LOXL1‐AS1 communicating with TIAR modulates vasculogenic mimicry in glioma via regulation of the miR‐374b‐5p/MMP14 axis

Abstract At present, growing evidence indicates that long non‐coding RNAs (lncRNAs) participate in the progression of glioma. The function of LOXL1‐AS1 in vasculogenic mimicry (VM) in glioma remains unclear. First, the expressions of TIAR, the lncRNA LOXL1‐AS1, miR‐374b‐5p and MMP14 were examined by qRT‐PCR and Western blot in both, glioma tissues and glioma cell lines. Proliferation, migration, invasion and tube formation assays were conducted to evaluate the roles of TIAR, LOXL1‐AS1, miR‐374b‐5p and MMP14 in malignant cellular behaviours in glioma cells. A nude mouse xenograft model and dual staining for CD34 and PAS were used to assess whether VM was affected by TIAR, LOXL1‐AS1 or miR‐374b‐5p in vivo. In this study, low levels of TIAR and high levels of LOXL1‐AS1 were found in glioma cells and tissues. TIAR downregulated the expression of LOXL1‐AS1 by destabilizing it. LOXL1‐AS1 acted like a miRNA sponge towards miR‐374b‐5p so that downregulation of the former greatly inhibited cell proliferation, migration, invasion and VM. Additionally, miR‐374b‐5p overexpression repressed malignant biological behaviours and VM in glioma by modifying MMP14. In summary, we demonstrated that TIAR combined with LOXL1‐AS1 modulates VM in glioma via the miR‐374b‐5p/MMP14 axis, revealing novel targets for glioma therapy.

lung cancer and colorectal cancer. 6-10 VM has a close association with tumour metastasis and is a predictor of poor clinical prognosis. 11,12 RNA-binding proteins (RBPs) participate in tumour development, playing roles in pre-mRNA splicing, translation and RNA stabilization. 13 Increased TIA1-related protein (TIAR) expression represses the proliferation of 293 cell lines and xenograft tumour growth. 14 Additionally, TIAR could prolong the survival of patients with astrocytoma and glioblastoma multiforme, while G3BP1 has the opposite effect. 15 However, the expression of TIAR in glioma and its possible association with progression have not yet been reported.
MicroRNAs (miRNAs) participate in the post-transcriptional processes of tumorigenesis by regulating the 3′-UTR of downstream target genes. miR-374b-5p downregulates ABCA8 and promotes carcinogenesis in hepatocellular carcinoma. 21 Moreover, MiR-374b-5p plays an antitumour role in pancreatic and cervical cancers. 22,23 Nevertheless, we know little about miR-374b-5p expression and its function in VM in gliomas.
Matrix metalloproteinase 14 (MMP14), a member of the membrane-type MMP family, is strongly associated with tumour metastasis. MMP14 also plays a VM-related role in gastric carcinoma, hepatocellular carcinoma and lung cancer. [24][25][26] This study aimed to investigate the expressions of TIAR, LOXL1-AS1 and miR-374b-5p in both, glioma tissues and glioma cells. Further, the functions of TIAR, LOXL1-AS1, miR-374b-5p and MMP14, and their interactions in modulating cellular behaviours and VM in glioma are yet to be established. Our study provides new potential therapeutic targets for glioma therapy.

| Clinical specimens
We obtained 37 tissues in total, according to the WHO classification of central nervous system tumours, including 13 low-grade glioma tissues (LGGTs; WHO I-II), 16 high-grade glioma tissues (HGGTs; WHO III-IV) and 8 normal brain tissues (NBTs). All tissues were from material discarded during surgery for glioma or traumatic brain injury. Ethical approval was obtained for the current research.

| Cell culture
Normal human astrocytes (NHA) along with HEK293T, U87 and U251 cell lines were purchased from the Shanghai Cell Bank affiliated to the Chinese Academy of Life Sciences. NHA were incubated in 1640 medium, while the HEK293T, U87 and U251 cells were incubated in high-glucose medium with 10% foetal bovine serum (Gibco).
The growth conditions were 37°C and 5% CO 2 , together with a certain degree of humidity.

| Quantitative real-time PCR (qRT-PCR)
After the total RNA was extracted according to the manufacturer's manual of Trizol reagent, TIAR, LOXL1-AS1, MMP14 and GAPDH expressions were determined using SYBR One Step RT-PCR kits (Takara). The detections of miR-374b-5p and U6 were performed using the miRNA First Strand cDNA Synthesis kit and the MicroRNA qPCR Kit (Sangon Biotech,). All primers were designed by Sangon Biotech and are presented in Table S1.

| Western blot analysis
After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (0.22 µm). The membranes were immersed in 5% non-fat milk at room temperature for 2 h and then incubated with primary antibodies at 4°C for 16-18 h. The primary antibodies were as follows: anti-TIAR (1:500, Proteintech), anti-MMP14 (1:1000, Proteintech) and anti-GAPDH (1:10000, Proteintech). Next, the membranes were incubated with secondary antibodies at room temperature for 2 h, and bands were detected using enhanced chemiluminescence (ECL) reagents from the ECL Detection System. The relative expressions of the proteins were calculated based on the internal reference, GAPDH. Lipofectamine 3000. To screen for stably expressing transfected cells, we added G418, puromycin or hygromycin B to the medium. The transfection efficiency was determined using qRT-PCR and Western blotting ( Figure S1). To identify the effects of TIAR on biological behaviour and VM in glioma, we divided the cells into five groups: control, TIAR(−)NC, TIAR(−), TIAR(+)NC and TIAR(+). To investigate the corresponding effects F I G U R E 1 TIA1-related protein (TIAR) plays a suppressive role in glioma. A-F. The data are displayed as mean ± standard deviation. A. The TIAR expression in glioma tissues is shown. n = 8, normal brain tissues (NBTs); n = 13, low-grade glioma tissues (LGGTs); n = 18, highgrade glioma tissues (HGGTs); **p < 0.01 vs. NBTs; ##p < 0.01 vs.

| RNA immunoprecipitation (RIP)
Following the manufacturer's protocol, we performed RIP assays in which the whole-cell lysate was incubated with magnetic beads from the EZ-Magna RIP kit (Millipore, Billerica, MA). The beads were conjugated with anti-Ago 2 antibody or normal mouse IgG. After incubation in proteinase K buffer, the immunoprecipitated RNA was extracted and analysed by qRT-PCR.

| Fluorescence in situ hybridization (FISH)
After blocking in prehybridization buffer, slides were disposed with PCR-grade proteinase-k (Roche Diagnostics, Germany). LOXL1-AS1 probe (GenePharma), which was constructed to confirm the localization of LOXL1-AS1 in glioma cells, was added to the hybridization solution.
Afterwards, the sections were stained with anti-digoxin rhodamine conjugate (Exon Biotech Inc.,) at 37°C for 1 h and then DAPI (Beyotime) for 2 min. All images were captured under a fluorescence microscope.

| Dual-luciferase reporter assays
To investigate the interplay of LOXL1-AS1 and miR-374b-5p, HEK293T cells were co-transfected with LOXL1-AS1-Wt or After 48 h, the relative luciferase activity was calculated and analysed using the Dual-Luciferase Reporter Assay System (Promega).

| Nascent RNA assay
Per the manufacturer's protocol, the expression of nascent LOXL1-AS1 was examined using the Click-iT Nascent RNA Capture Kit (Life Technologies Corporation). In general, we used 0.2 mM of ethylene uridine (EU) ribonucleotide homologs to mark the nascent RNA, and the RNA so labelled was released from the magnetic beads and collected. Finally, the nascent RNA expression was determined using qRT-PCR.

| RNA stability assay
After transfection with TIAR(+) and its NC, HEK293 cells were treated with Actinomycin D (5 mg/ml, Sigma-Aldrich), and the total RNA was obtained in real time and detected using qRT-PCR.

| Cell proliferation assay
To assess the cell viability, cells were resuspended and seeded into a 96-well plate. The Cell Counting Kit-8 reagent was used per the instructions of the manufacturer. The cell viability was determined using a microplate reader.

| Cell migration and invasion assays
Migration assay: After re-suspension in a serum-free medium, approximately 1 × 10 5 U87 or U251 cells were seeded into a transwell. Simultaneously, the cell-culture medium was added to the side beneath the transwell chamber. Next, the cells were fixed and stained with 10% Giemsa in phosphate buffer at room temperature overnight. Then, cell numbers were counted in three random fields under a microscope. Modification for invasion assay: We coated 70 μl of Matrigel solution (Corning) with F I G U R E 2 LOXL1-AS1 functions as an oncogene in glioma. A-G. The data are displayed as mean ± standard deviation. A. LOXL1-AS1 expression in glioma tissues; n = 8, normal brain tissues (NBTs); n = 13, low-grade glioma tissues (LGGTs); n = 18, high-grade glioma tissues (HGGTs); **p < 0.01 vs. NBTs; ##p < 0.01 vs.

| Tube formation assay
The 3D model tube was formed using a gel. After U87 and U251 cells were successfully transfected, we resuspended the cells and seeded them into a 24-well plate pre-coated with Matrigel. Tube formation was then observed and analysed in three random fields under a microscope immediately after incubation at 37°C for nearly 6 h.

| CD34 and PAS dual staining
Tissue specimens were fixed in 4% paraformaldehyde and embedded in paraffin, microtomed, and de-paraffinized in xylene and graded ethanol solutions. Specimens were then placed in a citrate antigen retrieval solution and heated in a microwave oven at a temperature controlled at close to the boiling point. After subsequent incubation with 3% hydrogen peroxide and goat serum, the specimens were incubated in CD34 primary antibody at a ratio of 1:100 at 4°C overnight. Subsequently, the specimens were incubated with the secondary antibody at room temperature for 20 min and stained with a DAB kit immediately. The PAS reaction was performed using periodic acid and Schiff staining reagents. The specimens were then permanently preserved with neutral resins and stained with haematoxylin. The number of VM tubes was counted and analysed using a normal microscope.

| Xenograft tumour in nude mice
Glioma cells were transfected and screened for stable expression. An pre-miR-374b-5p and TIAR(+)+LOXL1-AS1(−)+pre-miR-374b-5p. Each group contained ten mice to ensure experimental accuracy. After transfection and re-suspension at a density of 2 × 10 6 /ml, 100 ml of cell suspension was injected subcutaneously into each mouse. The volume of tumours was measured and calculated every four days using the following formula: volume (mm 3 ) = length × width 2 /2. All mice were sacrificed after 44 days. We implanted 1 × 10 5 cells into the right striatum of nude mice in the orthotopic inoculation experiments.
Survival time was analysed using the Kaplan-Meier survival curve.
Importantly, this research was approved by a panel of experts on laboratory animal care and was conducted following the standards for the care and handling of laboratory animals.

| Statistical analysis
All experimental data shown as mean ± standard deviation were analysed using SPSS 22.0. Statistical significance was set at p < 0.05.

| TIAR shows low expression levels in glioma tissues and cells, and its overexpression represses VM by glioma cells
To detect TIAR expression, Western blot analysis was performed.
The results demonstrated that TIAR expression was lower in glioma tissues and cells than those in NBTs ( Figure 1A) and NHA cells ( Figure 1B). To investigate the role of TIAR in glioma, we evaluated the effects of TIAR overexpression and knockdown on cell proliferation, migration, invasion and VM. We observed that MMP14 was

LOXL1-AS1 expression was higher in glioma tissues and cells than in
NBTs and NHA, and the higher the grade of glioma, the higher the expression of LOXL1-AS1 (Figure 2A,B). The FISH experiment confirmed that LOXL1-AS1 was located in the cytoplasm of the NHA and glioma cells ( Figure 2D). To explore the mechanism of action of LOXL1-AS1 F I G U R E 3 TIA1-related protein (TIAR) negatively modulates the stability of LOXL1-AS1 in glioma. A-I. The data are displayed as mean ± standard deviation (n = 3, each group). A. LOXL1-AS1 expression is regulated by TIAR. negative controls (NC); **p < 0.

| miR-374b-5p has an anti-tumour role in glioma, and miR-374b-5p overexpression suppresses VM by glioma cells
miR-374b-5p was expressed at lower levels in glioma tissues and cells than in NBTs and NHA ( Figure 4A,B). To evaluate the function of miR-374b-5p in glioma, we evaluated the effects of pre-miR-374b-5p and anti-miR-374b-5p on cell behaviour and VM potential in glioma.
As shown in Figure 4C, MMP14 was downregulated in the pre-miR-374b-5p group but upregulated in the anti-miR-374b-5p group compared with their NC groups. Moreover, cell proliferation, migration, invasion and VM were suppressed in the pre-miR-374b-5p group but promoted in the anti-miR-374b-5p group in comparison with their NC groups ( Figure 4D-F).

LOXL1-AS1 expression was decreased in the pre-miR-374b-5p
group but increased in the anti-miR-374b-5p group in comparison with the pre-NC and anti-NC groups respectively ( Figure 5B). Figure 5C shows that the MMP14 expression was reduced in the LOXL1-AS1(−)+pre-miR-374b-5p group compared with those of their NCs. Furthermore, the dual-luciferase reporter assay demonstrated that miR-374b-5p binds to LOXL1-AS1 at its 3′-UTR ( Figure 5D,E). To further investigate this interaction, we evaluated the effects of LOXL1-AS1 and miR-374b-5p on cell proliferation, migration, invasion and VM. As shown in Figure 5F-H, cell proliferation, migration, invasion and VM in glioma (henceforth, 'malignant cell behaviour') were greatly inhibited in the LOXL1-AS1(−)+pre-miR-374b-5p group compared with those of the NCs.

| MMP14 has an oncogenic role in gliomas and exerts facilitation in VM
As shown in Figure S3A

| DISCUSS ION
In this study, we showed that TIAR exerted anti-tumour effects, and inhibited the invasion in ovarian cancer. 28 A recent study suggested that the lncRNA MT1JP communicating with TIAR posttranscriptionally regulates P53 in tumours. 29 In our study, TIAR was expressed at low levels in glioma. TIAR overexpression remarkably inhibited proliferation, metastasis and tube formation, while its knockdown tended to act in the opposite direction, confirming its tumour suppression role in glioma.
Malfunctions of lncRNAs play a unique role in oncogenesis and progression. A recent study identified the promotion of lnc_000231 in cervical cancer, which acts by interacting with miR-497-5p. 30 The lncRNA ATB binds to EZH2 and downregulates the expression of DAB2IP, CDH1, LATS2, FOXC1 and CDX1, thus facilitating the progression of ovarian cancer. 31 Furthermore, our study showed that LOXL1-AS1 is highly expressed in glioma tissues and cells, and its downregulation repressed the proliferation, metastasis and VM ability of these cells. These results confirmed that LOXL1-AS1 has an aggressive role in glioma. Silencing LOXL1-AS1 suppresses cell proliferation in glioblastoma, 32 which is consistent with our findings. Moreover, LOXL1-AS1 enhances the proliferation and invasion in medulloblastoma 33 and negatively modulates miR-3128, resulting in attenuation of the malignancy of H1299 and A549 lung cancer cells. 34 However, the relationship between TIAR and LOXL1-AS1 in regulating VM formation in glioma has not been explored.
Several studies have reported that RBPs can positively or negatively affect RNA stability, resulting in tumour growth. For example, LARP1 enhances the stability of BCL2 but attenuates the stability of BIK, leading to the malignant progression of ovarian cancer. 35 Linc-00313, which is stabilized by UPF1, regulates miR-342-3p and miR-485-5p, eventually accelerating the progression of glioblastoma. 36 In colorectal cancer, MBNL1 destabilizes Snail and inhibits the epithelial-to-mesenchymal transition and metastasis of tumour cells. 37 In our study, LOXL1-AS1 expression was reduced in TIARoverexpressing glioma cells and increased in TIAR-knockdown glioma cells. Moreover, the results showed that TIAR communicated sponge miR-486-5p, and its suppression downregulates the malignant progression of colorectal cancer. 39 The lncRNA EBLN3P, functioning as a competitive sponge of miRNA-144-3p, positively modulates DOCK4 in the ceRNA pathway and facilitates adverse processes in liver cancer. 40 To confirm the function of LOXL1-AS1 in glioma, we used Starbase and predicted miR-374b-5p as a target of LOXL1-AS1.
These results indicate that LOXL1-AS1 exerts sponge-like effect on  46 In this study, miR-374b-5p was downregulated in glioma, which revealed its function as a tumour suppressor. Similarly, miR-374b-5p targets FOXP1 and exerts a protective role in non-small-cell lung cancer and ovarian cancer. 47,48 Additionally, both miR-374b-5p and miR-454-3p exert inhibitory effects by regulating ZEB2 in bladder cancer. 49 Moreover, miR-374b targets FOXM1, and its overexpression mediates adverse effects in cervical cancer. 50 We also proved that MMP14 promotes malignant cell behaviours in gliomas. Likewise, MMP14 is highly expressed in gliomas where it acts as a mediator of migration. 51 We also noticed that miR-374b-5p restrained MMP14 by binding to its 3′UTRs according to the dual-luciferase reporter assay and Western blot, leading to the inhibition of VM by glioma cells. Similarly, MMP2 and MMP9 function as the key mediator of VM in glioma, while the suppression of Tenascin-c attenuates AKT phosphorylation, and downregulates MMP2 and MMP9 expression, thus repressing VM in gliomas. 52 Additionally, the upregulation of MMP14 reduced the inhibitory effects of overexpressed miR-374b-5p in glioma cells.
Overall, our study shows for the first time that TIAR plays a tumour-suppressive role, while LOXL1-AS1 plays an aggressive role in glioma. TIAR downregulates LOXL1-AS1 by reducing its stability.
LOXL1-AS1 overexpression reversed the anti-tumour effects of TIAR overexpression in glioma cells. Moreover, LOXL1-AS1 knockdown upregulated miR-374b-5p, leading to the downregulation of MMP14, eventually suppressing malignant cell behaviour and VM in glioma cells. Our study shows that the TIAR/LOXL1-AS1/miR-374b-5p/ MMP14 axis has significant effects in regulating VM in glioma, which could help reveal novel targets for glioma therapy.

ACK N OWLED G EM ENTS
Our research is supported by Liaoning Science and Technology Plan Project (ME86); outstanding scientific fund of Shengjing hospital (M0214, M0167).

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

DATA AVA I L A B I L I T Y S TAT E M E N T
We confirm that we will share the data underlying the findings reported in this manuscript and allow researchers to verify the results presented, replicate the analysis, and conduct secondary analyses.