LncRNA PVT1 promotes tumorigenesis of glioblastoma by recruiting COPS5 to deubiquitinate and stabilize TRIM24.

LncRNA PVT1 has been implicated in numerous pathophysiological processes and diseases, especially cancers. However, the role and mechanism of PVT1 in the tumorigenesis of glioblastoma remain unclear. We investigated the alteration of PVT1 and its key functions in glioblastoma. PVT1 was upregulated and associated with poor prognosis in glioblastoma. We demonstrated that PVT1 silencing suppressed cell proliferation, colony formation, and orthotopic xenograft tumor growth. Mechanistic investigations found that PVT1 interacted with TRIM24 directly and increased its protein stability. PVT1 recruited COPS5 to deubiquitinate TRIM24; reciprocally, PVT1 depletion impaired the interaction between COPS5 and TRIM24, resulting in decreased expression of TRIM24. PVT1, TRIM24, and COPS5 coordinately contributed to the activation of STAT3 signaling and malignant phenotype of glioblastoma. Collectively, this study elucidates the essential role of PVT1 in the tumorigenesis of glioblastoma, which provides candidacy therapeutic target for glioblastoma treatment.

Introduction

Glioblastoma (GBM) is the most common primary malignant brain tumor of the central nervous system. Current mainstay treatments for GBM are maximum feasible surgical resection, followed by radiotherapy and adjuvant temozolomide chemotherapy. Although targeted therapy, immunotherapy, biotherapy, and tumor treatment fields have achieved great advances in GBM treatment, the median overall survival remains low at approximately 15 months. Recently, molecular classifications have ushered the diagnosis and treatment of glioma into a new era. Therefore, it is of great necessity to foster a major effort to investigate the complex gene regulation networks and identify novel therapeutic targets for GBM.

Long noncoding RNAs (lncRNAs) are a kind of RNA transcripting more than 200 nucleotides without protein-coding potential. LncRNAs are involved in several biological processes, such as cell proliferation, differentiation and apoptosis, inflammation, autophagy, and immunity. LncRNAs act as oncogenes or tumor suppressors via their participation in various cellular processes including chromatin remodeling, alternative splicing, RNA decay, and epigenetic modification. Aberrant expression of several lncRNAs has been found in GBM, such as NEAT1, HOTAIR, TUG1, FOXM1-AS, MALAT1, NBAT1, GAS5, MEG3, and CASC2. As these lncRNAs play an essential role in the tumorigenesis of GBM through regulating tumor cell growth, invasion, migration, stemness, angiogenesis, and drug resistance, they are of therapeutic interest.,

The plasmacytoma variant translocation 1 gene (PVT1), which encodes an lncRNA and is located at 8q24.21, has been identified as an oncogene in several cancers. PVT1 recruits EZH2 to the LATS2 promoter region to inhibit transcription and promote proliferation of non-small cell lung cancer tumor cells. In hepatocellular carcinoma, PVT1 has been demonstrated to bind and stabilize NOP2, which promotes tumor cell proliferation and maintains stemness. In gastric cancer, PVT1 has been shown to bind the FOXM1 promoter region to promote FOXM1 expression, and FOXM1 can also bind the PVT1 promoter region to promote the transcription of the latter, thus forming a positive feedback pathway to promote tumor growth and invasion. PVT1 recruits EZH2 to the promoter region of miR-200b, which increases methylation and inhibits the transcription of miR-200b, thus promoting the occurrence and development of cervical cancer. PVT1 regulates histone methylation of the promoter of ANGPTL4 by binding PRC2, thus promoting cell growth and migration of cholangiocarcinoma. While PVT1 has been well characterized in certain cancers, the precise mechanism and clinical significance of PVT1 in GBM have not been described.

In this study, we found that PVT1 was significantly upregulated and associated with poor prognosis in GBM patients. We confirmed that PVT1 promoted the proliferation, colony formation, and tumor growth in orthotopic mouse xenograft models. Furthermore, we demonstrated that PVT1 recruited COPS5 to deubiquitinate and stabilize TRIM24, which activated STAT3 signaling and malignant biological behaviors in glioma cells. Based on our results, manipulation of the PVT1/TRIM24/COPS5 pathway may yield a novel treatment strategy for GBM.

Results

PVT1 is elevated in GBM tissues and associated with the poor prognosis of GBM patients

To identify the roles of PVT1 in GBM, we first assessed the expression of PVT1 in clinical specimens of GBM patients. Compared with normal brain tissues, PVT1 was highly expressed in clinical GBM samples (Figure 1A). To support our results, we downloaded and analyzed Gene Expression Omnibus and The Cancer Genome Atlas (TCGA) RNA sequencing datasets. In the GSE4290 dataset, 23 normal brain tissues and 153 glioma tumors were included, and comparing normal brain tissues and low-grade glioma (LGG), PVT1 expression was significantly elevated in high-grade glioma (HGG) (Figure 1B). In the GSE108476 dataset, PVT1 was expressed at the highest levels in GBM compared with normal brain tissues and other glioma subtypes (Figure 1C). Furthermore, PVT1 was significantly upregulated in GBM tissues in TCGA dataset (Figure 1D). To further evaluate the relationship between PVT1 expression and GBM patient survival, we performed Kaplan-Meier survival analysis in our GBM samples and found that GBM patients with high PVT1 level had a statistically significant poor prognosis compared with those with low expression of PVT1 (Figure 1E). In addition, Kaplan-Meier analysis using publicly available dataset GSE43378, GSE108476, and TCGA also demonstrated that patients with high PVT1 level frequently had shorter overall survival time (Figures 1F, S1A, and S1B). We also assessed the relevance of PVT1 expression with GBM patient's clinical characteristics. The results revealed that PVT1 expression was associated with necrosis changes (p = 0.03), recurrence (p = 0.035), and survival (p = 0.024) (Table S3). These findings suggest that increased PVT1 expression is correlated with advanced GBM progression and poor prognosis, which indicates that PVT1 plays an oncogenic role in GBM development.

Figure 1

PVT1 is elevated in GBM tissues and associated with poor prognosis of GBM patients

(A) The expression level of PVT1 is significantly higher in clinical GBM specimens compared with normal brain tissues. (B) PVT1 level in normal brain tissues, LGG, and HGG from GSE4290 dataset. (C) PVT1 level in normal brain tissues and different glioma subtypes from GSE108476 dataset. (D) PVT1 is significantly upregulated in GBM tissues in TCGA dataset. (E) Kaplan-Meier analysis of GBM patients at Renji Hospital. (F) Kaplan-Meier analysis of the publicly available dataset GSE43378. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. p values were calculated using two-tailed Student's t tests. The cutoff value for Kaplan-Meier analysis is the median of PVT1 expression level.

PVT1 promotes glioma cell proliferation and tumorigenesis in vitro and in vivo

To investigate the potential effect of PVT1 on the pathogenesis of GBM, we first detected PVT1 mRNA level in normal human astrocytes (NHAs) and our available glioma cell lines. Compared with NHAs, LN229 showed low PVT1 expression, U87 had moderate PVT1 transcription, while T98-G, U251, and U373 exhibited high PVT1 level (Figure 2A). Subsequently, U251 and U373 were selected for depletion of PVT1, LN229 was selected for overexpression of PVT1, and the efficiencies were confirmed by real-time quantitative polymerase chain reaction (qPCR) (Figures S2A and S2B). The further experiments found that knockdown of endogenous PVT1 significantly inhibited cell growth in U251 and U373 compared with the controls (Figures 2B, 2D, 2E, S2C–S2E), while overexpression of PVT1 in LN229 (Figures 2C, 2F, and 2G) accelerated cell growth as measured via CCK-8 assays and colony formation assays. To further validate whether PVT1 is essential for tumorigenesis, U251 transfected with normal control shRNA (shNC) or shPVT1(sh1#, sh2#) was intracranially implanted separately. Compared with the control xenograft models, knockdown of PVT1 significantly reduced glioma tumor growth (Figures 2H and 2I). These data strongly indicate that PVT1 is necessary for glioma cell growth and tumorigenesis.

Figure 2

PVT1 promotes glioma cell proliferation and tumorigenesis in vitro and in vivo

(A) PVT1 level in NHA and glioma cells. (B) Knockdown of PVT1 suppressed cell proliferation in U251 cells via CCK-8 assays. (C) Overexpression of PVT1 promotes cell proliferation in LN229 cells via CCK-8 assays. (D and E) Knockdown of PVT1 inhibits colony formation ability in U251 cells. (F and G) Overexpression of PVT1 promotes colony formation ability in LN229 cells. Scale bars, 10 mm. (H and I) Representative bioluminescence images and hematoxylin and eosin (H&E)-stained images of xenografts' brains with indicated U251 cells expressing PVT1 shNC, PVT1sh1#, or PVT1sh2#. Scale bars, 2 mm. n = 5. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

PVT1 interacts with TRIM24 in glioma cells

Accumulating evidence supports that lncRNAs bind proteins to regulate various biological processes. To seek out potential PVT1-interacting candidates, mass spectrometry of proteins pulled down by PVT1 was performed (Figure 3A). LS-MS mass spectrometric analyses identified that PVT1 may interact with TRIM24. In our previous study, we also found that TRIM24 played an oncogenic role in the development of GBM via increasing the level of p-STAT3 and activating the STAT3 signaling pathway. Thus, we hypothesized that PVT1 interacts with TRIM24 to promote the progression of glioma. We found that PVT1 was primarily localized in the nucleus via nucleo-cytoplasmic separation assay, which was consistent with TRIM24 (Figure 3B). To validate whether TRIM24 binds PVT1, we performed RNA pull-down using the HA MS2bp-MS2bs system (Figure 3C) and detected that PVT1 co-precipitates exogenous and endogenous TRIM24 (Figures 3D and 3E). Next, we performed RNA immunoprecipitation (RIP)-qPCR using an anti-FLAG antibody in U251 transfected with FLAG-tagged TRIM24 and detected the enrichment of PVT1 with the anti-FLAG antibody compared with the control (Figure 3F). Furthermore, to identify the interacting domain of TRIM24 regulating its association with PVT1, we constructed truncated fragments and transfected them into U251 separately (Figure 3G). All except Δ1 and Δ1.1 were not able to interact with PVT1, suggesting that the coiled-coil domain of TRIM24 is required for interacting with PVT1 (Figures 3H and 3I). To further identify the region of PVT1 that binds TRIM24, several truncated fragments were generated (Figure 3J). After transfected into U251 separately, both D2 and D3 were able to interact with TRIM24 (Figure 3K), suggesting that exon 5 and exon 6 are essential for PVT1 to bind TRIM24, whereas, PVT1 D1 did not contain the TRIM24 binding region. Taken together, these data suggest that PVT1 binds TRIM24 in glioma cells.

Figure 3

PVT1 interacts with TRIM24 in glioma cells

(A) Mass spectrum of proteins pulled down by PVT1 was performed. The pull-down precipitates were separated via SDS-PAGE and visualized with silver staining. The arrows indicate target bands for mass spectrum analysis. (B) The detection of PVT1 location via nucleo-cytoplasmic separation assay in U251 and U373 cells. (C) Schematics of the HA MS2bp-MS2bs RNA pull-down system. (D and E) Immunoblotting for the detection of FLAG-tagged TRIM24 or endogenous TRIM24 pull-down by PVT1 in U251 cells. (F) RT-PCR for the detection of PVT1 and U6 by RIP with antibody against FLAG in U251 cells. (G) Schematics of TRIM24 WT and truncated constructs. (H and I) PVT1 interacts with TRIM24 with Δ1 and Δ1.1. (J) Schematics of PVT1 WT and truncated constructs. (K) TRIM24 interacts with PVT1 with exon 5 and exon 6.

PVT1 stabilizes TRIM24 protein

As shown in Figure 3E, overexpression of PVT1 increased TRIM24 protein level. To further investigate the role of PVT1 in regulating the expression of TRIM24, we first assessed TRIM24 mRNA and protein expression in PVT1 overexpressing and knockdown glioma cells. The results demonstrated that knockdown of PVT1 dramatically decreased TRIM24 protein level in U251 and U373, while overexpression of PVT1 markedly increased TRIM24 protein level in U251 and LN229 (Figure 4A). However, intervention of PVT1 expression had no effect on TRIM24 mRNA expression (Figures 4B and 4C). Therefore, we hypothesized that PVT1 may mediate TRIM24 protein stability. To test this hypothesis, we treated glioma cells with translation inhibitor cycloheximide (CHX) at indicated times. Compared with controls, TRIM24 protein levels were markedly decreased after treatment with CHX for 12 h in U251 and U373. Knockdown of PVT1 significantly increased TRIM24 degradation compared with the controls (Figures 4D–4G). Furthermore, PVT1 D1, which does not contain the TRIM24 interacting region, was used as negative control. We found that overexpression of WT PVT1, but not vector or PVT1 D1, significantly increased the stability of TRIM24 protein (Figures 4H and 4I). Collectively, these results demonstrate that PVT1 regulates TRIM24 protein stability in glioma cells.

Figure 4

PVT1 regulates TRIM24 protein stability

(A) Knockdown of PVT1 decreases TRIM24 protein expression in U251 and U373 cells, while overexpression of PVT1 elevates TRIM24 protein expression in U251 and LN229 cells. (B and C) Real-time qPCR analysis on the effects of intervention of PVT1 expression on TRIM24 mRNA expression. (D and F) Effects of PVT1 knockdown on TRIM24 degradation in U251 and U373 cells. (E and G) Quantification of TRIM24 protein levels in (D and F). (H) Effects of overexpression of PVT1 WT and D1 truncated fragment on TRIM24 degradation in U251 cells. (I) Quantification of TRIM24 protein levels in (H). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

PVT1 promotes TRIM24-dependent glioma progression

As mentioned above, we demonstrated that TRIM24 promoted the progression of glioma via increasing the level of p-STAT3 and activating STAT3 signaling in our previous study. Given that PVT1 bound TRIM24 to stabilize and upregulate its protein level, we further detected the effects of PVT1 on p-STAT3 expression. Knockdown of endogenous PVT1 significantly decreased STAT3 phosphorylation, while overexpression of PVT1 markedly increased the level of p-STAT3 (Figure S3A), which suggest that PVT1 regulates STAT3 activity in glioma. To further investigate whether PVT1 functioned in a TRIM24-dependent manner in GBM, we re-introduced TRIM24 after PVT1 deletion in glioma cells and demonstrated that re-introduction of TRIM24 restored the inhibitory effects on cell proliferation, colony formation, and tumorigenesis caused by PVT1 deletion (Figures 5A, 5C, 5D, 5G, 5H, and S3B–S3D). We also found that overexpression of PVT1 promoted LN229 cell growth, which nevertheless was impaired by simultaneous knockout of TRIM24 (Figures 5B, 5E, and 5F). Moreover, re-introduction of TRIM24 reversed PVT1 depletion-inhibited p-STAT3 level (Figure 5I, left and S3E), while knockout of TRIM24 reduced the upregulation of PVT1 overexpression on p-STAT3 level (Figure 5I, right). These data support that TRIM24 is critical for tumorigenesis of GBM regulated by PVT1.

Figure 5

PVT1 promotes glioma progression depending on TRIM24

(A) Re-expression of TRIM24 after PVT1 deletion in U251 cells restores cell proliferation. (B) Knockout of TRIM24 after overexpression of PVT1 in LN229 cells impairs cell proliferation. (C and D) Re-expression of TRIM24 after PVT1 deletion in U251 cells restores colony formation ability. (E and F) Knockout of TRIM24 after overexpression of PVT1 in LN229 cells impaired colony formation ability. Scale bars, 10 mm. (G and H) Representative bioluminescence images and H&E-stained images of xenografts' brains with indicated U251 cells stably transfected with PVT1sh or TRIM24OE. Scale bars, 2 mm. n = 5. (I) WB analysis of the expression of p-STAT3 after transfection with PVT1sh or TRIM24OE in U251 and LN229 cells. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Identification of COPS5 as a candidate deubiquitinase recruited by PVT1 for TRIM24

To determine potential mechanisms by which PVT1 regulates the expression level of TRIM24 protein, we systematically identified deubiquitinases (DUBs) that may regulate TRIM24 protein stability. Performing a DUB overexpression screen in 293T cells, we focused on eight candidate DUBs whose overexpression led to upregulation of TRIM24 protein. Twenty-nine human DUBs spanned four subfamilies (Figure 6A). TRIM24 protein levels were determined by western blot (WB) analysis (Figures 6B and S4A). Among the eight candidate TRIM24-regulating DUBs, USP4, USP37, COPS5, and COPS6 drew our attention. Compared with control and overexpression of the other four DUBs (USP3, USP12, USP42, and USP47), TRIM24 protein levels were markedly increased after treatment with CHX for 12 h in 293T cells (Figures 6C and S4B). In addition, to determine candidate DUBs for TRIM24, we transfected expression vectors encoding USP4, USP37, COPS5, and COPS6; ubiquitin and FLAG-tagged TRIM24 into 293T cells. Cells were treated with MG132, and TRIM24 protein was purified with anti-FLAG antibody. Subsequently, TRIM24 ubiquitination was analyzed. The results showed that USP4, COPS5, and COPS6 markedly reduced the level of ubiquitinated TRIM24 (Figure 6D), whereas the overexpression of USP4, COPS5, and COPS6 had no effect on TRIM24 mRNA expression (Figure 6E). Moreover, we performed RNA pull-down using the HA MS2bp-MS2bs system with MYC-tagged USP4, COPS5, and COPS6 and detected that among the three DUBs that affect ubiquitination of TRIM24, PVT1 only interacted with COPS5 (Figures 6F and S5). Taken together, these data suggest that COPS5 is recruited by PVT1 and forms a complex with TRIM24 to further regulate its protein expression.

Figure 6

Identification of COPS5 as a candidate deubiquitinase recruited by PVT1 for TRIM24

(A) Classification of the 29 human DUBs included in the identification of DUBs that may regulate TRIM24 protein stability and ubiquitination. (B) The quantification of TRIM24 protein levels upon overexpression of each DUB in 293T cells. Twenty-nine human DUBs were individually transfected into 293T cells; endogenous TRIM24 was detected by WB, and protein bands were quantified using the ImageJ software. (C) Effects of overexpression of eight candidate TRIM24-regulating DUBs on TRIM24 degradation in 293T cells. (D) Ubiquitination assay measures the ubiquitinated TRIM24 after transfection with USP4, USP37, COPS5, and COPS6 in 293T cells. (E) qRT-PCR analysis of the effects of the overexpression of three candidate TRIM24 ubiquitination-regulating on TRIM24 mRNA expression. (F) Immunoblotting for the detection of MYC-tagged COPS5 pull-down by PVT1 in 293T cells. USP, ubiquitin-specific proteases; JAMM, JAMM/MPN metalloproteases; UCH, ubiquitin carboxylterminal hydrolases; OTU, otubain proteases.

COPS5 interacts with and deubiquitinates TRIM24 depending on PVT1

To validate COPS5 as the candidate deubiquitinase for TRIM24, we first assessed the TRIM24 mRNA and protein expression levels after intervention COPS5 expression in glioma cells. Knockdown or overexpression of COPS5 had no effect on TRIM24 mRNA expression; however, TRIM24 protein level was correlated to changes in COPS5 expression (Figures S6A–S6C). Protein co-immunoprecipitation (coIP) analysis was performed with FLAG-tagged TRIM24 and MYC-tagged COPS5 co-transfected into U251. Ectopic COPS5 could be detected in TRIM24 immunoprecipitates using anti-FLAG antibody, while ectopic TRIM24 could be immunoprecipitated by COPS5 using anti-MYC antibody (Figure 7A). Moreover, an interaction between COPS5 and TRIM24 was found between endogenous proteins in U251 cells (Figure 7B). In addition, immunofluorescence microscopy of glioma cells confirmed that COPS5 co-localized with TRIM24 (Figures 7C and S6D). We then simultaneously transfected the FLAG-tagged TRIM24 and MYC-tagged COPS5 into U251 cells, and the cell lysates collected were treated with RNaseA, followed by dual coIP assays with either anti-FLAG or anti-MYC antibodies. The results demonstrated that TRIM24 and COPS5 protein could be co-immunoprecipitated reciprocally in the normal samples but not in RNaseA-treated samples (Figures 7D and 7E). Furthermore, we found that knockdown of PVT1 enhances COPS5 attenuated TRIM24 degradation (Figures 7F and S5E). Finally, we found that PVT1 deletion markedly reversed the amount of ubiquitinated TRIM24 reduced by COPS5 (Figure 7G), while knockdown of COPS5 restored the amount of ubiquitinated TRIM24 impaired by PVT1 (Figure 7H). Importantly, these findings demonstrate that TRIM24 is a direct substrate of COPS5, and complex formation between TRIM24 and COPS5 depends on PVT1.

Figure 7

COPS5 interacts with and deubiquitinates TRIM24 depending on PVT1

(A) Immunoblotting for the detection of exogenous MYC-tagged COPS5 and FLAG-tagged TRIM24 immunoprecipitated by anti-FLAG or anti-MYC in U251 cells. (B) Immunoblotting for the detection of endogenous COPS5 and TRIM24 immunoprecipitated by TRIM24-specific or COPS5-specific antibody in U251 cells. (C) Representative images of co-localization of TRIM24 with COPS5 in U251 cells. Scale bar, 40 μm. (D) Immunoblotting for the detection of exogenous MYC-tagged COPS5 and FLAG-tagged TRIM24 immunoprecipitated by anti-FLAG antibody with or without RNaseA treatment in U251 cells. (E) Immunoblotting for the detection of exogenous FLAG-tagged TRIM24 and MYC-tagged COPS5 immunoprecipitated by anti-MYC antibody with or without RNaseA treatment in U251 cells. (F) PVT1 knockdown enhances COPS5 attenuated TRIM24 degradation. (G) PVT1 knockdown reverses the amount of ubiquitinated TRIM24 reduced by COPS5. (H) COPS5 knockdown restores the amount of ubiquitinated TRIM24 impaired by PVT1.

The TRIM24/PVT1/COPS5 complex promotes glioma progression through activating the STAT3 pathway

As COPS5 plays a crucial role in the TRIM24/PVT1/COPS5 complex, we therefore evaluated the effect of COPS5 on glioma progression. The results showed that overexpression of COPS5 promoted cell proliferation, colony formation, and tumorigenesis of glioma, while PVT1 knockdown or TRIM24 knockout inhibited the development of glioma caused by overexpression of COPS5 (Figures 8A–8F). Moreover, the overexpression of COPS5 increased p-STAT3 level, while PVT1 knockdown or TRIM24 knockout caused a dramatic decrease in COPS5 overexpression-increased p-STAT3 level (Figure 8G). These results support that the TRIM24/PVT1/COPS5 complex promotes glioma progression through activation of the STAT3 pathway.

Figure 8

The TRIM24/PVT1/COPS5 complex promotes glioma progression through activating STAT3 pathway

(A–D) Effects of overexpression of COPS5 and knockdown of PVT1 or knockout of TRIM24 on cell proliferation (A and B) and colony formation ability (C and D). Scale bars, 10 mm (in U251 and U373 cells compared with control). (E and F) Representative bioluminescence images and H&E-stained images of xenografts' brains with indicated U251 cells stably transfected with COPS5OE, PVT1sh, or TRIM24sg. Scale bars, 2 mm. n = 5. (G) WB analysis of the expression of p-STAT3 after overexpression of COPS5 and knockdown of PVT1 or knockout of TRIM24 in U251 and U373 cells. (H) Schematic model illustrates the mechanism by which PVT1 recruits COPS5 to deubiquitinate and stabilize TRIM24 so as to promote tumorigenesis of GBM. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Discussion

LncRNAs are essential regulators for various cancers.,, Previous studies have demonstrated that lncRNAs interact with cellular macromolecules, such as DNA, several types of RNAs, and proteins, to perform their functions.18, 19, 20 In this study, we elucidated PVT1 as a crucial modulator in the tumorigenesis of GBM. PVT1 forms a trimeric complex with TRIM24 and COPS5 through specific motifs to deubiquitinate and stabilize TRIM24, which results in the activation of STAT3 and GBM progression (Figure 8H).

The role of PVT1 in development of glioma

In this study, we revealed that PVT1 was frequently upregulated in GBM tissues, and that patients with high PVT1 expression had a poor clinical prognosis. A systematic analysis of public databases confirmed our results. Simultaneously, we demonstrated that depletion of PVT1 inhibited glioma cell proliferation, colony formation, and tumorigenesis. At present, few studies have reported that PVT1 promotes the occurrence and development of glioma.21, 22, 23 We found that PVT1 promoted the malignant phenotype of glioma using in vitro and in vivo experiments, which was consistent with previous research results. These results suggest that PVT1 may act as a proto-oncogene in GBM development, which is similar to its role in lung cancer, breast cancer, digestive system tumors, cervical cancer, and ovarian cancer.10, 11, 12, 13, 14 It is worth noting that the current regulatory mechanism of PVT1 in glioma mainly focuses on its miRNA sponge role. In this study, we investigated a novel regulatory mechanism of PVT1 in GBM.

The role of TRIM24 in various tumors including glioma

Our data revealed that PVT1 regulated GBM through TRIM24, also known as transcription intermediary factor 1 α (TIF1α), which is a member of the TIF1 subfamily of tripartite motif (TRIM) superfamily; TRIM28 (TIF1β) and TRIM33 (TIF1γ) are other subfamily members. TRIM24 regulates the development of various tumors as an oncogene or tumor suppressor gene. In a hepatocarcinoma mouse model, TRIM24 suppresses the development of tumors via inhibition of VL30. Conversely, in prostate cancer, TRIM24 binds the androgen receptor (AR), thus improving the transcription efficacy of AR and promoting malignant progression of prostate cancer. TRIM24 is also protected by TRIM28 from SPOP-mediated degradation and promotes prostate cancer progression. In addition, TRIM24 has been revealed to promote the progression of breast cancer, cervical cancer, head and neck squamous cell carcinoma, and renal cell carcinoma and is positively correlated with tumor stage and poor prognosis.28, 29, 30, 31 In glioma, TRIM24 acts as a transcriptional coactivator of STAT3, activating the STAT3 downstream pathway to promote the progression of glioma. Meanwhile, TRIM24 regulates GBM stemness and invasiveness via activating the expression of SOX2. Moreover, TRIM24 also mediates the resistance of glioma cells to temozolomide (TMZ), and patients with high expression of TRIM24 were not sensitive to TMZ treatment.

PVT1 promotes tumorigenesis of GBM in a TRIM24-dependent manner

Here, we demonstrated that PVT1 bound TRIM24 and regulated its protein stability. We demonstrated that PVT1 interacted with the coiled-coil domain of TRIM24 through exon 5 and exon 6 directly. PVT1 knockdown reduced TRIM24 protein stability and decreased phosphorylation of STAT3. Consistent with this observation, overexpression of TRIM24 restored PVT1 knockdown-inhibited p-STAT3 expression, cell proliferation, colony formation, and tumorigenesis of glioma. These lines of evidence suggest that PVT1 promotes glioma progression relying on TRIM24.

The mechanism of the TRIM24/PVT1/COPS5 complex promoting glioma progression

Our results further demonstrated that PVT1 enhanced TRIM24 protein stability by recruiting COPS5. Current studies have confirmed that COPS5 has metalloproteinase, phosphokinase, and deubiquitination enzyme activity and plays an important role in numerous biological processes, such as signaling pathway transduction, cell cycle, apoptosis, cell proliferation, angiogenesis, and DNA damage and repair. COPS5 binds P27 protein via the PBD domain and induces P27 to translocate to the cytoplasm and accelerates its degradation, thus promoting the cell cycle from G1 to S phase transition and cell proliferation. Meanwhile, COPS5 mediates the phosphorylation of P53 protein and promotes P53 translocation to the cytoplasm, which accelerates its degradation, so as to regulate the process of cell apoptosis. Moreover, COPS5 inhibits the ubiquitin-dependent degradation of HIF-L and enhances its stability, thus regulating angiogenesis. Furthermore, COPS5 reduces ROS and elevates DDR activities, which leads to inhibition of DNA damage and repair. Various studies have confirmed that COPS5 is highly expressed in ovarian cancer, breast cancer, lung cancer, hepatocellular carcinoma, and osteosarcoma and is associated with tumor progression and poor prognosis.38, 39, 40, 41, 42 In this study, we revealed that COPS5 regulated the expression of TRIM24 protein at the post-transcriptional level rather than at the transcriptional level. Through coIP experiments, we confirmed that COPS5 interacted with TRIM24, which was dependent on PVT1. Moreover, PVT1 knockdown impaired the ability of COPS5 to deubiquitinate and stabilize TRIM24 protein. Consistent with previous studies, we demonstrated that overexpression of COPS5 promoted cell proliferation, colony formation, and tumorigenesis of glioma, which would be weakened by the knockdown of PVT1 or the knockout of TRIM24.

Conclusions

Taken together, this study provides a novel mechanism of how PVT1 regulates the progression of GBM. Our results elucidate that PVT1 activates the STAT3 pathway via recruiting COPS5 to deubiquitinate and stabilize TRIM24 protein. These findings shed new light on the oncogenic role of the PVT1/TRIM24/COPS5 complex, and these molecules may become novel potential therapeutic targets for GBM in future.

Materials and methods

Human tissues and ethical application

Forty-nine GBM tissues and six normal brain tissue samples were obtained from patients following operations at Renji Hospital, School of Medicine, Shanghai Jiao Tong University (Shanghai, China). Two experienced pathologists defined histological and pathological diagnostics of GBM independently. All GBM patients received neither chemotherapy nor radiotherapy before surgery. All tissues were stored at −80°C. Ethical approval was granted from the Ethics Committee of Renji Hospital (no. 2017-058), and written informed consent was obtained from the patients before collecting samples.

Cell culture

The 293T, NHA, and human glioma cell lines U87, T98G, A172, LN229, U251, and U373 were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). These cells were cultured in DMEM medium (Gibco, NY, USA) with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere with 5% CO2. All cell lines were authenticated using STR DNA fingerprinting by Shanghai Biowing Applied Biotechnology (Shanghai, China). Mycoplasma PCR testing was performed using LookOut Mycoplasma PCR Detection kit (Sigma Aldrich) every month.

Cell proliferation and colony formation assays

Cell proliferation potential was detected using CCK-8 assay. In brief, 1 × 103 glioma cells were seeded in quadruplicate in a 96-well plate and cultured for 24 h. Then, 10 μL of CCK-8 solution (Beyotime, Shanghai, China) was added to each well and incubated for 3 h. The optical density values at 450 nm were measured with a microplate reader (BioTek, Winooski, VT, USA). For colony formation assay, 0.5 × 103 glioma cells were seeded into 6-well plate and cultured for 2 weeks. Colonies were fixed with 100% methanol and stained with 1% crystal violet solution. The visible colony numbers on each well were counted, and data were analyzed.

Plasmids and transfection

PVT1 cDNA was amplified by PCR from U251 cells, sequenced, and subcloned into pcDNA3.1 and pLVX-Puro vectors (Clontech). FLAG-TRIM24 was a gift from Michelle Barton (Addgene, plasmid no. 28138), MS2-HB was a gift from Marian Waterman (Addgene plasmid, no. 35573), and pSL-MS2-12X was a gift from Robert Singer (Addgene, plasmid no. 27119). COPS5, other DUBs, and COPS5 knockdown plasmids were purchased from the DNA library of the School of Medicine, Shanghai Jiao Tong University. Subsequently, pLVX-PVT1-MS2-12X and its truncated constructs were derived from pSL-MS2-12X, and truncated constructs of TRIM24 were subcloned and inserted into pcDNA3.1-FLAG. FUGW-H1-Syndecan shRNA vector was a gift from Sally Temple (Addgene, plasmid no. 40623).

shRNA knockdown, sgRNA knockout, and transfection assays

shRNAs were designed using the web-based software provided by Invitrogen (http://rnaidesigner.invitrogen.com/rnaiexpress/). sgRNA sequences were designed using the MIT online tool (http://crispr.mit.edu) and purchased from HuaGene Biotech (Shanghai, China); the specific sequences are listed in Table S1. Lentiviruses were produced by co-transfecting packaging plasmids into 293T cells using Lipofectamine 2000 reagent according to the manufacturer's instructions (no. 52758, Invitrogen). At 48 h after transfection, viruses were concentrated by ultracentrifugation and collected. After transduction with indicated viruses (supplemented with 8 μg/mL of polybrene), cells were collected, and then, the efficacy of knockdown of target genes was validated by real-time qPCR or western blotting assay.

RNA isolation and real-time qPCR

Total RNA was isolated from the tissues and cells with TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions. Reverse transcription was performed with Reverse Transcription Kit (Takara), and real-time qPCR was performed with the Power SYBR Green Master Mix (Life Technologies) on the Applied Biosystems StepOne Plus Real-Time Thermal Cycling Block. Comparative quantification was assessed using 2-(ΔΔCt) method. Primers are listed in Table S2.

Antibodies for WB assay

The antibodies used for western blotting were FLAG (no. F3165, Sigma Aldrich), HA (no. 66006-1-Ig, Proteintech), MYC (no. 2276, Cell Signaling Technology), His (no. 2365, Cell Signaling Technology), β-actin (no. 66009-1-Ig, Proteintech), TRIM24 (no. 14208-1-AP, Proteintech), COPS5 (no. SC-13157, Santa Cruz Biotechnology), STAT3 (H-190) (no. SC-7179, Santa Cruz Biotechnology), and phospho-STAT3 (Y705) (D3A7) (no. 9145, Cell Signaling Technology).

RIP and RNA pull-down assays

RIP assays were performed using the EZ-Magna RIP Kit (Millipore) according to the manufacturer's protocol. In brief, 5 μg of FLAG, HA, and IgG control antibodies (Millipore) were used to pull down RNAs. The co-precipitated RNAs were extracted and subsequently analyzed by qPCR. The PCR products were further confirmed by agarose gel electrophoresis. The HA-MS2bp-MS2bs system was used to perform RNA pull-down assays.

Mass spectrometric analyses

After RNA pull-down assays, PVT1-associated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver-stained. Then, the specific band around 120 KDa were excised and analyzed by LC-MS/MS using Q Exactive Plus (Thermo Fisher Scientific) at Jiyun Biotech. (Shanghai, China). The raw data were processed by MAXQUANT software and searched against the UniProt database.

CoIP assay

Cells were lysed in IP lysis/wash buffer. The immune complex was prepared using anti-FLAG, anti-Myc, anti–TRIM24, anti-COPS5 (sc-13157, Santa Cruz Biotechnology), or control IgG (Millipore) antibodies and was subsequently captured using the Pierce Classic IP Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Subsequently, the samples were analyzed by WB.

Ubiquitination assay

Ubiquitination assay was performed as described previously. In brief, the indicated plasmids were co-transfected into cells; after 48 h, cells were treated with MG-132 for 6 h before collection. Then, cells were lysed using the denaturing buffer (6 M guanidine-HCl, 100 mM Na2HPO4/NaH2PO4, and 10 mM imidazole) and incubated with nickel beads for 3 h. The precipitated proteins were analyzed by WB analysis.

In vivo tumorigenesis assay

All animal experiments were approved by Shanghai Jiao Tong University Institutional Animal Care and Use Committee. Athymic (NCr-nu/nu) female mice at age 6–8 weeks (SLAC, Shanghai, China) were used and fed under standard pathogen-free conditions. The glioma cells (1 × 106) were stereotactically implanted into the brain of the animals. Mice were killed when dysneuria developed. Tumor volumes were measured and assessed as (W2 × L)/2, (W < L), via hematoxylin and eosin staining and bioluminescence imaging on IVIS Lumina imaging station (Caliper Life Sciences).

Statistical analysis

Sample size and statistics were provided in the result section and figure legends. GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA) and SPSS version 17.0 Software (SPSS) were used for all statistical analyses. Student's t test was performed to analyze unpaired comparisons, while one-way analysis of variance with the Newman-Keuls post-hoc test was performed for multiple comparisons. Kaplan-Meier survival probability analysis was carried out using log rank tests. Chi-square test or Fisher's exact test was performed to analyze relevant factors. Statistical significance was indicated by p < 0.05 (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).