Tumor specimens were obtained from patients who were initially diagnosed with GBM and underwent their first surgical procedure between December 2016 and May 2020 at Shanghai Changzheng Hospital, Naval Medical University (Shanghai, China). The median age of patients was 49 (range, 34-65), comprising 15 men and 15 women. Normal brain tissues were obtained from craniocerebral trauma patients who received intracranial decompression therapy between January 2018 and May 2020 at the aforementioned hospital. The median age of patients was 41 years (range, 29-57). The present study was approved (approval no. CZEC2018-032) by the Independent Ethics Committee of Shanghai Changzheng Hospital (Shanghai, China). Written informed consent was obtained from all patients or their legal guardians indicating their understanding of the risks and benefits.

lncRNA microarray data were obtained from the authors' previous study (GEO dataset: GSE51146) (21). Differentially expressed lncRNAs were identified by using Volcano Plot filtering. The threshold of upregulated and downregulated lncRNAs was P<0.05 and a fold change >2.

Human cell lines U87MG (RRID: CVCL_0022), U251MG (RRID: CVCL_0021) and 293T (DSMZ no. ACC 635) were purchased from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). In order to authenticate these cell lines, STR profiling was conducted in the year 2020; the identities of these cell lines were confirmed and authenticated. The U87 cell line used in the present study is most probably an ATCC-originating version of the U87 MG cell line. All these cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (both from Hyclone; Cytiva) and 5% CO₂ at 37°C.

Total RNA was extracted from tissues and cells using TRIzol reagent (Wuhan Servicebio Technology Co., Ltd.). RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using FastStart Universal SYBR Green Master (Roche Diagnostics) on the RT-PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: The procedure involved 40 cycles, which consisted of denaturation at 98°C for 10 sec, annealing at 58°C for 25 sec and extension at 68°C for 30 sec. GADPH was marked as an internal control. The primers used were as follows: GAPDH forward, 5′-GGA AGC TTG TCA TCA ATG GAA ATC-3′ and reverse, 5′-TGA TGA CCC TTT TGG CTC CC-3′; UBD forward, 5′-CAA TGC TTC CTG CCT CTG TGT-3′ and reverse, 5′-GGG TAA GGT GGA TGG TCT TCT CT-3′; and UBDP1 forward, 5′-TGG CTG CTA AAA TGG AGT GAA GA-3′ and reverse, 5′-AGG TGA GGT GGA TGG TCT TCT T-3′. The expression levels were determined using the 2^−ΔΔCq method (22).

The cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed with PBS, treated with pepsin (1% in 10 mM HCl), and dehydrated with 70, 85 and 100% ethanol. The cells were then air-dried and incubated in a hybridization buffer containing the FISH probe for 5 min at 73°C in a water bath. The hybridization was performed for 12 h at 37°C. After washing and dehydrating the cell slides, they were counterstained with DAPI (2.5 μg/ml). The RNA FISH probe for UBDP1 was designed and synthesized by Shanghai GenePharma Co., Ltd., and its sequence was 5′-FAM-CCT ACC TTC TTC ACT CCA TTT TAG CAG CCA-FAM-3′.

The gene sequences and target sequences of UBD and UBDP1 were constructed into the GV657/GV248 vector plasmid (Shanghai GeneChem Co., Ltd.) by PCR amplification and double enzyme digestion (restriction enzymes, New England BioLabs, Inc.). Thus, GV657/UBD-Overexpression, GV657/UBDP1-Overexpression, GV248/UBD-Knockdown and GV248/UBDP1-Knockdown plasmids were obtained. GV657/GV248 vector plasmids (Shanghai GeneChem Co., Ltd.) were used as negative control (NC). The sequences of PCR amplification primers were as follows: UBD overexpression forward, 5′-CGG AAT TCA TGG CTC CCA ATG CTT CCT-3′ and reverse, 5′-CGG GAT CCT CAC CCT CCA ATA CAA TAA CAT GCC A-3′; UBD knockdown forward, 5′-CCG GCG AGA CTA AGA CGG GTA TAA TCT CGA GAT TAT ACC CGT CTT AGT CTC GTT TTT G-3′ and reverse, 5′-AAT TCA AAA ACG AGA CTA AGA CGG GTA TAA TCT CGA GAT TAT ACC CGT CTT AGT CTC G-3′; UBDP1 overexpression forward, 5′-CGG AAT TCG TTG GTG ATA CCT ACT TTC ACT GAG-3′ and reverse, 5′-CGG GAT CCG CAT CTC TCT ACC CCT GGG-3′; and UBDP1 knockdown forward, 5′-CCG GTC TGG TGG AAA CAT GTG ATC AAG CTT CAT CAC ATG TTT CCA CCA GAT TTT TG-3′ and reverse, 5′-AAT TCA AAA ATC TGG TGG AAA CAT GTG ATG AAG CTT GAT CAC ATG TTT CCA CCA GA-3′.

The gene sequences and target sequences of miR-6072 and miR-6818-3p were constructed into the GV251/GV249 vector plasmid (Shanghai GeneChem Co., Ltd.) by PCR amplification and double enzyme digestion (restriction enzymes, New England BioLabs, Inc.). Thus, the GV251/miR-6072-Overexpression, GV251/miR-6818-3p-Overexpression, GV249/miR-6072-Inhibition and GV249/miR-6818-3p-Inhibition plasmids were obtained. GV251/GV249 vector plasmids (Shanghai GeneChem Co., Ltd.) were used as NC. The sequences of PCR amplification primers were as follows: MiR-6072 overexpression forward, 5′-TGT GGA AAG GAC GCG GGA TCA GAT GCA CAG GAC TGG GCA C-3′ and reverse, 5′-CAG CGG TTT AAA CTT AAG CTA AAA AAT AAG AAC TAC TCT ATG-3′; miR-6072 inhibition forward, 5′-GCT AAA AAT CCT CAT CAC ACT GCA CCT TAG G-3′ and reverse, 5′-ATC CCT AAG GTG CAG TGT GAT GAG GAT TTT T-3′; miR-6818-3p overexpression forward, 5′-ACG GGC CCT CTA GAC TCG AGT GTT GGT TGT GTA AGA TTT C-3′ and reverse, 5′-TTA AAC TTA AGC TTG GTA CCT ACT GAC TGT ACC AGA TGC-3′; and miR-6818-3p inhibition forward, 5′-GCT AAA AAT TGT CTC TTG TTC CTC ACA CAG G-3′ and reverse, 5′-ATC CCT GTG TGA GGA ACA AGA GAC AAT TTT T-3′. The vectors and DNA fragments were then connected using T4 ligase from New England BioLabs, Inc.

U87 and U251 cell lines were cultured to a density of 2×10⁵ cells for transfection purposes. Transfection was performed using 50 pmol of either miRNA mimics or inhibitors. Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) was employed as the transfection reagent, and 5 μl was administered to each well of a 6-well plate, as per the manufacturer's instructions. To prepare the transfection mix, Lipofectamine^® 3000 was first diluted in Opti-MEM Medium (Gibco; Thermo Fisher Scientific, Inc.) and thoroughly mixed. Separately, a master mix of the DNA was prepared by diluting the desired amount of DNA also in Opti-MEM Medium and mixing until being homogeneous. The diluted DNA was then combined with the previously diluted Lipofectamine^® 3000, using a 1:1 ratio, to form a DNA-lipid complex. This complex was allowed to incubate for 20 min at room temperature (37°C) to facilitate the formation of a stable DNA-lipid complex. Subsequently, the complex was administered to the cultured cells. Following transfection, the cells were incubated at 37°C for a period of 48 h prior to conducting any subsequent experiments. For lentiviral transduction, the vectors (GL132/GV248; Shanghai GenePharma Co., Ltd.) were co-transfected with helper in the 2nd generation transfection system into 293T cells. The 293T cells were cultured in DMEM supplemented with 10% FBS at a temperature of 37°C and a CO₂ concentration of 5%. Following a 48-h incubation period at 37°C, the supernatant of the 293T cells were collected and the lentivirus was concentrated by subjecting to centrifugation at 25,000 x g for 2 h at 4°C. The harvested lentiviral vectors were then introduced into the target GBM cells (U87/U251) for transduction. Subsequently, U87/U251 cell lines were plated into a 6-well plate and the cells were cultured until they reach 80% confluence. To generate stable cell lines, GBM cells were transduced with these lentiviral vectors in a milieu containing polybrene (Shanghai GenePharma Co., Ltd.) at a concentration of 5 μg/ml. Then, lentivirus was added and co-cultured with the cells at 37°C for 24 h (Multiplicity of infection, 10). After that, the medium was replaced, and then culture continued in a 5% CO₂ and 37°C incubator for another 48 h. Following a 72-h incubation period, cells underwent a selection process using 2 μg/ml of puromycin (Shanghai GeneChem Co., Ltd.), over a course of 3 days.

Cell lysates were prepared with RIPA buffer (Thermo Fisher Scientific, Inc.). The proteins were separated using 10% SDS-PAGE and then transferred to PVDF membranes (EMD Millipore), and a total of 25 μg protein were loaded per lane. After 2 h of blocking using 5% milk at room temperature, membranes were incubated overnight at 4°C with different primary antibodies (FAT10 Rabbit mAb; 1:1,000; cat. no. 76194; Cell Signaling Technology, Inc.). The membranes were then incubated at room temperature for 1 h with secondary antibodies (Anti-rabbit IgG HRP-linked antibody; 1:10,000; cat. no. 7074; Cell Signaling Technology, Inc.; RRID: AB_2099233), and the bands were detected using a chemiluminescence imager (Syngene) and analyzed with Image Lab™ Software (version 5.1; Bio-Rad Laboratories, Inc.). GAPDH (1:1,000; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.) served as the control.

The proliferation ability of the cells was tested using the Cell Counting Kit-8 assay (CCK-8; Shanghai Yeasen Biotechnology Co., Ltd.), according to the manufacturer's instructions. Cells were seeded in a 96-well plate at a density of 1,000-2,000 each and cultured at 37°C. At the indicated time points (0, 24, 48, 72 and 96 h), 10 μl CCK-8 was added to the plate for 4 h. The optical density at 450 nm was measured consecutively using a microplate reader (Bio-Rad Laboratories, Inc.). Each experiment was performed thrice.

The migration assays were performed using the uncoated plates in Transwell chambers (8-μm diameter pores; Corning, Inc.), while Matrigel-coated plates were used for invasion assays at 37°C for 1 h. The upper chambers contained 2×10⁴ cells/well in serum-free medium, while FBS with 10% serum was loaded into the lower chamber. After 24 h of incubation at 37°C with 5% CO₂ in the humidified incubator, the cells were stained with 0.4% crystal violet at room temperature for 10 min. The remaining cells in the upper chamber were removed with cotton swabs, while the migratory or invasive cells in the lower chamber were counted under a light microscope.

The miRanda tool (http://www.microRNA.org/) was used to predict binding sites between miRNAs, UBD mRNA, and UBDP1, with Score and Energy thresholds of >140 and <-20, respectively. The screened miRNAs targeting UBD and UBDP1 were subsequently matched against databases, including miRWalk 3.0 (http://mirwalk.umm.uni-heidelberg.de/), miRDB (https://mirdb.org/), ENCORI (https://rnasysu.com/encori/), microRNA (https://mirbase.org/), Tarbase (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=tarbase/index) and miRNet (https://www.mirnet.ca/). Then, the miRNA(s) shared by the maximum number of databases and binding with maximum similarity to the UBD mRNA and UBDP1 sequences were identified.

The dual luciferase reporter plasmids [GM-1013FL02 + UBDP1 mutant (MT)/wild-type (WT), GM-1013FL02 + UBD MT/WT] were designed and synthesized by Shanghai GenePharma Co., Ltd. Co-transfection was conducted with Lipofectamine^® 3000 for 48 h. The activities of the firefly and Renilla luciferases were analyzed using a dual luciferase assay kit (cat. no. E1910; Promega Corporation), according to the manufacturer's instructions. The assay was conducted independently in triplicate.

Nude female BALB/cA (RRID: MGI:2160349) mice (age, 4 weeks-old) were purchased from the Shanghai Jihui Laboratory Animal Care Co., Ltd. The nude mice were kept in separate ventilated cages within a controlled environment that was free from pathogens, and they were provided with free access to food and water. The mice were subjected to a 12/12-h light/dark cycle, with the temperature maintained between 20-25°C and humidity levels ranging from 50-80%. At the time of tumor cell injection, each mouse weighed ~18±1.75 g and was 6 weeks-old. They were divided into three groups (n=9 in each group). The mice were subjected to anesthesia through intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). Subsequently, they were secured in a stereotaxic frame (Kopf Instruments) and placed on a heating pad to ensure temperature maintenance. After careful sterilization, 2 μl PBS containing 1×10⁷ U87 cells were injected using a Hamilton micro-syringe in the mice's parietooccipital median region with the depth 2-3 mm, the whole injection period lasting for 2 min. After injection, the health and behavior of every nude mouse was monitored on a daily basis. The tumor burden in the mice began to adversely affect their quality of life, causing severe difficulties in fundamental activities such as drinking, eating and moving, thereby severely diminishing their comfort. Consequently, to minimize any further distress, all mice were compassionately euthanized using an intraperitoneal injection of pentobarbital (150 mg/kg). This was followed by a secondary verification of euthanasia, employing cervical dislocation to confirm the cessation of life. In total, 60 days after implantation, all mice were euthanized by intraperitoneal injection of pentobarbital and cervical dislocation. Cardiac arrest was then employed to confirm death by the examination of pulse palpation. The overall survival time from tumor implantation to the death of mice was then recorded. Their whole brains were removed when the mice were euthanized and immediately processed for histological evaluation. In the present investigation, the maximum tumor diameter was found to be <2 cm, and the maximum tumor volume was <2,000 mm³. All mice experiments were performed according to the Institutional Guidelines for the Care and Use of Laboratory Animals of the Naval Medical University and were approved by the Animal Care and Experimental Committee of Naval Medical University (approval no. CZEC2018-032; Hefei, China).

The IHC assay was performed as previously described (23). The primary antibody used was Ki-67 Rabbit mAb (1:100; cat. no. 9027; Cell Signaling Technology, Inc.). The tumor tissues sections (5.0-μm-thick) were stained with hematoxylin and eosin (cat. no. G1003; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 5 and 7 min, respectively.

All statistical analyses were performed using GraphPad Prism 6.0 (Dotmatics) and SPSS (RRID:SCR_002865) 18.0 software (IBM Corp.). The unpaired Student's t-test was used to compare the two groups. A one-way ANOVA test followed by Turkey's post hoc test was performed to analyze data among multiple groups. Pearson correlation analysis was applied to determine the linear relationship between the expression of different genes. The survival curves were calculated by the Kaplan-Meier method, and a log-rank test detected the difference. Univariate and multivariate survival analyses used the Cox hazard regression model; univariate analysis factors with P<0.1 were included in the multivariate analysis. Data were presented as the mean ± standard deviation (SD). P<0.05 was considered to indicate a statistically significant difference.

From the authors' previous microarray data (GSE51146), differentially expressed lncRNAs in GBM were searched and eight upregulated and 29 downregulated pseudogenes were identified (Fig. 1A and Table SI). Among these dysregulated pseudogenes, UBDP1 was selected due to its significant expression shifts and unexplored functions in glioma (Fig. 1B). RT-qPCR was then utilized to assess UBDP1 expression in 30 GBM and 15 normal brain tissue samples, confirming a substantial upregulation of UBDP1 in GBM (Fig. 1C). Furthermore, analysis of the UBDP1 gene disclosed a pseudogene with 75% sequence similarity to UBD mRNA, which was also found to be significantly overexpressed in GBM (Fig. 1C), with its levels exhibiting a strong positive correlation with UBDP1 (Fig. 1D).

To elucidate the clinical significance of UBDP1 in GBM, clinical data from patients with GBM were collected, and patients were categorized based on their UBDP1 expression level (high and low) (Table I). When analyzed using the Kaplan-Meier curve and Cox hazard regression model, elevated UBDP1 expression significantly associated with shorter overall survival times (Fig. 1E) and presented as a potential independent risk factor (Tables II and SII). Consistently, high expression of UBD also associated with poor patient prognosis (Fig. 1F). These findings suggested that both UBDP1 and UBD could potentially drive GBM progression.

To investigate the biological roles of UBDP1 and UBD in glioma, stable UBDP1- and UBD-overexpressing or knocking down cell lines [U87 and U251 (RRID: CVCL_1G29) cells] were established, validating their expressions using RT-qPCR and western blotting. Then, a CCK-8 assay was performed to determine the effect of UBDP1 and UBD on proliferation. Overexpression of UBDP1 and UBD increased the proliferation potential of glioma cells, whereas the knockdown of UBDP1 and UBD significantly impeded this capacity (Fig. 2A and B). Furthermore, Transwell and Matrigel assays were conducted to examine whether UBDP1 and UBD are involved in glioma migration and invasion. As indicated in Fig. 2C-F, upregulation of UBDP1 and UBD significantly accelerated glioma cell migration and invasion compared with the control group. By contrast, knockdown of UBDP1 and UBD decreased the number of migratory and invasive cells. These results demonstrated the tumor-promoting capacities of UBDP1 and UBD in glioma cells.

To determine the in vivo function of UBDP1 and UBD, U87 cells, either overexpressing UBDP1 or UBD or serving as a NC, were injected into the brains of nude mice. As expected, elevated levels of UBDP1 and UBD associated with reduced overall survival in xenograft mice relative to the NC group (Fig. 3B). To access tumor characteristics in vivo, additional five mice from each group were euthanized two weeks post-injection. H&E staining of xenograft sections revealed that mice bearing UBDP1 or UBD overexpressing U87 cells exhibited significantly enhanced tumor growth than mice in control group (Fig. 3A). Consistently, an IHC assay confirmed higher Ki-67 levels in xenograft samples overexpressing UBDP1 or UBD compared with the control group (Fig. 3C and D). Collectively, these results suggested that both UBDP1 and UBD contribute to the oncogenic progression of glioma.

The localization of UBDP1 in glioma cells was investigated using FISH and immunofluorescence assays. As demonstrated in Fig. 4A, UBDP1, similar to UBD, was predominantly found in the cytoplasm of U251 and U87 cells, suggesting its potential role as a miRNA sponge (Fig. 4A) (24,25). Several potential miRNA targets for UBDP1 were predicted using online databases (Table SIII) and two targets, miR-6072 and miR-6818-3p, were found to interact with both UBDP1 and UBD mRNA (Fig. 4B and C). To evaluate the influence of these miRNAs on UBD expression, 293T cells were transfected with miRNA mimics or inhibitors, and alterations in UBD protein levels were assessed via western blotting. Compared with the control, miR-6072 mimics were associated with a reduction in UBD levels, whereas miR-6072 inhibitors resulted in increased UBD expression. However, miR-6818-3p mimics presented conflicting results (Fig. 4D). Dual-luciferase reporter gene assays further confirmed that both UBDP1 and UBD were direct targets of miR-6072 (Fig. 4E and F). These findings suggested that miR-6072 plays a pivotal role in the competitive endogenous RNA network involving UBDP1 and UBD.

To verify the hypothesis that UBDP1 functions as a competitive endogenous RNA, sequestering UBD from miRNA-6072 induced degradation, expression of UBD was analyzed in U87 and U251 cells transfected with either a miRNA-6072 mimic or an inhibitor. The results revealed that overexpression of miRNA-6072 significantly decreased the level of UBD in both glioma cells, whereas its inhibition increased the UBD expression (Fig. 5A). Additionally, the UBD expression was detected in U87 and U251 cells transfected with UBDP1 overexpression or knockdown vector and it was found that the level of UBD was markedly elevated in UBDP1-overexpressing glioma cells and notably decreased in UBDP1-knockdown cells (Fig. 5B). Moreover, co-transfection of UBDP1 and miR-6072 demonstrated that miR-6072 reversed the protein-level upregulation of UBD mediated by UBDP1 (Fig. 5C). UBDP1 was overexpressed in U87 and U251 glioma cells to determine the role of the UBD-miR-6072-UBDP1 network in glioma progression. Transfection with miR-6072 mimics counteracted the increased proliferative ability of UBDP1-overexpressing cells (Fig. 5D). Similarly, miR-6072 mimics diminished the enhanced invasion and migration capabilities prompted by UBDP1 (Fig. 5E and F). These results confirmed that UBDP1 can competitively bind to miR-6072.