Upregulation of long noncoding RNA zinc finger antisense 1 enhances epithelial–mesenchymal transition in vitro and predicts poor prognosis in glioma

Abstract

Increasing evidence indicates that long noncoding RNAs play important roles in development and progression of various cancers. Zinc finger antisense 1 is a novel long noncoding RNA whose clinical significance, biological function, and underlying mechanism are still undetermined in glioma. In this study, we reported that zinc finger antisense 1 expression was markedly upregulated in glioma and tightly correlated with clinical stage. Moreover, patients with high zinc finger antisense 1 expression had shorter survival. Multivariate Cox regression analysis provided a clue that, probably, zinc finger antisense 1 level could serve as an independent prognostic factor for glioma. Functionally, zinc finger antisense 1 acted as an oncogene in glioma because its knockdown could promote apoptosis and significantly inhibit cell proliferation, migration, and invasion. Furthermore, zinc finger antisense 1 silencing could result in cell cycle arrest at the G0/G1 phase and correspondingly decrease the percentage of S phase cells in both U87 and U251 cell lines. Moreover, it was found that silenced zinc finger antisense 1 could impair migration and invasion by inhibiting the epithelial–mesenchymal transition through reducing the expression of MMP2, MMP9, N-cadherin, Integrin β1, ZEB1, Twist, and Snail as well as increasing E-cadherin level in glioma. Taken together, our data identified that zinc finger antisense 1 might act as a valuable prognostic biomarker and potential therapeutic target for glioma.

Introduction

Glioma is the most serious form of primary tumors in adult central nervous system (CNS). As the most common type of glioma, glioblastoma multiforme (GBM) carries a poor prognosis, with the median survival of merely approximately 15 months.^1–3 Despite advances in combination treatments, including surgical resection followed by radiation therapy and chemotherapy, the average survival of malignant glioma patients has only slightly been improved in the past decades.⁴ Similar histological features of glioma may exhibit different clinical characteristics and responses to therapies.⁵ Therefore, it is urgent to identify novel biomarkers that are specific to different histopathological types of gliomas, which may be helpful in developing more rational and effective regimens for their treatment.

The long noncoding RNAs (lncRNAs) were once considered as a class of transcriptional noise without any functions.⁶ However, in recent years, high-throughput transcriptome analysis has revealed that the vast majority (98%) of the human genome can be transcribed into noncoding RNAs.⁷ Among them, lncRNAs have acquired extensive attention, recently, as new regulators in many biological processes, such as transcriptional regulation,⁸ differentiation,⁹ immune response,¹⁰ metabolism,¹¹ cell growth, and especially tumorigenesis.^12,13 Moreover, since an increasing number of reports have indicated that their abnormal expression is frequently observed in human cancers, some lncRNAs have been validated as biomarkers for diagnostic, metastasis, recurrence, and poor prognosis,^14–17 such as MALAT-1 for lung, glioma, gastric, and prostate cancer;^18–21 HOTAIR for breast, ovarian, pancreatic, and colorectal cancer;^22–25 and CCAT2 for oral squamous cell carcinoma and bladder cancer.^26,27

Zinc finger antisense 1 (ZFAS1) locus is host to three C/D-box small nucleolar RNAs (snoRNAs), and its transcription initiates from the antisense strand near the 5′ end of the protein-coding gene Znfx1.²⁸ Initially, lncRNA ZFAS1 was identified as a new tumor suppressor gene in human breast cancer.²⁸ However, recent studies have shown that ZFAS1 was amplified in hepatocellular carcinoma, colorectal and gastric cancer, and associated with their poor prognosis.^25,29,30 However, the implication of ZFAS1 in glioma progression was still unclear. In this study, we investigated the expression of lncRNA ZFAS1 in glioma tissues as well as in normal brain tissues and further explored the relationship between ZFAS1 and clinicopathological features as well as prognosis of glioma patients. Moreover, we examined the biological roles and underlying molecular mechanisms of ZFAS1 in regulation of cell viability, cell cycle, apoptosis, migration, and invasion in vitro.

Materials and methods

Tissue collection

A total of 69 glioma tissues (45 cases of astrocytomas, 7 cases of oligodendrogliomas, 3 cases of ependymomas, 2 cases of choroid plexus tumors, and 8 cases of others) were obtained from patients who had undergone surgical treatment without chemotherapy or radiotherapy at the First Affiliated Hospital of Nanchang University (Nanchang, Jiangxi, China) between 2013 and 2014. A total of 15 normal brain tissues from patients with cerebral trauma were used as control. All patients had experienced a follow-up period lasting 48 months from the date of surgical resection. Overall survival (OS) was defined as the time length between the date of the initial surgical operation and death or the last follow-up. All procedures were approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (Ethical Approval No. 2010-015; 12 March 2010), with written informed consent obtained from every participant.

Cell culture and small interfering RNA interference

HS683, T98G, U87, and U251 human glioma cell lines were purchased from the Cell Bank of the Shanghai Branch of Chinese Academy of Sciences and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). All cells were maintained at a humidified 37°C incubator with 5% CO₂. ZFAS1-specific small interfering RNA (named siRNA-ZFAS1) targeting ZFAS1: 5′-CAAACACCTTTACACGCTA-3′ and negative control (NC, a nonspecific scramble siRNA) were synthesized by RiboBio (Guangzhou, China). About siRNA interference, the cells (60% confluent) were first transfected with 100 nM siRNA using 5 µL Lipo-RNAiMAX following the manufacturer’s instruction (Invitrogen, USA). Then, the efficacy of siRNA-ZFAS1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) analysis.

RNA extraction

Total RNA was extracted from tissues and glioma cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction. Concentration and purity of RNA were determined spectrophotometrically by measuring its optical density (A260/280 > 2.0; A260/230 > 1.8) using a NanoDrop ND-1000 (Thermo Fisher Scientific, USA).

RT-PCR

RT-PCR was performed using SYBR Green real-time PCR kit (TaKaRa, Dalian, China) on a LightCycler480 (Roche, USA) to detect the expression of lncRNA ZFAS1, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene for normalization of lncRNA expression levels. The relative expression levels of target lncRNAs were determined by the equation 2^−ΔΔCT. The primers for ZFAS1 were as follows: 5′-ACGTGCAGACATCTACAACCT-3′ and 5′-TACTTCCAACACCCGCAT-3′.²⁹ The primers for GAPDH were as follows: 5′-CCCATCACCATCTTCCAGGAG-3′ and 5′-GTTGTCATGGATGACCTTGGC-3′.

Cell proliferation assays

The effect of siRNA-ZFAS1 on the proliferation of cells was evaluated by the CellTiter 96 aqueous one solution reagent (MTS) assay. U87 and U251 cells were plated in 96-well plates and divided into four groups, with 3 × 10³ cells per well and six wells for each group. Cells were attached and transfected with NC and siRNA-ZFAS1 before being cultured for 24, 48, 72, and 96 h. The medium was replaced by 100 µL MTS and DMEM medium with the ratio of 1:9. Following the incubation at 37°C for 30 min, cell viability was obtained by measuring the absorbance at the wavelength of 490 nm. All experiments were performed at least three times.

Colony formation assay

U87 and U251 cells were transfected with the NC or siRNA-ZFAS1 in six-well plates with 1 × 10³ cells per well. The plates were incubated for 1–2 weeks at 37°C in 5% CO₂ atmosphere until colonies were formed. Cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde for 20 min before being stained with 0.1% crystal violet. At last, a microscope was used to count the (number of) colonies containing more than 50 cells. The same procedure was performed in triplicate.

Wound healing assay

U87 and U251 cells were plated in six-well plates and transfected with 100 nM NC and siRNA-ZFAS1 using 6 µL Lipo-RNAiMAX when reaching 80%–90% density. A wound was made using a 1 mL plastic pipette tip before being washed twice with PBS. Subsequently, the medium was replaced by DMEM with 2% FBS. The size of the wound was measured under a microscope at 0 and 24 h after wounding.

Transwell invasion assay

Cell invasion assay was carried out using the 24-well transwell chamber (Corning, USA). Matrigel (BD Biosciences, USA) was diluted to 1:8 with cold DMEM without FBS and coated in the upper compartment chamber. Different groups of cells (2 × 10⁴) were plated onto the upper wells with 100 µL serum-free DMEM and the bottom chamber was filled with DMEM containing 10% FBS. In the experiment, 3 × 10⁶ cells were suspended in serum-free medium and added into the insert. At the same time, 500 µL of medium with 10% FBS was added to the lower well of the plate. After the incubation at 37°C for 24 h, noninvasive cells on the top chambers were gently removed with cotton wool. Invasive cells on the bottom surface were fixed with 4% paraformaldehyde for 0.5 h and stained with 0.1% crystal violet for 2 h, with their number counted under a light microscope. The experiment for each group was performed in triplicate and results were averaged.

Analysis of apoptosis and cell cycle arrest

U87 and U251 cells were seeded in six-well plates. After growing for 24 h, they were then transiently transfected with the siRNA-ZFAS1 or NC and harvested by trypsinization after 48 h followed by resuspension in 75% ethanol and fixed overnight at 4°C. The fixed cells were washed twice with PBS, resuspended in 200 µL PBS with 10 µL propidium iodide (PI) and addition of 5 µL RNase (Beyotime Institute of Biotechnology, Shanghai, China), and then incubated for 15 min at room temperature in the dark. Afterward, cell cycle was analyzed by flow cytometer after vortexing.

The apoptosis assay was performed using a kit (BD Biosciences) according to the manufacturer’s instructions. Cells were plated onto six-well plates and left overnight for attachment. They were then transiently transfected with the siRNA-ZFAS1 or NC before trypsinization and then suspended in PBS with a cell density of 1 × 10⁶/mL. A total of 195 µL binding buffer, 5 µL Annexin V-FITC, and 5 µL PI were added to the suspension and the cells were incubated for additional 15 min at room temperature in the dark. Then, cell samples were incubated on ice in the dark and measured using a flow cytometer, with total sample volume added upto 300 µL with binding buffer.

Western blot assay and antibodies

U87 and U251 cells (NC and siRNA-ZFAS1) were transfected for 72 h and harvested. Total protein was extracted using cold radioimmunoprecipitation assay buffer (RIPA) buffer with protease inhibitor cocktail tablet (Roche) and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich, USA) freshly added for 30 min on ice, and centrifuged at 12,000g for 15 min at 4°C to be clarified. According to the manufacturer’s instructions, protein concentrations were detected by BCA protein assay kit (Thermo Fisher Scientific, USA). The protein extracts (40 µg/lane) were then subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for separation and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA).Membranes were blocked with 5% nonfat milk for 1.5 h at room temperature and subsequently incubated with primary antibodies at 4°C overnight. After the incubation with the secondary antibodies (1:5000; Jackson ImmunoResearch, USA) at room temperature for 2.5 h, the specific protein was visualized using Enhanced chemiluminescence (ECL; GE Healthcare, USA) and quantified by Quantity One software (Bio-Rad Laboratories, USA). Antibodies against MMP2, MMP9, E-cadherin, N-cadherin, and Integrin β1 were obtained from the Cell Signaling Technology (USA; 1:1000). Antibody to β-actin was purchased from Sigma-Aldrich (USA; 1:4000). All the Western blots were performed at least three times.

Statistical analysis

Statistical analyses were mainly conducted using SPSS 20.0 software (IBM SPSS, USA). Data were presented as mean ± standard deviation (SD) from at least three separate experiments. Differential expression of ZFAS1 between glioma and normal brain tissues was detected by independent two samples t test. A chi-square test was applied to determine the association of ZFAS1 levels with clinicopathologic features. Survival analysis was carried out with the GraphPad Software Version 6.0. (GraphPad Software Inc., San Diego, CA, USA). The hazard ratio (HR) was estimated via a Cox regression model and OS curves were plotted from Kaplan–Meier estimates, while log-rank test was conducted to compare the survival distributions between two groups. Differences were considered statistically significant if p < 0.05.

Results

Overexpression of lncRNA ZFAS1 correlates with histological malignancy of gliomas

To investigate the potential roles of ZFAS1 in the development and progression of glioma, we first evaluated its expression level in glioma tissues (n = 69) and normal brain tissues (n = 15) by qRT-PCR, discovering that ZFAS1 expression was significantly elevated in glioma tissues compared with normal brain tissues (p = 0.017; Figure 1(a)). Furthermore, we explored the relationship between ZFAS1 expression and histological malignancy in 30 cases of low-grade (I–II) and 39 cases of high-grade (III–IV) glioma tissues. As shown in Figure 1(b), with the increase of ZFAS1 expression, the malignancy of glioma exhibited an increasing tendency (p = 0.0262).

Figure 1.

Upregulation of ZFAS1 was correlated with poor prognosis in glioma patients. (a) ZFAS1 expression was analyzed in 69 human glioma tissues and 15 normal brain tissues by qRT-PCR. (b) The difference in expression of ZFAS1 in low-grade (I–II) and high-grade (III–IV) glioma tissues. (c) With the median value as a reference, patients with glioma were divided into ZFAS1 high-expression and low-expression group, and Kaplan–Meier analysis was performed to investigate the relationship of ZFAS1 expression level with prognosis of 69 patients with glioma (p = 0.0117).

[Figure omitted. See PDF]

In order to determine the clinical significance of ZFAS1 in glioma, we investigated the correlation of ZFAS1 expression with clinicopathological parameters (Table 1), observing that increased ZFAS1 expression level was significantly associated with advanced tumor grade of glioma (p = 0.0184), but no correlation with age, gender, or tumor location was examined. All the data suggested that ZFAS1 might play an important role in glioma progression.

Table 1.

Correlation between lncRNA ZFAS1 expression and clinicopathological features of glioma patients.

Clinical characteristic	Number of patients	Number of patients		p value
		High expression (n = 27)	Low expression (n = 42)
Age (years)
≥45	45	17	28	0.7526
<45	24	10	14
Sex
Male	38	17	21	0.2907
Female	31	10	21
Clinical stage
Low grades I–II	30	7	23	0.0184*
High grades III–IV	39	20	19
Tumor location
Frontal	20	7	13	0.6882
Parietal	6	2	4
Occipital	14	5	9
Temporal	17	6	11
Others	12	7	5

lncRNA: long noncoding RNAs; ZFAS1: zinc finger antisense 1.

*

The values had statistically significant differences.

(p < 0.05)

Upregulation of lncRNA ZFAS1 is associated with poor prognosis of glioma patients

To further elucidate the correlation of ZFAS1 expression with prognosis of glioma patients, Kaplan–Meier analysis and log-rank test were performed. Remarkably, patients with high ZFAS1 expression had notably shorter OS time than those with low ZFAS1 expression (HR = 1.878, 95% confidence interval (CI) = 1.203–3.734, p = 0.0117; Figure 1(c)). In addition, univariate analysis identified that ZFAS1 expression level (HR = 1.915, 95% CI = 1.136–3.226, p = 0.015) and the clinical stage (HR = 2.043, 95% CI = 1.214–3.437, p = 0.007) were associated with prognosis. Multivariable Cox regression analysis confirmed that the highly expressed ZFAS1 (HR = 1.918, 95% CI = 1.059–3.473, p = 0.032) and the clinical stage (HR = 1.904, 95% CI = 1.076–3.369, p = 0.027) were independent prognostic factors for OS of glioma patients (Table 2). These results suggested that upregulated ZFAS1 might serve as an independent prognostic marker for glioma patients.

Table 2.

Univariate and multivariate analyses of OS in 69 glioma patients by Cox regression analysis.

Variable	Univariate analysis		Multivariate analysis
	HR (95% CI)	p value	HR (95% CI)	p value
Age (<45 vs ≥45 years)	0.885 (0.516–1.516)	0.656	0.777 (0.397–1.518)	0.460
Sex (female vs male)	1.189 (0.712–1.986)	0.509	1.201 (0.685–2.104)	0.523
Clinical stage (III–IV vs I–II)	2.043 (1.214–3.437)	0.007**	1.904 (1.076–3.369)	0.027*
Tumor location		0.801		0.425
Parietal vs frontal	1.052 (0.391–2.829)	0.920	1.208 (0.420–3.475)	0.727
Occipital vs frontal	1.459 (0.689–3.091)	0.324	1.715 (0.696–4.227)	0.241
Temporal vs frontal	1.183 (0.600–2.334)	0.627	1.282 (0.626–2.627)	0.497
Others vs frontal	0.872 (0.405–1.878)	0.727	0.720 (0.284–1.822)	0.488
ZFAS1 expression (high vs low)	1.915 (1.136–3.226)	0.015*	1.918 (1.059–3.473)	0.032*

OS: overall survival; HR: hazard ratio; CI: confidence interval; ZFAS1: zinc finger antisense 1.

*

The values had statistically significant differences.

(p < 0.05)

Silencing of lncRNA ZFAS1 expression in glioma cells by siRNA

To investigate the functional role of ZFAS1 in glioma cells, first, RT-PCR was performed to detect the expression of ZFAS1 in normal brain tissues and four glioma cell lines (HS683, T98G, U87, and U251). As shown in Figure 2(a), ZFAS1 expression level was significantly higher in three high-degree glioblastoma cell lines (T98G, U87, and U251) compared with the low-degree glioma cell line (HS683) and normal brain tissues. Then siRNA-ZFAS1 was transfected into U87 and U251 cells, which had the highest levels of ZFAS1, leading to a significant reduction of ZFAS1 expression in these two cell lines (p < 0.001 as compared with NC, Figure 2(b) and (c)).

Figure 2.

Silencing effect of the siRNA-ZFAS1 in U87 and U251 cells. (a) Expression of lncRNA ZFAS1 in normal brain and four glioma cell lines as determined by RT-PCR. (b and c) siRNA-ZFAS1 decreased the expression of ZFAS1 in U87 and U251 cells (*p < 0.05, ***p < 0.001).

[Figure omitted. See PDF]

Knockdown of lncRNA ZFAS1 could inhibit proliferation by arresting cell cycle and increasing apoptosis of glioma cells

To further examine whether ZFAS1 was involved in glioma progression, functional analyses were performed in vitro. Compared with NC groups, siRNA-ZFAS1 significantly inhibited cell proliferation ability in both U87 and U251 cell lines based on the MTS assay (p < 0.01 for U87, p < 0.05 for U251; Figure 3(a)). Similarly, the colony formation assay revealed that ZFAS1 silencing drastically decreased the number of colonies formed in U87 and U251 cells in contrast with NC groups (Figure 3(b)).

Figure 3.

Effects of ZFAS1 knockdown on glioma cell proliferation in vitro. (a) MTS assay demonstrated that the proliferation activity of the U87 and U251 NC groups were significantly higher compared with cells transfected in siRNA-ZFAS1 at 96 h (**p < 0.01 for U87, *p < 0.05 for U251). (b) Colony-forming growth assays were performed to determine the proliferation of U87 and U251 cells after silencing of ZFAS1.

[Figure omitted. See PDF]

In order to explore the potential role of ZFAS1 in cell proliferation, we further examined the effect of ZFAS1 knockdown on cell cycle and apoptosis in U87 and U251 cells by flow cytometry. Our data revealed that the proportion of cells dramatically increased in G0/G1 phase and decreased in the S phase both in U87 and U251 cell lines after siRNA-ZFAS1 transfection. Since induction of apoptosis was critical for effective tumor regression, we next measured apoptosis by Annexin V-FITC/PI staining, confirming that ZFAS1 silencing significantly induced apoptosis in U87 and U251 cells (p < 0.05 for U87, p < 0.01 for U251; Figure 4). These findings suggested that ZFAS1 might promote cell proliferation via accelerating cell cycle progression as well as inhibiting apoptosis in glioma.

Figure 4.

Effect of ZFAS1 silencing on cell cycle and apoptosis in U87 and U251 cells. (a) Effects of ZFAS1 knockdown on cell cycle. Downregulating ZFAS1 expression caused the percentage of cells to increase in G0/G1 phase and decrease in S phase obviously. (b) ZFAS1 silencing dramatically induced apoptosis in U87 and U251 cells (*p < 0.05, **p < 0.01).

[Figure omitted. See PDF]

Silencing of lncRNA ZFAS1 inhibited U87 and U251 cell migration and invasion

In the wound healing assay, compared with nontransfected cells, cell migration after 24 h of incubation was inhibited in cells transfected with siRNA-ZFAS1 (Figure 5(a)). Then we performed transwell migration assay, showing that most of U87 and U251 cells invaded from top chambers to the bottom chamber in control and NC groups but not in the siRNA-ZFAS1 groups (Figure 5(b)). Collectively, these findings indicated that ZFAS1 might play a functional role in the regulation of glioma cell migration and invasion capacity.

Figure 5.

Downregulation of ZFAS1 inhibited the migration and invasion of glioma cells. (a) Effects of ZFAS1 knockdown on the migration of the U87 and U251 cells (**p < 0.01 for U87, *p < 0.05 for U251). (b) In transwell migration assay, most of U87 and U251 cells invaded from top chambers to the bottom chamber in the control and NC groups but not in the siRNA-ZFAS1 groups (*p < 0.05 for U87, *p < 0.05 for U251).

[Figure omitted. See PDF]

Knockdown of lncRNA ZFAS1 suppressed migration and invasion of glioma cells by inhibiting epithelial–mesenchymal transition signaling pathway

During tumorigenesis, the epithelial–mesenchymal transition (EMT) plays a crucial role in metastasis, invasion, and expansion motility of cancer cells. Therefore, to further investigate the possible mechanism by which ZFAS1 promoted glioma progression, we examined the expression of several EMT markers, such as MMP2, MMP9, E-cadherin, N-cadherin, Integrin β1, ZEB1, Twist, and Snail by Western blot. As shown in Figure 6, we found that the expression levels of MMP2, MMP9, N-cadherin, Integrin β1, ZEB1, Twist, and Snail were significantly reduced in the siRNA-ZFAS1 groups, while E-cadherin expression greatly increased after siRNA-ZFAS1 transfection.

Figure 6.

ZFAS1 activated the EMT pathway in the glioma cell lines. (a and b) Western blot analysis was performed to determine the expressions of MMP2, MMP9, N-cadherin, E-cadherin, Integrin β1, ZEB1, Twist, and Snail in U87 and U251 cell lines. All experiments were repeated for three times, independently.

[Figure omitted. See PDF]

Basing on the above findings, we then examined whether ZFAS1 regulated EMT in glioma patients. Pearson’s correlation analysis was used to study the relationship between ZFAS1 expression and EMT markers in 69 glioma tissues, finding that the expression level of E-cadherin was negatively correlated with ZFAS1 expression, while other EMT markers, such as N-cadherin, Integrin β1, ZEB1, and Twist were positively correlated with ZFAS1 expression (Figure 7). These results implied that ZFAS1 might be able to promote the migration and invasion of malignant glioma by enhancing EMT progression.

Figure 7.

Correlations of ZFAS1 expression with EMT markers in glioma tissues. (a) ZFAS1 expression is negatively correlated with E-cadherin mRNA levels. (b–e) ZFAS1 is positively correlated with N-cadherin, Integrin β1, ZEB1, and Twist expression in 69 glioma tissues.

[Figure omitted. See PDF]

Discussion

A growing interest toward lncRNAs had drawn abundant attention as a potentially novel and crucial regulator of gene expression and cellular processes. Moreover, dysregulated lncRNAs were observed in numerous cancer types, suggesting that abnormal lncRNA expression could mark the spectrum of tumor progression and act as a major contributor to tumorigenesis.^13,31,32 In previous studies, it was shown that ZFAS1 was highly expressed in hepatocellular carcinoma, colorectal cancer, and breast cancer, with its functions as oncogenes or tumor suppressors.^25,28,29 However, little was known about the pathological role of lncRNA ZFAS1 in glioma.

In this study, we investigated the function of ZFAS1 in glioma for the first time and detected that ZFAS1 was remarkably upregulated in glioma tissues compared with normal brain tissues. Importantly, this phenomenon was correlated with a shorter survival, suggesting that the expression of ZFAS1 may be used as a diagnostic and prognostic marker for glioma, which was compatible with the research results by Li et al.²⁹ and Nie et al.³⁰ in hepatocellular carcinoma and gastric cancer patients. Since the clinical prognostic biomarkers were required to be highly constant and reproducible, but we conducted a relative quantitative method (qRT-PCR) in our research. Therefore, it is essential for us to validate the accuracy of the result by absolute quantitative method in the future.

The function and mechanism of ZFAS1 was examined in the biological process of glioma. We had identified that RNA interference (RNAi)-mediated suppression of ZFAS1 significantly inhibited cell proliferation in vitro according to the results of MTS and colony formation assays. Furthermore, knockdown of ZFAS1 drastically impaired migration, invasion, and increased apoptosis in glioma cells. Nie et al. had similar findings that silencing ZFAS1 inhibited gastric cancer cells proliferation and induced apoptosis in vitro, as well as suppressed tumorigenicity of gastric cancer in vivo.³⁰

Previous studies showed that the dysregulation of cell cycle progression and division was a principal element in the development and progression of cancer. The lncRNAs might exert a significant role in cell cycle progression via modulating the expression of critical cell cycle regulators, such as cyclins and cyclin-dependent kinases (CDKs), as well as mediating p53-dependent cell cycle control.^33,34 Our study revealed that knockdown of ZFAS1 caused the G0/G1 phase arrest and correspondingly decreased the percentage of S phase using flow cytometry in glioma cells. Concordant with our results, ZFAS1 in colorectal cancer cells was related to the G0/G1 phase arrest by acting as an interacting partner of CDK1, as confirmed by pull-down experiment and RNA immunoprecipitation. However, silencing ZFAS1 did not influence the levels of CDK1 but could markedly reduce the cyclin B1.²⁵ Actually, the impact of ZFAS1 in the cell cycle and apoptosis had not been fully clarified in our study, which needed to be elucidated by further well-designed studies in the future.

To date, the molecular mechanisms by which lncRNAs promoted tumor migration and invasion were not fully understood. Li et al. reported that amplification of ZFAS1 could promote metastasis through increasing the expression of ZEB1, MMP14, and MMP16 in hepatocellular carcinoma.²⁹ In this study, we found that blocking the expression of ZFAS1 remarkably decreased migration and invasion ability of glioma cells, suggesting the important role of ZFAS1 in glioma metastasis. The EMT process had been exhibited to play a vital role in cancer metastasis.³⁵ In this study, by evaluating the expression of major markers of EMT, we observed that knockdown of ZFAS1 greatly decreased the expression of MMP2, MMP9, N-cadherin, Integrin β1, ZEB1, Twist, and Snail, as well as markedly upregulated E-cadherin level, suggesting that ZFAS1 might promote glioma metastasis via inducing EMT.

In conclusion, our study reported for the first time that ZFAS1 was greatly upregulated in glioma tissues and associated with histological malignancy of glioma. Furthermore, overexpression of ZFAS1 was tightly correlated with a poor prognostic outcome, suggesting that it might be used as an underlying prognostic biomarker and a new potential therapeutic target for the treatment of human gliomas.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was partially supported by the National Natural Science Foundation of China (No.81503166).