Long non-coding RNA RNCR3 promotes glioma progression involving the Akt/GSK-3β pathway

Increasing evidence has confirmed that long non-coding RNAs (lncRNAs) serve critical roles in the development of a large number of human malignancies, including glioma. Several previously published studies have reported that the lncRNA retinal non-coding RNA3 (RNCR3; also termed LINC00599) exerts important roles in certain human malignancies; however, the precise biological role and underlying molecular mechanisms of RNCR3 in the development of glioma are yet to be fully elucidated. In the present study, it was revealed that the expression of RNCR3 was increased in glioma tissues compared with in corresponding adjacent normal tissues. Furthermore, increased levels of RNCR3 expression were associated with tumor progression and poor survival rates of patients with glioma. In addition, the U87 and U251 cell lines were selected to investigate the biological function and potential mechanisms of RNCR3 in glioma, and it was observed that RNCR3 knockdown led to an impairment of the proliferative and invasive abilities of cells; furthermore, G1 phase arrest was induced in glioma cells in vitro. Finally, the results of western blot analyses revealed that knockdown of RNCR3 led to a decrease in the expression levels of phosphorylated Akt and glycogen synthase kinase-3β (GSK-3β), without any clear effect on the expression levels of total Akt and GSK-3β. Collectively, these results suggested that RNCR3 is able to regulate cell proliferation, the cell cycle and cell invasion in glioma, potentially via the Akt/GSK-3β signaling pathway.


Introduction
Glioma is the most common and most aggressive primary malignant tumor arising in the central nervous system of adults globally (1). It is also one of the main causes of cancerassociated mortality (2). Although certain progress has been made regarding effective early diagnosis of the disease, the majority of patients with glioma have progressed to the advanced stages by the time of diagnosis (3). Intensive research has been conducted to examine the biological mechanisms underlying the progression of glioma; however, the prognosis of patients in this category remains rather poor (4,5).
With the rapid development of genome and transcriptome sequencing technologies, numerous novel non-protein-coding RNAs have been identified (6). Whereas >80% of the genome is transcribed into mRNA transcripts, only ~2% of the genome codes for proteins (6,7). Long non-coding RNAs (lncRNAs) comprise a class of non-protein-coding RNAs that are >200 nucleotides in length (8). LncRNAs serve key regulatory roles in a variety of biological and pathological processes, including tumorigenesis, transcription regulation and epigenetic post-transcription regulation (9)(10)(11).
Retinal noncoding RNA3 (RNCR3, also termed LINC00599) is a lncRNA that is transcribed from the intergenic regions of the genome, and is highly conserved in mammals (12). A number of studies have reported that RNCR3 exerts important regulatory functions in cell proliferation and differentiation, and the process of atherosclerosis (13,14). Regarding human cancers, Tian et al (15) reported that RNCR promoted prostate cancer development by regulating microRNA (miR)-185-5p. However, the biological functions and molecular mechanisms of RNCR3 in glioma are yet to be fully elucidated.
In the present study, RNCR3 expression levels in glioma tissues and in paired normal tissues were evaluated, following which the biological function of RNCR3 in glioma cell lines, and the underlying mechanisms of RNCR3 in glioma, were further investigated. To the best of our knowledge, the present study is the first to demonstrate that RNCR3 functions as an oncogene in glioma development.

Materials and methods
Clinical samples. The present study was approved by the Ethics and Research Committees of the Sunshine Union Hospital of Shandong Province (Weifang, China), and performed in accordance with the principles of the Declaration of Helsinki. A total of 54 pairs of glioma tissue samples and paired adjacent normal tissues were obtained from patients undergoing resection at the Department of Neurosurgery of Sunshine Union Hospital between January 2005 and December 2010. The patients included 30 males and 24 females with a mean age of 53.9 years (range, 41-74 years). All the patients were pathologically confirmed, and none of the patients had received preoperative chemotherapy or radiation therapy. The tissues were collected during surgery and stored immediately in liquid nitrogen (-196˚C). Written informed consent was collected from all subjects. The clinical characteristics of all the patients are summarized in Table I.
Cell culture. A human astroglia cell line, HA, was acquired from BeNa Culture Collection. Two human glioma cell lines, SHG-44 and U251, and a glioblastoma of unknown origin, U87 (cat. no. HTB14) were purchased from the American Type Culture Collection. Cell lines were authenticated via short tandem repeat cell authentication profiling. All the cell lines were maintained in Gibco™ DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA), and 50 U/ml penicillin and 0.1 mg/ml streptomycin (Biowest). All the cell cultures were incubated at 37˚C with 5% CO 2 .
RNA extraction and reverse transcription-quantitative PCR (RT-qPCR). Following the manufacturer's protocol, TRIzol ® reagent (Thermo Fisher Scientific, Inc.) was used to isolate the RNA of tissues or cells, and the isolated RNA was subsequently reverse-transcribed using an Invitrogen™ PrimeScript RT Reagent kit (Thermo Fisher Scientific, Inc.). SYBR Premix Ex Taq™ reagent (Takara Biotechnology Co., Ltd.) was used for the qPCR assay, and qPCR was performed using an Applied Biosystems™ ABI PRISM 7500 PCR system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. PCRs were performed in a total volume of 20 µl with 10 µl 2x SYBR premix ex-taq, 5 µl cDNA, 0.8 µl primers (2.5 µM) and 4.2 µl ddH 2 O). The PCR thermocycling conditions were; denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 55˚C for 15 sec and 72˚C for 10 sec with a final extension step at 60˚C for 1 min. Melting curves of the amplified products were analyzed at the end of each PCR to confirm that only one product was amplified and detected. Expression was calculated via the relative quantification cycle (Cq) value and was normalized to the expression of the internal control gene GAPDH. Relative expression of RNCR3 was calculated using the 2 -ΔΔCq method (16). The primer sequences for RNCR3 and GAPDH are presented in Table II. The mean value was selected as the cut-off between high and low RNCR3 expression in patients with glioma. The relative expression of control groups was normalized to 1.
RNCR3 short hairpin RNA (shRNA). RNCR3 shRNA (sh-RNCR3) was purchased from Shanghai GenePharma Co. Ltd. And transfected into cells at a final concentration of 50 nM. An Invitrogen™ Lipofectamine ® 3000 kit (Thermo Fisher Scientific, Inc.) was used to perform the transfections, according to the manufacturer's protocol. RT-qPCR was subsequently used to evaluate the knockdown efficiency. For all experiments involving transfected cells, cells were transfected for 48 h before subsequent experiments were performed.
Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was examined every 24 h, according to the manufacturer's protocol. Cells were placed into the 96-well plates at a density of ~3,000 cells/well. Subsequently, 10 µl CCK-8 (Dojindo Molecular technologies, Inc.) was added, and the cells were incubated at 37˚C for a further 2 h. Finally, the absorbance at 450 nm was determined using a spectrometer.
Cell cycle assay. Cells were collected, washed 3 times with cold PBS, and then fixed in 70% ethanol at 4˚C overnight. Subsequently, the cells were washed, re-suspended, and incubated in a solution of 10 µg/ml RNase and 1 mg/ml propidium iodide (Sigma-Aldrich; Merck KGaA) at 37˚C for 30 min in the dark. Finally, a FACSCalibur™ Cell Analyzer with ModFit version 5.0 (both BD Biosciences) was used to analyze the cells and the resultant flow cytometry data.
Cell invasion assay. The invasive ability of the cells was determined using BD 24-well Transwell ® chambers (Costar; Corning Inc.) pre-coated with Matrigel™ coating, according to the manufacturer's protocol. Briefly, 1x10 5 cells suspended in 200 µl serum-free medium were seeded into the upper chamber, and 800 µl DMEM supplemented with 10% FBS was placed into the lower chamber. After incubating the cells for 18 h at 37˚C, cells on the lower chamber membranes were fixed with 4% formaldehyde 15 min at room temperature and stained with 1% crystal violet at room temperature for 10 min. Cells in five randomly selected fields of the membrane were counted under a light microscope (magnification, x40).
Western blot analysis. RIPA buffer (Beyotime Institute of Biotechnology) was used to extract proteins from the cells. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit. The proteins (30 µg per lane) were resolved on a 10% SDS-PAGE and then electrophoretically transferred to polyvinylidene fluoride membranes (Bio-Rad   ) was used to detect the intensities of the immunoblots, which were visualized following exposure to X-ray film. Protein expression levels were quantified relative to β-actin levels using ImageJ (version 1.8.0 National Institutes of Health).

Statistical analysis.
Experimental data from at least three independent experiments are presented as the mean ± standard deviation. SPSS 18.0 software (SPSS, Inc.) was used to perform the statistical analyses. The associations between the levels of RNCR3 and clinicopathological factors was assessed by χ 2 test. Differences between multiple groups were evaluated by one-way analysis of variance followed by Tukey's post hoc test. Differences between two groups were evaluated using an unpaired or paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Expression of RNCR3 is increased in glioma tissues and cell lines.
RT-qPCR analysis was performed to measure the relative expression of RNCR3 in 54 pairs of glioma tissue and adjacent normal tissue, we have used the normal tissues as  control for normalization. The results obtained suggested that RNCR3 expression was markedly higher in glioma tissues compared with the corresponding adjacent normal tissues (Fig. 1A). RNCR3 expression was further examined in cell lines: A human astroglia cell line (HA), two human glioma cell lines (SHG-44 and U251), and a glioblastoma of unknown origin (U87). These experiments revealed that the expression of RNCR3 was higher in the glioma cell lines compared with that in the HA cell line (Fig. 1B).

Upregulated expression of RNCR3 is associated with progression of the disease, and poor prognosis of patients with glioma.
Subsequently, the correlation between RNCR3 expression levels and clinicopathological factors in 66 glioma individuals was investigated (Table I). This analysis revealed that an elevated expression of RNCR3 was associated with tumor size and clinical stage, although no significant correlations were identified between RNCR3 expression and age and gender. In addition, KaplanMeier survival curves suggested that glioma patients who had higher expression levels of RNCR3 in their glioma tissues had significantly poorer survival rates compared with glioma patients who had correspondingly lower expression levels of RNCR3 in their glioma tissues (Fig. 1C).
Knockdown of RNCR3 inhibits glioma cell proliferation. The results of the RT-qPCR assay confirmed that the RNCR3 expression levels of U87 and U251 cells transfected with sh-RNCR3 were lower compared with that of the control untransfected cells, or those U87 and U251 cells that were transfected with NC shRNA ( Fig. 2A and B). RNCR3 knockdown led to a repression of cell proliferation in the U87 (Fig. 2C) and U251 (Fig. 2D) glioma cell lines.

Knockdown of RNCR3 induces G 1 phase arrest of glioma cells.
The results of the cell cycle assay experiments revealed that knocked-down RNCR3 induced the G 1 phase arrest of U87 and U251 cells (Fig. 3). Fig. 4, the cell invasion assays confirmed that RNCR3 knockdown repressed cell invasion in the U87 and U251 cells.

Knockdown of RNCR3 inhibits cell invasion in the glioma cell lines. As shown in
Knockdown of RNCR3 inhibits the Akt/GSK-3β pathway in glioma cells. As has been well established, the Akt/GSK-3β signaling pathway is constitutively active in a large number of different types of human cancer. To further investigate whether RNCR3 regulates glioma development through Akt/GSK-3β pathway activation, western blot assays were performed to evaluate the levels of total and phosphorylated Akt and GSK-3β in the glioma cells. As shown in Fig. 5, silencing of RNCR3 did not exert a clear influence on the total level of Akt and GSK-3β, but the levels of phosphorylated Akt and GSK-3β were significantly reduced. These results indicated that overexpression of RNCR3 activated the Akt/GSK-3β pathway in human glioma cells.

Discussion
In view of the increasing number of studies that are being published focusing on the functional attributes of lncRNAs, emerging evidence has indicated that lncRNAs have important roles in various physiological and pathological processes, including cell proliferation, apoptosis and differentiation, and the development of different types of cancer (9,17,18). LncRNAs fulfill a range of different roles, including the regulation of gene transcription in basal transcription machinery, post-transcriptional regulation of RNA splicing and epigenetic regulation (19). For example, the lncRNA small ubiquitin-like modifier 1 pseudogene 3 promoted breast cancer progression by negatively regulating miR-320a (9). LncRNA highly upregulated in liver cancer   triggers autophagy by stabilizing sirtuin-1, and impairs the chemosensitivity of hepatocellular carcinoma cells (18). LncRNA HOxA transcript at the distal tip enhances tumorigenesis and metastasis in esophageal squamous carcinoma cells through the transcriptional and post-transcriptional regulation of homeobox protein A13 (19).
In the present study it was revealed for the first time, to the best of our knowledge, that the expression of lncRNA RNCR3 was upregulated in glioma tissues and cell lines. Additionally, the increased expression levels of RNCR3 in glioma tissues were associated with tumor progression and poor survival rates in patients with glioma. Furthermore, RNCR3 knockdown inhibited the proliferative and invasive abilities, and induced the G 1 phase arrest of glioma cells. Finally, the present study suggested that the effects of RNCR3 on cell proliferation, the cell cycle and cellular invasion may involve regulation of the Akt/GSK-3β signaling pathway.
A number of studies have suggested that aberrant signaling of the Akt pathway contributes to cell proliferation and invasion in various human malignancies (20). The results of the present study suggested that RNCR3 knockdown led to an inhibition of the phosphorylation of Akt, although there was no clear influence on the total levels of Akt. Akt phosphorylation has previously been reported to inhibit the activity of GSK-3β, which is an important downstream target protein of Akt (20). These results are consistent with previous studies, which reported on the function of Akt/GSK-3β in glioma (21,22). In addition, a large number of lncRNAs have been reported to regulate human cancers via actions on the Akt/GSK-3β pathway. For example, the lncRNA lung cancer-associated transcript 1 was revealed to enhance proliferation and invasion in clear cell renal cell carcinoma via the Akt/GSK-3β pathway (23). The lncRNA urothelial carcinoma-associated 1 (UCA1) was identified to promote tumorigenesis by regulating the AKT/GSK-3β pathway in cholangiocarcinoma (24). LncRNA UCA1, induced by SP1 transcription factor, was also shown to enhance cell proliferation by recruiting enhancer of zeste 2 polycomb repressive complex 2 subunit, the catalytic subunit of the polycomb repressive complex 2, and activating the Akt signaling pathway in gastric cancer (25).
In conclusion, the present study demonstrated that the expression level of RNCR3 was increased in glioma tissues and in cell lines, and that RNCR3 may therefore serve as a novel prognostic indicator for patients with glioma. Silencing RNCR3 in glioma cells led to a suppression of the proliferative and invasive abilities of the cells, and induced cell cycle arrest involving the Akt/GSK3β pathway.