Long Noncoding RNA LINC00958 Accelerates Gliomagenesis Through Regulating miR-203/CDK2

Introduction

Glioma is the most aggressive malignancy, accounting for 80% of malignant adult primary brain tumors (Armento et al., 2017; Rossmeisl, 2017). Although many advanced treatment technologies have been developed for clinical therapy of glioma, prognosis of glioma patients is still poor (Zhao et al., 2017). According to histopathological grades of World Health Organization (WHO), glioma is categorized as I–IV grades. The obstacle for clinical therapy of glioma patients is due to both infiltrative metastasis and the resistance toward chemoradiotherapy. The 5-year survival rate of glioma patients is extremely low, which is <10% with grade IV (Reiss et al., 2017). Therefore, it is very necessary to develop effective treatments for glioma.

Long noncoding RNAs (lncRNAs) are type of transcripts with length longer than 200 nucleotides (Cimadamore et al., 2017; Kapinova et al., 2018). Previous experience mistakenly considers that noncoding RNAs (ncRNAs) constitute a major part of human transcriptome, acting as transcriptional “noise” (Malhotra et al., 2017). However, recent researches have powerfully proved that lncRNAs are involved in various critical biological processes. In the series of tumors, lncRNAs regulate proliferation, apoptosis, metastasis, and drug resistance. For instance, lncRNA DANCR is significantly upregulated in glioma tissues and cell lines and high expression is correlated with advanced tumor grade, and results show crucial roles of DANCR/miR-634/RAB1A axis in the progression of glioma (Xu et al., 2018).

Emerging evidence has illustrated the important role of lncRNAs in multiple diseases, especially cancer. Long intergenic noncoding RNA 00958 (LINC00958) is a novel identified oncogenic gene in cancer (Seitz et al., 2017). In this study, we investigate the role of lncRNA LINC00958 in lncRNA LINC00958 and explore the underlying molecular mechanism.

Materials and Methods

Patients and specimens

A total of 35 paired glioma tissue samples and the matched adjacent normal brain tissues were surgically excised and reserved under −80°C for use at The Second Affiliated Hospital of Hebei Medical University between November 2015 and December 2016. None of these samples had ever received chemotherapy or radiotherapy before this excision. Tissue samples were identified by experienced pathologists according to the WHO classification to be I–IV histopathological grade. This study was approved by the Ethics Committee of The Second Affiliated Hospital of Hebei Medical University. Each patient gave written informed consent before surgery.

Cell lines and culture

Human glioma cell lines (SHG44, U87, U251, and A172) and normal human astrocytes (NHAs) were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Gibco) at 37°C in a humidified incubator containing 5% CO₂.

Cell transfection

All the oligonucleotides were purchased from GenePharma (Shanghai, China), including siRNA targeting LINC00958, miR-203 mimic, miR-203 inhibitor, and its negative controls. Oligonucleotide sequences included si-LINC00958-1, 5′-GTGACTAGCTTAAACTAAATT-3′; si-LINC00958-2, 5′-GAGGTACCCAATAGTTTCATT-3′; and si-LINC00958-3, 5′-GTACCCAAGTTATTCAGGATT-3′. For the transfection, glioma cells (1.0 × 10⁶ cells per well) were transfected with siRNAs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

RNA extraction and RT-PCR

Total RNA was extracted from glioma tissue sample and cells using Trizol reagent (Invitrogen). cDNA was reversely transcribed from RNA (1 μg) using M-MLV reverse transcriptase (Invitrogen). Real-time PCR was performed by Power SYBR Green PCR Master Mix (Applied Biosystems). Primers used in the section were as follows: LINC00958, 5′-CCATTGAAGATACCACGCTGC-3′ (forward), 5′-GGTTGTTGCCCAGGGTAGTG-3′ (reverse); miR-203, 5′-CGGTAGTCTGATACTGTAA-3′ (forward), 5′-GTGCTCCGAAGGGGGT-3′ (reverse); and GAPDH 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward), 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse). GAPDH acted as endogenous controls. Each expression level was calculated with the 2^−ΔΔCt method and every datum was performed in triplicate.

Glioma proliferation assay

The proliferation ability of glioma was measured using Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. In brief, cells were cultured in 96-well plates at a density of 1000 cells/well. The absorbance was measured at 450 nm to determine the cell viability every 24 h. The experiments were independently repeated three times.

Transwell invasion assay

Transwell assay was performed for the glioma invasion. Cells were cultured at about 80% confluence and starved in basal medium without FBS. Invasion assay was carried out using the BD BioCoat Tumor Invasion System (BD Biosciences). About 1 × 10⁵ glioma cells were seeded into the upper chamber. After 24 h, the invaded cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The invaded number of cells was counted in five randomly selected microscopic views and photographed.

Flow cytometry cycle analysis

For glioma cell cycle analysis, flow cytometry was performed. After 40 h of transfection, glioma cells were collected and fixed in 75% ethanol overnight at 4°C and then incubated with RNase A for 30 min at 37°C. Then, cells were stained with propidium iodide and the stained cells (1 × 10⁵ cells) were analyzed by flow cytometry (FACScan; BD Biosciences).

Dual luciferase reporter assay

For luciferase assay, U251 cells were cotransfected with miR-203 or pcDNA3.1-luc vector containing LINC00958 and CDK2 wild type or mutant 3′-untranslated region (3′-UTR) using Lipofectamine 2000 according to the manufacturer's protocol. Cells were seeded in 24-well plates at 60% confluence for 24 h. After 48 h, cells were collected and examined for β-galactosidase and luciferase activities using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.

Xenograft in vivo assay

The experimental animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The program was approved by the Animal Care and Use Committee of the Second Affiliated Hospital of Hebei Medical University. In brief, the male BALB/c (6-week-old, 10 mice) nude mice were obtained from Cancer Institute of Chinese. About 1 × 10⁷ U87 cells were subcutaneously injected into the flank of mice. Tumor growth was determined by caliper measurements. Tumor volume was calculated according to the following formula: 0.5 × (longest diameter × shortest diameter²).

Western blot

Total protein was extracted from tissue for Western blot analysis as previously described. The protein lysates added with protease inhibitor were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Then, the protein lysates were transferred to 0.22 mm nitrocellulose membranes. The membranes were incubated with primary antibody (anti-CDK2, 1:1000 dilution; Cell Signaling Technology). Then, the membrane was probed with horseradish peroxidase-conjugated secondary antibodies. Lastly, the blots were measured using an enhanced chemiluminescence detection system (Pierce, Rockford, IL). Anti-GAPDH antibody was used to monitor the loading amount.

Statistical analysis

All measurement data are presented as the mean ± standard deviation. Independent sample t-test and one-way ANOVA were used to analyze the difference between two groups. p < 0.05 was considered as statistical significance. Statistical analysis was performed using SPSS software and graphed using GraphPad software.

Results

lncRNA LINC00958 was upregulated in glioma tissue and indicated poor prognosis

To investigate the role of lncRNA LINC00958, RT-PCR assay was performed in 35 paired glioma tissue samples and the matched adjacent normal brain tissues. The clinicopathological characteristics of 35 glioma patients are given in Table 1. Results presented that lncRNA LINC00958 expression was significantly upregulated in glioma tissue (Fig. 1A). According to the median value, the expression levels of lncRNA LINC00958 were divided into high-expression group (20 cases) and low-expression group (15 cases) (Fig. 1B). For the prognosis of patients, Kaplan–Meier and log-rank tests indicated that the patients with higher LINC00958 expression presented poor prognosis than those with low LINC00958 expression (Fig. 1C). In summary, these results concluded that LINC00958 overexpression indicated poor prognosis of glioma patients.

LINC00958 knockdown suppressed proliferation and invasion and induced cell cycle arrest at G0/G1 phase in vitro

Our primary researches indicated that LINC00958 was upregulated in glioma tissue and indicated poor prognosis. To verify the biological role of LINC00958 on glioma tumorigenesis, loss-of-functional experiments in vitro were performed. Expression levels of LINC00958 were significantly upregulated in human glioma cell lines (SHG44, U87, U251, and A172) compared with those of NHA cells (Fig. 2A). The transfection of interfering oligonucleotides targeting LINC00958 markedly decreased the expression of LINC00958 in U87 and U251 cell lines (Fig. 2B). CCK-8 assay showed that LINC00958 knockdown suppressed the proliferation ability of U87 and U251 cells compared with negative control transfection (Fig. 2C). Transwell assay showed that LINC00958 knockdown reduced the invasive ability of U87 and U251 cells compared with negative control transfection (Fig. 2D, E). Flow cytometry assay showed that LINC00958 knockdown induced the cell cycle arrest at G0/G1 phase (Fig. 2F, G). In summary, these results concluded that LINC00958 knockdown suppressed proliferation and invasion and induced the cell cycle arrest at G0/G1 phase, suggesting the tumor-promoting function of LINC00958 in glioma tumorigenesis.

LINC00958 knockdown suppressed the tumor growth of glioma in vivo

The loss-of-function experiments in vitro had indicated the tumor inhibition of LINC00958 knockdown. To investigate the role of LINC00958 on glioma cell growth, the in vivo experiment was performed using U87 cells (Fig. 3A). LINC00958 knockdown significantly decreased the tumor volume compared with empty control group (Fig. 3B). Besides, LINC00958 knockdown significantly downregulated tumor weight compared with the empty control group (Fig. 3C). RT-PCR showed that the interfering oligonucleotides transfection decreased the LINC00958 expression level in mice tissue in vivo (Fig. 3D). Therefore, the xenograft assay revealed that LINC00958 knockdown suppressed glioma tumor growth in vivo.

miR-203 acted as a downstream miRNA of LINC00958

Now that the physiopathologic role of LINC00958 had been verified, we investigated the underlying mechanism of LINC00958 during glioma oncogenesis. Bioinformatics prediction tools revealed that miR-203 had complementary binding sites targeting with 3′-UTRs of LINC00958 (Fig. 4A). Luciferase reporter assay confirmed that miR-203 combined with LINC00958 with decreasing luciferase activity, indicating molecular interaction (Fig. 4B). In glioma cell lines, RT-PCR showed that miR-203 expression levels were significantly decreased compared with those of NHAs (Fig. 4C). In glioma tissue, RT-PCR presented that miR-203 expression was significantly downregulated compared with that of adjacent normal tissue (Fig. 4D). In 20 glioma patient samples, Pearson's correlation showed that LINC00958 was negatively correlated with miR-203 expression (Fig. 4E). Thus, results concluded that miR-203 acted as a downstream molecular of LINC00958.

CDK2 acted as the target protein of miR-203 and LINC00958

RT-PCR showed that CDK2 mRNA expression was significantly upregulated in glioma cell lines (U87 and U251) (Fig. 5A). Then, we performed bioinformatics prediction tools to discover the downstream target protein of miR-203 and LINC00958. Fortunately, bioinformatics programs and luciferase reporter assay revealed that miR-203 had the complementary binding sites targeting with 3′-UTR of CDK2 (Fig. 5B). In U87 cells, the transfection of miR-203 inhibitor significantly upregulated the CDK2 mRNA expression, whereas the transfection of si-LINC00958 downregulated the CDK2 mRNA expression (Fig. 5C). In 20 cases of glioma patient samples, Pearson's correlation showed that CDK2 was negatively correlated with miR-203 expression (Fig. 5D). Similarly, Western blot showed that si-LINC00958 transfection decreased the CDK2 protein expression, and miR-203 inhibitor transfection increased CDK2 protein expression (Fig. 5E, F). Overall, the aforementioned results concluded that CDK2 acted as the target protein of miR-203 and LINC00958, constructing the LINC00958/miR-203/CDK2 axis.

Discussion

Glioma is the most prevalent malignant brain tumors, causing serious lethality worldwide (Glenn et al., 2017). Although great attempts have been made to improve the therapeutic effects, the long-term prognosis of glioma patients is still pessimistic. In this research, we investigate the pathophysiological role of lncRNA LINC00958 in glioma tumorigenesis.

With rapid development of next-generation sequencing and bioinformatics technology, increasing quantity of lncRNAs is identified and their aberrant expression has been verified to be associated with the progression in various types of cancers (Hu et al., 2017; Lemler et al., 2017). lncRNA LINC00958 has been reported to act as a candidate oncogene in bladder cancer, regulating the viability and migration and binding proteins involved in translation in post-transcriptional modification (Seitz et al., 2017). In our research, we find that lncRNA LINC00958 is significantly upregulated in glioma tissue samples and cell lines. Besides, the patients with higher LINC00958 expression present poor prognosis than those with low LINC00958 expression. Therefore, these pieces of evidence prove that lncRNA LINC00958 might act as a risk factor in glioma.

In the efforts of researchers, it was definitely indicated that the regulatory mechanism of lncRNA involved in the glioma tumorigenesis covers a series of pathophysiological aspects, including proliferation, invasion, metastasis, and chemoresistance (Ayers and Vandesompele, 2017; Meng et al., 2017; Li et al., 2018). Our study and experiments found that lncRNA LINC00958 knockdown suppresses the proliferation ability and invasive ability, and induces cell cycle arrest at G0/G1 phase of U87 and U251 cells in vitro. Meanwhile, xenograft assay revealed that LINC00958 knockdown suppressed glioma tumor growth in vivo. Thus, we conclude that LINC00958 regulates glioma carcinogenesis through modulating proliferation, invasion, and cell cycle regulation.

Before this study, numerous lncRNAs have been validated to participate in the tumorigenesis of glioma (Cui et al., 2017; Matjasic et al., 2017; Yang et al., 2018). For example, lncRNA HOXA11-AS is significantly upregulated in glioma tissues and cell lines compared with that in adjacent normal brain tissues and NHAs; moreover, HOXA11-AS knockdown inhibited glioma cell proliferation, migration, and invasion in vitro, and tumor growth in vivo through miR-214-3p/EZH2 axis (Xu et al., 2017). For another example, lncRNA PLAC2 induces cell cycle arrest by targeting ribosomal protein L36 in glioma and blocks cell cycle progression in glioma through a mechanism involving STAT1 (Hu et al., 2018).

Up to now, the most canonical regulatory mechanism of lncRNAs is the competing endogenous RNA (ceRNA) (Gao et al., 2017b; Qin et al., 2017). The theory demonstrates that lncRNAs harbor miRNAs and counteract their abundance, acting as a “sponge” to absorb miRNAs (Xue et al., 2017). For instance, lncRNA HOTTIP is upregulated in glioma cells treated by hypoxia and promotes the hypoxia-induced epithelial–mesenchymal transition by regulating the miR-101/ZEB1 axis (Zhang et al., 2017). In our study, we found that lncRNA LINC00958 was upregulated in glioma tissue and cells; moreover, LINC00958 promoted CDK2 expression through miR-203. Therefore, lncRNA LINC00958 functions as miR-203 “sponge” to accelerate CDK2 expression. Cyclin-dependent kinase (CDK) is a group of important cellular process regulators controlling the cell cycle checkpoint (Li et al., 2017; Wang et al., 2017). It has been reported that CDK2 participates in the gliomagenesis through post-transcriptional repression mediated by lncRNA HSP90AA1-IT1-miR-885-5p-CDK2 signaling axis (Gao et al., 2017a).

Taken together, our study investigates and validates the carcinogenic role of lncRNA LINC00958 in the gliomagenesis. LINC00958 promotes gliomagenesis through miR-203/CDK2 axis, acting as a ceRNA of miRNA. This novel finding triggers a new perspective for clinical treatment of glioma and might provide a therapeutic strategy.

Acknowledgment

This work was supported by Medical Research Center of The Second Affiliated Hospital of Hebei Medical University.

Disclosure Statement

No competing financial interests exist.