An upstream open reading frame regulates vasculogenic mimicry of glioma via ZNRD1‐AS1/miR‐499a‐5p/ELF1/EMI1 pathway

Abstract Increasing evidence has suggested that gliomas can supply blood through vasculogenic mimicry. In this study, the expression and function of ZNRD1‐AS1‐144aa‐uORF (144aa‐uORF) and some non‐coding RNAs in gliomas were assessed. Real‐time quantitative PCR or Western blot was used to discover the expression of 144aa‐uORF, ZNRD1‐AS1, miR‐499a‐5p, ELF1 and EMI1 in gliomas. In addition, RIP and RNA pull‐down assays were applied to explore the interrelationship between 144aa‐uORF and ZNRD1‐AS1. The role of the 144aa‐uORF\ZNRD1‐AS1\miR‐499a‐5p\ELF1\EMI1 axis in vasculogenic mimicry formation of gliomas was analysed. This study illustrates the reduced expression of the 144aa‐uORF in glioma tissues and cells. Up‐regulation of 144aa‐uORF inhibits proliferation, migration, invasion and vasculogenic mimicry formation within glioma cells. The up‐regulated 144aa‐uORF can increase the degradation of ZNRD1‐AS1 through the nonsense‐mediated RNA decay (NMD) pathway. Knockdown of ZNRD1‐AS1 inhibits vasculogenic mimicry in glioma cells by modulating miR‐499a‐5p. At the same time, miR‐499a‐5p is down‐regulated and has a tumour‐suppressive effect in gliomas. In addition, ZNRD1‐AS1 serves as a competitive endogenous RNA (ceRNA) and regulates the expression of ELF1 by binding to miR‐499a‐5p. Notably, ELF1 binds to the promoter region of EMI1 and up‐regulates EMI1 expression, while simultaneously promoting vasculogenic mimicry in glioma cells. This study suggests that the 144aa‐uORF\ZNRD1‐AS1\miR‐499a‐5p\ELF1\EMI1 axis takes key part in regulating the formation of vasculogenic mimicry in gliomas and may provide a potential target for glioma treatment.


| INTRODUC TI ON
Gliomas occupy 80% of the malignant primary tumours of the human central nervous system, and their prognosis is extremely poor. 1 Glioma is an angiogenic solid tumour; its malignant progression relies on the formation of blood vessels in tumour tissue. 2 Although some advances have been made in anti-angiogenic therapy applied to glioma, 3 the effect is still insufficient to be relied upon as a primary therapy. 4 One of the limiting factors of anti-angiogenesis is related to the generation of vasculogenic mimicry (VM) in glioma cells. The generation of VM is correlated with the pathological grade of a given glioma and promotes the proliferation, migration and invasion in glioma cells. 5 Therefore, a new mechanism to reveal the VM of glioma cells contributes to the study of the developmental mechanisms of glioma and may contribute new ideas to the remedy for glioma.
At the translational level, the regulation of gene expression depends primarily on the specific structure and function of the 5′ and 3′ untranslated regions (UTR) of mRNA. 6 Upstream open reading frames (uORFs), commonly found in the 5′UTR of eukaryotic genes, are of importance in the initiation of translation. 7 Studies have reported that uORFs are present in 44%-49% of human genes and uORFs in a gene activate nonsense-mediated RNA decay (NMD), which promotes the degradation of the gene. 8,9 In this study, an ORF finder was used to predict the presence of a uORF. This was accomplished by encoding a length of 144 amino acids in the 5′UTR of ZNRD1-AS1. The main point of our study was to explore the regulatory mechanism and effect of ZNRD1-AS1-144aa-uORF (144aa-uORF) on VM in glioma.
A long non-coding RNA (lncRNA) is an RNA >200 nucleotides in length and participates in the development of tumours such as glioma. 10 ZNRD1 (Zinc ribbon domain-containing 1) is located on chromosome 6p21.3. In its upstream region, there is a lncRNA gene that encodes the ZNRD1 antisense RNA, ZNRD1-AS1, which is located on chromosome 6p22.1. Emerging evidence has confirmed that ZNRD1-AS1 expression is great in lung cancer tissues, and that ZNRD1-AS1 and its functional Cis-eQTL promote lung cancer. 11 Presently, there is no report on the expression of ZNRD1-AS1 in glioma and its involvement in functional regulation.
MicroRNAs (miRNAs/miRs), which are comprised of 18-25 nucleotides, are a kind of small non-coding RNA molecules. As regulators of gene expression, miRNAs cause mRNA degradation and/or inhibit protein translation by binding to the 3′UTR of the target gene directly. 12,13 miR-449a-5p is located on the chromosome 5q11.2.
Recent studies have reported that miR-499a-5p acted a tumour-suppressive role in gliomas, inhibited migration and invasion of glioma cells and promoted apoptosis. 14 There is a class of RNA molecules that regulate the action of miRNAs in RNA regulatory networks. For instance, transcripts, such as those from lncRNA, may regulate each other's expression levels and functions through competitive binding of miRNA recognition elements to miRNA. These RNA molecules are called competitive endogenous RNA (ceRNA). 15 Therefore, whether ZNRD1-AS1, which is abnormally expressed in glioma cells, can act as a ceRNA to regulate glioma VM, has attracted the attention of the authors. E74 like ETS transcription factor 1 (ELF1), which belongs to the ETS family, is located in the 13q13.3-13q14.11 region. Its encoded protein is mainly expressed in lymphocytes, and it acts as an enhancer that regulates the transcriptional expression of various genes as well as the development of breast cancer, melanoma and cervical cancer. [16][17][18] ELF1 expression is abnormal in many tumours. For example, in endometrial and ovarian cancer, ELF1 is vastly expressed and has a positive correlation with pathological grades and poor prognosis. 19,20 Early mitotic inhibitor-1 (EMI1), also named as FBXO5 (F-box only protein 5), is a key cell cycle regulator. Studies have proposed that, in hepatocellular carcinoma tissues and cells, EMI1 was excessive, promoted cells proliferation and was negatively correlated with the patients' prognosis. 21 In breast cancer tissues and cells, expression of EMI1 was increased and was positively associated with poor prognoses. 22,23 It was highly expressed in ovarian clear cell carcinoma tissues and participated in the regulation of its development and progression. 24 At present, the role of ELF1 and EMI1 in VM formation has many unknowns.
Our study first identified the endogenous expression of ZNRD1-AS1-144aa-uORF, ZNRD1-AS1, miR-499a-5p, ELF1 and EMI1 in tissues and cells of glioma and further studied the above-mentioned intermolecular regulatory relationship, and the role and mechanism of biological behaviours such as VM formation in gliomas.
The research aims to provide a new basis for the occurrence and development of glioma and also point out fresh targets for molecular-targeted glioma therapy.

| Human tissue samples
Human glioma tissues and normal brain specimens were taken from at Shengjing Hospital of China Medical University. We received informed consent from the participating patients, and this experiment was approved by the Ethics Committee of Shengjing Hospital of China Medical University. Specimens were cryopreserved in liquid nitrogen instantly after surgery. According to the WHO classification of tumours in the central nervous system (2007) by neuropathologists, the obtained glioma tissues were classified into low-grade glioma (WHO I-II: n = 12) and high-grade glioma (WHO III-IV: n = 12).
Negative controls were those from normal brain tissue obtained from patients with traumatic brain surgery without any significant brain and brain diseases.

| Cell culture
The Cell Resource Center of the Shanghai Institute of Biological Sciences provides paid for human glioma cell lines (U87 and U251) and human embryonic kidney cell line (HEK293T). About 10% foetal bovine serum (FBS, Gibco) was added to Dulbecco's modified Eagle medium (DMEM) for culture of U87 and HEK293T cells, and 10% foetal bovine serum was added to DMEM/F12 medium for culture of U251 cells. The humidified incubator was set to 37°C with 5% CO2 and was applied to cell culture.

| RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA of brain tissue samples and HA, U87 and U251 cells were extracted by TRIzol reagent (Life Technologies Corporation). The Nanodrop spectrophotometer (ND-100, Thermo) was adjusted to a ratio of 260/280 nm for measuring RNA concentration. Primers for ZNRD1-AS1, ELF1, EMI1, GAPDH, miR-499a-5p and U6 were designed and synthesized by Thermo Fisher. qRT-PCR detection of ZNRD1-AS1, ELF1, EMI1 and GAPDH was achieved utilizing a one-step SYBR PrimeScript RT-PCR kit (Takara Bio, Inc). cDNA from miRNA was synthesized by utilizing TaqMan miRNA Reverse Transcription kit (Applied Biosystems). qRT-PCR detection of miR-499a-5p and U6 (Applied Biosystems) was achieved utilizing TaqMan Universal Master Mix II. After the expression levels were normalized, the relative quantification (2 −ΔΔC t ) was calculated. The sequences of primers and probes were listed in Table S1.

| Western blot analysis
RIPA buffer is used to lyse cells on ice to obtain total protein. The SDS-PAGE gel was used for electrophoresis of an equal amount of various proteins, followed by electrophoresis of the target protein onto the PVDF membrane. Incubate in Tween-Tris buffered saline (TTBS) embracing 5% skim milk for 2 hours at room temperature then incubate with primary antibody as follows: ZNRD1-AS1-144aa-uORF (1:1000, Beijing Protein Innovation), ELF1 (1:500, Santa Cruz), EMI1 (1:500, Proteintech) and GAPDH (1:5000, Proteintech). After washing with TTBS three times, it was placed in a secondary antibody (goat anti-rabbit or goat anti-mouse, 1:4000; Proteintech Group).

| In vitro VM tube formation assay
About 350 μL of Matrigel solution was placed in each well of a 24-well plate and placed at 37°C until it solidified. About 500 μL of serumfree medium containing 3 × 10 5 cells was injected into all wells and nurtured at 37°C. After 24 hours, photographs were obtained under an inverted microscope (Olympus), and three fields of view were randomly selected to calculate the number of VM tube structures.

| CD34-periodic acid-schiff (PAS) dual staining
Paraffin-embedded tissue sections were deparaffinized using xylene, soaked in gradient ethanol and then heated to boiling in an EDTA antigen non-masking reagent. The cooled tissue specimens were wetted with peroxide, blocked with goat serum and nurtured at 4°C using CD34 primary monoclonal antibody (1:50, Proteintech).
After 16 hours, tissue specimens were incubated with the secondary antibody for at 37°C 10 minutes. Staining was performed using a DAB kit (MaiXin Biotech), a periodic acid solution, a Schiff solution and haematoxylin. The VM density was calculated by randomly selecting three regions under the microscope.

| Nascent RNA capture
The nascent was detected using a Click-iT ® NascentRNA Capture Kit (Thermo Fisher Scientific) according to the instructions. Briefly, nascent RNA was labelled with 0.2 mmol/L 5-ethynyl uridine (EU), followed by magnetic beads to capture EU-neonatal RNA. qRT-PCR was performed to detect target RNA.

| RNA stability assay
For the purpose of inhibiting the synthesis of de novo RNA synthesis, actinomycin D (5 μg/mL) was added to the cell culture medium. Total RNA was obtained at 0, 1, 2, 3, 4 and 5 hours, and its content was assessed through qRT-PCR. The ratio of the level of RNA at a certain point in time to the level of zero time-point is used to determine the half-life.

| Statistical analysis
All data were expressed as mean ± SD and from at least three independent experiments. Statistical analysis of Student's t test or oneway ANOVA was executed by GraphPad Prism v5.01 (GraphPad Software) software. When P < .05, the difference was deemed to be statistically significant.

| miR-499a-5p is reduced in glioma tissues and cells, and ZNRD1-AS1 binds to miR-499a-5p to regulate VM formation
The results of miRNA microarray analysis confirmed that miR-499a-5p was significantly up-regulated in glioma cells with ZNRD1-AS1 knockdown, indicating that miR-499a-5p may be involved in the regulation of glioma cells induced by ZNRD1-AS1 ( Figure S1). The statistics confirmed that the expression of miR-499a-5p in glioma tissues and cells was higher than in NBTs and NHA ( Figure 3A,B).

| ELF1 is up-regulated in glioma tissues and cells, and miR-499a-5p binds to ELF1 to regulate VM formation
In glioma tissues and cells, the ELF1 expression was detected by qRT-PCR and Western blot. As shown in Figure 4A group was not statistically significant ( Figure 4I).

| ZNRD1-AS1 competitively binds to miR-499a-5p and negatively regulates the inhibitory effect of miR-499a-5p on ELF1
To verify whether ELF1 participates in the management of gli-
The data suggest 144aa-uORF played a role as a tumour suppressor gene. Similar to our results, the low expression of NR2C2-47aa-uORF in gliomas inhibited cell malignant biological behaviour, but promoted apoptosis. 26 LncRNAs are a sort of non-coding RNAs with multiple regulatory functions. Earlier studies have shown that lncRNAs participate in regulating the occurrence, development and F I G U R E 7 The stable expressing cells were used for tumour xenografts study in vivo. A, The nude mice sample tumour from respective group was shown. B, Tumour growth curves in nude mice were shown. Tumour volume was calculated every 7 d after injection, and tumour was excised after 46 d; data are presented as mean ± SD (n = 8, each group). * P < .05, ** P < .01 vs ZNRD1-AS1(−)NC+144aa-uORF(+) NC+ELF1(+)NC group, # P < .05 vs ZNRD1-AS1(−) group, & P < .05 vs 144aa-uORF(+) group, & P < .05 vs ELF1(−) group. C, The survival curves of nude mice that were injected into the right striatum were shown (n = 8, each group). D, CD34-PAS staining was used to detect the VM in xenografted tumour. Data are presented as mean ± SD (n = 3, each group). Scale bars indicate 20 μm. * P < .05, ** P < .01 vs ZNRD1-AS1(−) NC+144aa-uORF(+)NC+ELF1(+)NC group, # P < .05 vs ZNRD1-AS1(−) group, & P < .05 vs 144aa-uORF(+) group, & P < .05 vs ELF1(−) group VM formation of various tumours, including gastric cancer, hepatocellular carcinoma and gliomas. [27][28][29] This study found that expression of ZNRD1-AS1 was elevated in glioma tissues and cells, and knockdown of ZNRD1-AS1 inhibited glioma cell VM. As was reported earlier, SNHG16 and linc00667 are increased in gliomas, which enhances cell VM formation ability. 30 35 The 5′UTR uORF can be regarded as a premature termination codon (PTC), 36,37 which promotes the degradation of lncRNA by activating NMD when the uORF is translated. 38 The NMD pathway acts as a regulatory mechanism for RNA by promoting the degradation of PTC-containing RNA. 39 The process of uORF activating NMD can be summarized as follows: when uORF is translated, its termination codon is recognized as a PTC by stagnate ribosomes, while factors such as UPF1 and SMG1 are recruited to stagnant ribosomes to form the SURF complex (SMG1-UPF1-eRF1-eRF3). Subsequently, UPF1 was activated by UPF2 and other factors to promote the degradation of RNA. 40 In this study, RIP and RNA pull-down showed that UPF1 binds to ZNRD1-AS1, indicating that ZNRD1-AS1 was the target of the NMD pathway. When 144aa-uORF is up-regulated, the respective inhibition of UPF1, UPF2 and SMG1 (key factors in the NMD pathway) reverses the inhibitory effect of 144aa-uORF up-regulation on ZNRD1-AS1. These results showed that the mechanism underlying the inhibition of the stability and expression of ZNRD1-AS1 by the 144aa-uORF was dependent on the NMD pathway. The above results suggested that ZNRD1-AS1-144aa-uORF could reduce the stability of ZNRD1-AS1 and its expression by activating the NMD pathway, thereby inhibiting the formation of cell VM. Related studies have clarified that the uORF of CDKN1A mRNA could activate the NMD pathway, reduce the stability of CDKN1A and inhibit its expression. 8 Our current data clarified that miR-499a-5p was decreased, and overexpression of miR-499a-5p could negatively regulate glioma cell proliferation, migration, invasion and VM. The knockdown of miR-499a-5p had the opposite effect. Some similar studies have found that miR-499a-5p acts as a tumour suppressor in a variety of cancers.
In oesophageal cancer, miR-499a-5p inhibits cell proliferation and promotes apoptosis. 41 miR-499a-5p is underexpressed in non-small cell lung cancer, and overexpression of miR-499a-5p inhibits cell proliferation and promotes apoptosis. 42 In hepatocellular carcinomas, overexpression of miR-499a-5p inhibits cell migration and invasion. 43 In our study, ZNRD1-AS1 and miR-499a-5p had opposite effects on the biological behaviour of glioma cells. The potential binding sites between miR-499a-5p and ZNRD1-AS1 were predicted by biological information analysis, and the binding effect was proved by a dual-luciferase reporter gene. We further proposed that ZNRD1-AS1 bound and negatively regulated miR-499a-5p expression in a RISCdependent manner. Knockdown of miR-499a-5p reversed the inhibitory effect of ZNRD1-AS1 knockdown on VM formation in glioma cells. ZNRD1-AS1 promoted glioma cell VM by targeting miR-499a-5p and inhibiting its expression. Another study reports that SNHG5 binds to miR-32 in gastric cancer and inhibits the expression of miR-32 in a RISC-dependent manner, enhancing the ability of cell proliferation and migration. 44 In the current experiment, it was elucidated that ELF1 was largely abundant in gliomas. Up-regulation of ELF1 enhanced the VM ability of glioma cell, whereas knockdown of ELF1 resulted in the opposite. The data argued that ELF1 might have a function in promoting oncogenic genes in gliomas. Interestingly, ELF1 is increased in nonsmall cell lung cancer (NSCLC) and further cell metastasis, invasion and VM. 45 The binding site between miR-499a-5p and ELF1 3′UTR was found by a dual-luciferase reporter gene. Up-regulation of miR-499a-5p decreased the expression of ELF1. Similarly, in osteosarcoma, miR-214-5p targets the ROCK1 3′UTR, and up-regulation of miR-214-5p reduces ROCK1 protein expression, thereby inhibiting cell proliferation and migration. 46 miR-486-3p directly targets DDR1 mRNA 3′-UTR to inhibit DDR1 expression, decreasing cell proliferation and enhancing apoptosis in oral cancer. 47 A large body of evidence suggests that mRNAs and non-coding RNAs (like lncRNAs, pseudogenes, and circRNAs) can act as ceRNAs; they can competitively bind to microRNAs through miRNA response elements (MREs) and induce the development of human diseases including cancer. 48,49 We predicted that ELF1 and ZNRD1-AS1 had the same MRE sequence: AGUCUUA (AGTCTTA), which competes to bind with miR-499a-5p. Therefore, ZNRD1-AS1 can be considered as a ceRNA to compete with miR-499a-5p, to reduce its negative regulation of ELF1 and promote the formation of VM in gliomas.
Similarly, FAM225A exerts a ceRNA effect and competes for binding to miR-590-3p and miR-1275, thereby reducing their inhibitory effect on ITGβ3 and promoting proliferation and invasion of nasopharyngeal carcinoma cells. 50 In gallbladder carcinoma, PVT1 competitively binds to miR-143, reduces miR-143's inhibitory effect on HK2 and promotes cell proliferation and migration. 51 The increasement of EMI1 contributes to the development of human solid tumours and chromosomal instability. 52 Our results revealed EMI1 expression increased in gliomas, and knockdown of EMI1 reduced cell VM formation. Similarly, EMI1 is abundant in squamous cell lung cancer tissues and cells, down-regulating EMI1 promotes apoptosis, and EMI1 is positively correlated with tumorigenesis and poor prognoses of squamous cell lung cancers. 53 Down-regulation of EMI1 interferes with DNA synthesis in varieties of tumour, such as glioblastoma and breast cancer, inhibiting cancer cell growth and enhancing its sensitivity to chemotherapy and radiation therapy. 54 JASPAR software predicted and confirmed by ChIP experiments that ELF1 bound to the EMI1 promoter region. Up-regulation of ELF1 enhanced EMI1 mRNA and protein expression, whereas down-regulation of ELF1 showed the opposite effects. A previous study indicates that in MCF-7 breast cancer cells, ELF1 plays a transcriptional regulatory role, binding to the human Pygopus2 promoter region and activating its expression, promoting breast cancer cell growth. 55 Our study confirmed that EMI1 was affected by ELF1 transcriptional regulation and was involved in the regulation of glioma cell VM mediated by ZRND1-AS1/miR-499a-5p/ELF1. We first proofed degradation of ZNRD1-AS1 by low expression ZNRD1-AS1-144aa-uORF was weakened though the NMD pathway, which increased the expression of ZNRD1-AS1, overexpressed ZNRD1-AS1 competitively bound to miR-499a-5p and reduced the negative regulation of ELF1 by miR-499a-5p. ELF1 expression was increased, and its transcriptional regulation activated EMI1 expression, promoting cell proliferation, migration, invasion and VM formation in gliomas. This study revealed a novel mechanism by which the ZNRD1-AS1-144aa-uORF regulated vasculogenic mimicry in the gliomas via the ZNRD1-AS1/miR-499a-5p/ELF1/EMI1 pathway, providing a new therapeutic strategy for gliomas.

ACK N OWLED G EM ENTS
This work was supported by grants from the Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.