mimics group in U251 and LN229 cells glioma cells was evaluated by using Western blot and qRT-PCR assay. G.
The KCTD20 protein and mRNA expression was analyzed after NEAT1 knockdown in U251 and LN229 cells. H. Western blot and qRT-PCR assays were used to determine the KCTD20 protein and mRNA expression of siNEAT1 and miR-324-5p inhibitor co-transfected U251 and LN229 cells. I. Scatter diagram exhibited a positive linear correlation of NEAT1 and KCTD20 in 43 paired glioma tissues by qRT-PCR (r=0.3732, P=0.0137). Values are shown as the mean ± standard errors of the mean based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001