U3 snoRNA‐mediated degradation of ZBTB7A regulates aerobic glycolysis in isocitrate dehydrogenase 1 wild‐type glioblastoma cells

Abstract Aims The isocitrate dehydrogenase (IDH) phenotype is associated with reprogrammed energy metabolism in glioblastoma (GBM) cells. Small nucleolar RNAs (snoRNAs) are known to exert an important regulatory role in the energy metabolism of tumor cells. The purpose of this study was to investigate the role of C/D box snoRNA U3 and transcription factor zinc finger and BTB domain‐containing 7A (ZBTB7A) in the regulation of aerobic glycolysis and the proliferative capacity of IDH1 wild‐type (IDH1WT) GBM cells. Methods Quantitative reverse transcription PCR and western blot assays were utilized to detect snoRNA U3 and ZBTB7A expression. U3 promoter methylation status was analyzed via bisulfite sequencing and methylation‐specific PCR. Seahorse XF glycolysis stress assays, lactate production and glucose consumption measurement assays, and cell viability assays were utilized to detect glycolysis and proliferation of IDH1WT GBM cells. Results We found that hypomethylation of the CpG island in the promoter region of U3 led to the upregulation of U3 expression in IDH1WT GBM cells, and the knockdown of U3 suppressed aerobic glycolysis and the proliferation ability of IDH1WT GBM cells. We found that small nucleolar‐derived RNA (sdRNA) U3‐miR, a small fragment produced by U3, was able to bind to the ZBTB4 3′UTR region and reduce ZBTB7A mRNA stability, thereby downregulating ZBTB7A protein expression. Furthermore, ZBTB7A transcriptionally inhibited the expression of hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA), which are key enzymes of aerobic glycolysis, by directly binding to the HK2 and LDHA promoter regions, thereby forming the U3/ZBTB7A/HK2 LDHA pathway that regulates aerobic glycolysis and proliferation of IDH1WT GBM cells. Conclusion U3 enhances aerobic glycolysis and proliferation in IDH1WT GBM cells via the U3/ZBTB7A/HK2 LDHA axis.


| INTRODUC TI ON
At present, glioblastoma (GBM) is the most malignant intracranial tumor, with high morbidity and mortality rates. 1 in isocitrate dehydrogenase1 (IDH1), which facilitates the classification of GBM into IDH1 wild-type (IDH1 WT ) and IDH1 mutant-type (IDH1 Mut ). IDH1 R132H (IDH1 R132H ) is the most common point mutation. [4][5][6] Active aerobic glycolysis, known as the Warburg effect, is a typical metabolic feature of GBM cells. Glycolysis can provide energy and raw materials for the rapid proliferation of GBM cells. 7,8 Studies have found that the aerobic glycolytic and proliferative capacity of IDH1 WT GBM cells are significantly higher than that of IDH1 Mut GBM cells, and IDH1 WT GBM has been associated with a shortened survival time compared with IDH1 Mut GBM. 6,9 Therefore, it is essential that the molecular mechanisms regulating aerobic gly- It has been reported that the methylation status of CpG islands in DNA promoter regions is closely linked with the expression of small nucleolar RNA (snoRNA). 11 snoRNAs, which are noncoding small RNAs, are located mainly in the nucleus and participate in the tumorigenesis of various tumors. 12,13 For example, SNORD44 is generally expressed at low levels in gliomas; however, the high expression of SNORD44 inhibits GBM proliferation, invasion, and migration and promotes apoptosis. 14 SNORD12B expression is elevated in GBM and promotes glycolipid metabolism and proliferation. 15 U3 belongs to the RNU3 nuclear small RNA family, which is located at the core of the nonhomologous recombinant palindrome sequence on the long arm of chromosome 17 and is closely correlated with tumorigenesis. 16,17 Currently, the differential expression of U3 in IDH1 WT and IDH1 Mut GBM and the mechanisms regulating aerobic glycolysis in IDH1 WT GBM remain unclear.
The transcription factor ZBTB7A (zinc finger and BTB domaincontaining 7A) is a member of the POK (POZ/BTB and Krüppel) protein family and is known to repress target genes by recruiting co-repressors. 18 In osteosarcoma, ZBTB7A represses linc00473 transcription and expression and regulates the sensitivity of osteosarcoma cells to cisplatin chemotherapy. 19 In melanocytes, ZBTB7A transcriptionally represses the expression of the key adhesion protein MCAM and regulates the migratory and invasive abilities of melanoma cells. 20 However, there have been no reports on the role of ZBTB7A in aerobic glycolysis and the proliferative capacity of IDH1 WT GBM cells. Hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA) are key enzymes that regulate aerobic glycolytic energy metabolism. Increased expression of HK2 and LDHA in GBM cells can promote the aerobic glycolytic capacity of cells, which in turn can promote cell proliferation, migration, and invasion. 7,8,21 In the present study, we demonstrated the differential expression of U3 and ZBTB7A in IDH1 WT and IDH1 Mut GBM tissues and cells, as well as the intermolecular interactions between them.
Furthermore, we explored the mechanism responsible for their effects on aerobic glycolysis and the proliferative capacity of IDH1 WT GBM cells. This study identified a new mechanism of tumorigenesis and explored new molecular targets for the treatment of IDH1 WT GBM from the perspective of aerobic glycolysis.

| Clinical specimens
The normal brain specimens and glioma specimens used in this study were collected from the Department of Neurosurgery, Shengjing  Table S1.
For more detailed information, refer to Supporting Information of Materials and Methods.

| Western blot
Western blot was performed as previously described. 22,23 For more detailed information about experimental procedure and antibodies information, please refer to Supporting Information of Materials and Methods.
The qRT-PCR and western blot were utilized to confirm transfection efficacy ( Figure S1). For more detailed information about experimental procedure, please refer to Supporting Information of Materials and Methods.

| Extracellular acidification rate
Extracellular acidification rate (ECAR) assay was performed as previously described. 22,23 For more detailed information about experimental procedure, please refer to Supporting Information of Materials and Methods.

| Glucose utilization and lactate production assays
Glucose utilization and lactate production was measured as previously described. 22,23 For more detailed information about experimental procedure, please refer to Supporting Information of Materials and Methods.

| CCK-8
Cell proliferation was assessed via CCK-8 assay as previously described. 22,23 For more detailed information about experimental procedure, please refer to Supporting Information of Materials and Methods.   Table S4.

| Northern blot
Northern blot procedure was performed with the Signosis High Sensitive miRNA Northern Blot Assay Kit (Signosis Inc., Santa Clara, CA, USA) following the manufacturer's protocol. Total RNA (20 μg) was separated on 10% acrylamide denaturing gel and then blotted onto a nylon Hybond N membrane and analyzed using oligonucleotides probe complementary to U3-miR.

| RNA immunoprecipitation assay
The RNA immunoprecipitation (RIP) assay was performed as previously described. 22

| Dual-luciferase reporter assay
Dual-luciferase reporter assay was performed as previously described. 22,23 For more detailed information about experimental procedure, please refer to Supporting Information of Materials and Methods.

| Tumor xenograft in nude mouse
Animal experiments were performed as previously described. 22,23 The stable transfected and expressing cells were utilized to establish xenograft models in nude mice. For more detailed information about experimental procedure, please refer to Supporting Information of Materials and Methods.

| Statistical analysis
All values are presented as mean ± standard deviation (SD). Each experiment was conducted three times independently. The normality of the data was analyzed using the Shapiro-Wilk test. The data (two groups) were analyzed using the Student's t-test. The data (more than two groups) were analyzed using one-way or two-way ANOVA analysis of variance followed by the Dunnett's multiple comparisons test or Sidak's multiple comparisons test. For data not normally distributed, nonparametric tests were used. Statistical analysis was conducted via GraphPad Prism v8.4, and p value <0.05 was considered statistically significant.

| The glycolytic capacity and proliferation of IDH1 WT GBM cells were significantly higher than those of IDH1 R132H GBM cells
Sanger sequencing was used to confirm that the GBM cell lines, U87 and U251 were IDH1 WT GBM cells ( Figure 1A). IDH1 R132H and IDH1 WT overexpression vectors were stably transfected into GBM cells, and western blot experiment detected the indicated proteins IDH1 R132H and IDH1 WT expression using IDH1 R132H and IDH1 WT antibodies ( Figure 1B). Subsequently, we analyzed the differences in aerobic glycolytic and proliferative capacity among control, empty vector (negative control, NC), IDH1 WT vector, and IDH1 R132H vector transfected U87 and U251 cells. The aerobic glycolytic capacity of IDH1 WT GBM cells was significantly higher than that of IDH1 R132H GBM cells, while overexpression of IDH1 WT did not increase aerobic glycolytic capacity in IDH1 WT GBM cells ( Figure 1C-E). Glucose consumption was significantly higher in IDH1 WT GBM cells than in IDH1 R132H GBM cells, and the overexpression of IDH1 WT increased glucose consumption ( Figure 1F). Similarly, IDH1 WT GBM cells had a significantly higher proliferative capacity than IDH1 R132H GBM cells, and the overexpression of IDH1 WT enhanced cell proliferation capacity ( Figure 1G,H). The above results demonstrated that IDH1 WT GBM cells had higher aerobic glycolytic and proliferative capacity when compared to IDH1 R132H GBM cells. Overexpression of IDH1 WT enhanced cell proliferation but did not increase aerobic glycolytic capacity.

| U3 was highly expressed in IDH1 WT GBM and U3 knockdown inhibited glycolytic capacity and proliferation
Based on data from Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cance r-pku.cn/index.html), we found that U3 was highly expressed in glioma ( Figure 2A). Using qRT-PCR experiments, we found that U3 expression was significantly elevated in glioma tissues, and U3 expression was higher in IDH1 WT glioma tissues when compared to IDH1 R132H glioma tissues ( Figure 2B). Similarly, qRT-PCR experiments revealed that U3 expression was significantly higher in IDH1 WT GBM cells when compared to normal human astrocyte (NHA) cells, and U3 expression was higher in IDH1 WT GBM cells than in IDH1 R132H GBM cells ( Figure 2C). Based on the above results, we constructed U3 knockdown U87 and U251 IDH1 WT GBM cells ( Figure S1A) to investigate their effect on the aerobic glycolytic and proliferative capacity of IDH1 WT GBM cells. Using western blot assay, we found that HK2 and LDHA expression decreased upon U3 knockdown ( Figure 2D), and the extracellular acidification rate (ECAR) assay and colorimetric quantification revealed a significant decrease in the aerobic glycolytic capacity of IDH WT GBM cells upon U3 knockdown ( Figure 2E-G). Colony formation and CCK-8 assays revealed that the proliferative capacity of IDH1 WT GBM cells was also reduced upon U3 knockdown ( Figure 2H,I). Similarly, we observed that U3 knockdown also inhibited glycolytic capacity and proliferation in IDH1 WT GBM cell that overexpressed IDH1 WT vector ( Figure S2).

| Hypomethylation of the CpG island in the promoter region of U3 promoted U3 expression in IDH1 WT GBM
Next, we analyzed the mechanisms that led to the differential expression of U3 in IDH1 WT and IDH1 R132H GBM cells. The CpG island F I G U R E 1 The isocitrate dehydrogenase 1 wild-type (IDH1 WT ) molecular phenotype is correlated with enhanced aerobic glycolysis and proliferation ability. (A) U87 and U251 glioblastoma (GBM) cell lines were identified as IDH1 WT via Sanger sequencing for the IDH1 codon 132. (B) Western blot analysis detected the indicated proteins in control, empty vector (negative control, NC), IDH1 WT vector, and IDH1 R132H vector transfected U87 and U251 cells using IDH1 R132H antibodies and IDH1 WT antibodies. (C) Aerobic glycolytic ability was measured using extracellular acidification rate (ECAR) assay in the abovementioned transfected U87 cell. (D) Aerobic glycolytic ability was measured using ECAR assay in the abovementioned transfected U251 cell. (E) Lactate production was measured in the abovementioned transfected cells. (F) Glucose consumption was measured in the abovementioned transfected cells. (G) Cell viability was analyzed in the abovementioned transfected cells using CCK-8 assay. (H) The proliferation ability of the abovementioned transfected cells was analyzed using a colony formation assay. **p < 0.01 versus NC group; **p < 0.01 versus IDH1 WT (+) group. Data are presented as the mean ± SD of three independent experiments per group, unless otherwise specified. Data were statistically analyzed using one-way analysis of variance (ANOVA). analysis website (http://www.uroge ne.org/methp rimer/) was used to identify the presence of two CpG islands in the U3 promoter region ( Figure 4A). We designed specific primers for pyrosequencing ( Figure 4B) to identify whether there was a difference in CpG1 and CpG2 methylation levels in IDH1 WT and IDH1 R132H GBM cells. We found that the CpG2 methylation status was not significantly different between IDH1 WT and IDH1 R132H GBM cells; however, CpG1, which is closer to the transcription start site (TSS), was hypomethylated in IDH1 WT GBM cells and hypermethylated in IDH1 R132H GBM cells ( Figure 4C). The methylation-specific PCR (MSP) assay also indicated that CpG1 methylation in IDH1 WT GBM cells was lower than that in IDH1 R132H GBM cells ( Figure 4D). At the tissue level, we found that CpG1 methylation in IDH1 WT glioma tissues was lower than that in normal brain tissues (NBTs), and the level of CpG1 methylation in F I G U R E 2 U3 expression was higher in IDH1 WT GBM, and the knockdown of U3 suppressed aerobic glycolysis and proliferation. (A) U3 expression in gliomas based on the GEPIA database (B) U3 expression was detected in normal brain tissues (NBTs, n = 10), IDH1 R132H GBM tissues (n = 10), and IDH1 WT GBM tissues (n = 10) using qRT-PCR. *p < 0.05 versus NBT group; **p < 0.01 versus NBT group; ## p < 0.01 versus IDH1 R132H GBM group. (C) U3 expression in normal human astrocytes (NHA), IDH1 R132H , and IDH1 WT GBM cells as determined by qRT-PCR. **p < 0.01 versus NHA; ## p < 0.01 versus IDH1 R132H GBM. (D) Hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA) protein expression was detected using western blot after U3 knockdown in IDH1 WT GBM cells. (E) Aerobic glycolytic ability was measured after U3 knockdown in IDH1 WT GBM cells using an ECAR assay. (F) Glucose consumption was measured after U3 knockdown in IDH1 WT GBM cells. (G) Lactate production was measured after U3 knockdown in IDH1 WT GBM cells. (H) Proliferation ability was detected after U3 knockdown in IDH1 WT GBM cells using a colony formation assay. (I) Cell viability was detected after U3 knockdown in IDH1 WT GBM cells using a CCK-8 assay. **p < 0.01 versus U3(−)NC group. Data are presented as the mean ± SD of three independent experiments per group, unless otherwise specified. Data were statistically analyzed using one-way ANOVA.

| U3 downregulated ZBTB7A expression via the formation of sdRNA U3-miR and enhanced glycolytic capacity and proliferation in IDH1 WT GBM cells
Several studies have revealed that snoRNA can form snoRNAderived RNA (sdRNA) via its stem-loop structure and is further processed by the Dicer enzyme and bound to the Ago2 protein to exert microRNA-like functions. 26,27 RT-PCR experiments revealed that U3 was able to form the short fragment, sdRNA U3-miR ( Figure 5A), and U3-miR had an increased expression in IDH1 WT GBM cells when compared to IDH1 R132H GBM cells ( Figure 5B). RIP experiments confirmed that U3-miR was able to bind to the Ago2 protein ( Figure 5C).
Knockdown of Dicer in IDH1 WT GBM cells ( Figure S1C,D) and northern blot experiments revealed no significant change in U3 expression, whereas U3-miR expression was significantly reduced ( Figure 5D).
Comparative sequence analysis revealed that U3-miR was highly similar to hsa-miR-496, and by searching and analyzing the starBase database (http://starb ase.sysu.edu.cn/), we found that ZBTB7A was the primary target gene ( Figure 5E). We further investigated whether U3-miR has a microRNA-like function in regulating ZBTB7A expression. We found that U3-miR possessed a sequence complementary to the 3′UTR of ZBTB7A, and the dual-luciferase assay demonstrated that U3-miR could bind to the 3′UTR of ZBTB7A ( Figure 5F). The actinomycin D assay revealed that ZBTB7A mRNA stability increased and ZBTB7A protein expression was elevated upon U3knockdown ( Figure 5G,H). The above results suggest that U3 promotes the degradation of ZBTB7A mRNA and downregulates ZBTB7A protein expression by forming the short fragment sdRNA U3-miR to perform microRNA-like functions. Western blot experiments revealed that ZBTB7A expression was lower in GBM tissues compared with NBTs and ZBTB7A protein expression was significantly lower in IDH1 WT GBM than in IDH1 R132H GBM ( Figure 5I). Furthermore, ZBTB7A protein expression was lower in GBM cells, and ZBTB7A was expressed at significantly lower levels in IDH1 WT GBM cells than in IDH1 R132H

| U3 enhanced glycolytic capacity and proliferation of IDH1 WT GBM cells via the regulation of ZBTB7A expression
On the basis of the knockdown of U3 expression, we interfered with the expression of ZBTB7A and observed cell glycolytic and proliferation capacity. We detected U3 and ZBTB7A expression via qRT-PCR or western blot ( Figure S3). Compared with the U3 knockdown alone group, overexpression of ZBTB7A after U3 knockdown significantly inhibited the expression of HK2 and LDHA proteins ( Figure 6A

| Knockdown of U3 inhibited the growth of subcutaneous xenograft IDH WT GBM tumor and prolonged the survival of nude mice
To further demonstrate the effect of U3 knockdown on the inhibition of IDH1 WT GBM progression, nude mice were randomly divided into four groups: control, U3(−), ZBTB7A(+), and U3(−) + ZBTB7A(+).
The subcutaneous xenograft tumor assay revealed that compared with the control group, the graft tumor volume decreased in the U3(−) and ZBTB7A(+) groups; however, the graft tumor volume was the smallest in the U3(−) + ZBTB7A(+) group ( Figure 8A,B). In addition, we injected IDH1 WT GBM cells into the right striatal area of nude mice and detected differences in the survival periods of these groups. Compared with the control group, the survival period of nude mice in the U3(−) and ZBTB7A(+) groups was significantly longer and the survival period in the U3(−) + ZBTB7A(+) group was the longest ( Figure 8C). Taken together, our results demonstrate the mechanism by which the U3/ZBTB7A/HK2 LDHA pathway promotes aerobic glycolysis and proliferation of IDH1 WT GBM cells ( Figure 8D).

| DISCUSS ION
In this study, we found that the aerobic glycolytic and proliferative capacity of IDH1 WT GBM cells were higher than those of IDH1 R132H It has been reported that IDH1 WT and IDH1 Mut molecular phenotypes are closely correlated with glioma tumorigenesis and patient prognosis. Patients with secondary GBM carrying IDH1 R132H generally have a better prognosis and longer overall survival than patients with IDH1 WT primary glioblastoma (31 months vs. 15 months). Studies have found that the IDH1 molecular phenotype is also closely related to glycolytic energy metabolism in glioma cells, and the aerobic glycolytic capacity of IDH1 WT GBM cells is significantly higher than that of IDH1 R132H GBM cells, 28,29 which is consistent with results obtained in this study. The differential expression of the LDHA protein, a key glycolytic enzyme, in different molecular phenotypes of IDH1 may lead to heterogeneity in glycolytic energy metabolism in glioma cells and may affect glioma cell migration, invasion, and proliferation. 25,30,31 We found that HK2 and LDHA expression was higher in IDH1 WT GBM cells than in IDH1 R132H GBM cells, and the glycolytic and proliferative capacity of IDH1 WT GBM cells were higher than those of IDH1 R132H GBM cells.
U3 belongs to the RNU3 gene family of noncoding snoRNAs that cause genomic instability and increase susceptibility to genetic rearrangements, which play crucial roles in tumorigenesis and tumor progression. 16 Studies have shown that U3A increases the sensitivity of breast cancer cells to 5-FU chemotherapy by upregulating UMPS expression. 17 Furthermore, U3 is highly expressed in osteosarcoma cells and promotes cellular resistance to doxorubicin chemotherapy. 32 It has also been reported that SNORD3B-1 is highly expressed in hepatocellular carcinoma and is utilized as a diagnostic molecular marker for early-stage hepatocellular carcinoma and AFP-negative hepatocellular carcinoma. 33 This study revealed higher U3 expression in IDH1 WT GBM cells than in IDH1 R132H GBM cells. Furthermore, the knockdown of U3 inhibited the aerobic glycolytic and proliferative capacity of IDH1 WT GBM cells, suggesting that U3 exerts a pro-oncogenic role via its involvement in abnormal energy metabolism in IDH1 WT GBM cells. snoRNAs can form short RNA fragments of sdRNA via stemloop structures. The sdRNAs formed by snoRNAs were found to be of either of two lengths of 17-19 nts or greater than 26 nts. 34,35 sdRNAs are processed by the Dicer enzyme and bind to the Ago2 protein to perform microRNA-like functions. 34,36,37 Studies have shown that SNORA-93 regulates pipox expression and promotes breast cancer cell invasion by generating miRNA-like sdRNA-93. 38 Furthermore, in prostate cancer, the high expression of sdRNA-D19b and sdRNA-A24 enhances prostate cancer cell proliferation, metastatic ability, and resistance to chemotherapy by regulating F I G U R E 7 ZBTB7A transcriptionally regulated HK2 and LDHA expression. (A) HK2 mRNA expression was analyzed after ZBTB7A overexpression or knockdown via qRT-PCR in IDH1 WT GBM cells. (B) LDHA mRNA expression was analyzed after ZBTB7A overexpression or knockdown via qRT-PCR in IDH1 WT GBM cells. **p < 0.01 versus ZBTB7A(+)NC group; ## p < 0.01 versus ZBTB7A(−)NC group. (C) Diagram showing ZBTB7A binding site of HK2 promoter (above). Chromatin immunoprecipitation (ChIP) assay revealed ZBTB7A bound to the HK2 promoter (below). (D) Schematic diagram of luciferase reporter construction and relative luciferase activity analyzed in cells co-transfected with pEX3-ZBTB7A or empty vector and the HK2 promoter (−1000 to 0 bp) (or HK2 promoter without the putative ZBTB7A binding site). (E) Diagram showing ZBTB7A binding site of LDHA promoter (above). ChIP assay revealed ZBTB7A bound to the LDHA promoter (below). (F) Schematic diagram of luciferase reporter construction and relative luciferase activity analyzed in cells co-transfected with pEX3-ZBTB7A or empty vector and the LDHA promoter (−1000 to 0 bp) (or LDHA promoter without the putative ZBTB7A binding site). **p < 0.01 versus pEX3 empty vector group. Data are presented as the mean ± SD of three independent experiments per group, unless otherwise specified. The data were statistically analyzed via one-way ANOVA. the expression of CD44 and CDK12. 39 In this study, we found that U3 could form sdRNA U3-miR in GBM cells. Consistent with parental U3 expression, U3-miR had an increased expression in IDH1 WT GBM cells when compared to IDH1 R132H GBM cells. We found that U3 sdRNA needs to be processed by the Dicer enzyme to form U3-miR, and U3-miR can then bind to the Ago2 protein to exert a microRNA-like function in downregulating ZBTB7A expression.
We also found that ZBTB7A expression was reduced in GBM and was significantly lower in IDH1 WT GBM than in IDH1 R132H GBM. In summary, this study demonstrated that U3 is highly expressed and ZBTB7A is expressed at low levels in IDH1 WT GBM. We found that U3 formed the sdRNA U3-miR, which functioned similarly to microRNA by binding to the ZBTB7A 3′UTR region to downregulate ZBTB7A expression. ZBTB7A transcriptionally repressed the expression of HK2 and LDHA and regulated the aerobic glycolytic and proliferative capacity of IDH1 WT GBM cells. We revealed a new mechanism by which the U3/ZBTB7A/HK2 LDHA pathway promotes tumorigenesis in IDH1 WT GBM cells, thus providing new targets and strategies for the treatment of IDH1 WT GBM.

AUTH O R CO NTR I B UTI O N S
XL, YX, WD, and YL involved in the study conception and design.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no relevant financial or nonfinancial interests to disclose.

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 from the corresponding author upon reasonable request.