TRIM22 promotes glioblastoma development by ubiquitinating Bcl-2

ABSTRACT

Glioblastoma (GBM) exhibits elevated TRIM22 expression correlated with tumor progression, as validated in TCGA/GEO databases. The effects of TRIM22 knockdown and overexpression on GBM proliferation were evaluated with cellular assays. TRIM22 was identified as a potential Bcl-2 activator via a ubiquitination microarray. Flow cytometry (FCM) was utilized to investigate cell apoptosis. Additionally, the expression levels of Bcl-2 and proteins associated with Bcl-2 were evaluated using Western blot analysis. The interaction and ubiquitination of TRIM22 and Bcl-2 were analyzed via immunoprecipitation (IP). TRIM22 overexpression is correlated with glioma progression, and TRIM22 deficiency inhibits GBM cell proliferation. FCM revealed that TRIM22 knockdown promotes GBM cell apoptosis. A TRIM22-overexpressing ubiquitination microarray identified TRIM22 as a potential activator of Bcl-2. Western blot analysis revealed that TRIM22 increases the protein expression levels of Bcl-2. Ubiquitination assays revealed that TRIM22 promotes the stability of Bcl-2 via nondegradative ubiquitination. IP experiments indicated that TRIM22 binds to Bcl-2. TRIM22 may significantly impact glioma progression by modulating Bcl-2. Previous studies have shown that knockdown of TRIM22 can enhance the sensitivity of temozolomide treatment, so TRIM22 is expected to become a new target for glioma immunotherapy.

1. Introduction

Glioblastoma (GBM) is the most prevalent and highly aggressive form of primary brain tumor, owing to its swift growth and expansion. Despite the application of surgery, radiation therapy, and pharmacological interventions, patients with high-grade GBM have a poor prognosis and low survival rates.¹ Glioblastoma stem cells (GSCs) within GBM contribute to rapid dissemination, a propensity for recurrence, and strong/robust resistance to most treatments.^2,3 In the past few years, advancements in genomics, transcriptomics, and genetics have led to notable improvements in the classification and management of gliomas.^4,5 New treatment approaches, such as oncolytic virus therapy, immunotherapy, focused ultrasound therapy, and vaccine therapy, have emerged as promising alternatives. Posttranslational modifications (PTMs) involve various biological processes, such as tumor metabolism, the tumor immune microenvironment (TME), and cancer stem cells (CSCs), which may serve as promising therapeutic targets for the treatment of cancer.

Ubiquitination (Ub) is one of the most important PTMs and involves a cascade of enzymes, namely, E1, E2, and E3, which covalently attach ubiquitin to target proteins. This process is intricately associated with a myriad of diseases, especially in cancer.^6,7 The diversity of ubiquitination is characterized by its quantity and configuration on the substrate, which can be classified into monoubiquitination, multiubiquitination, and branched ubiquitination.^7,8 In the polyubiquitin chain, ubiquitin can be linked through 7 lysine residues (K6, K11, K27, K29, K33, K48, and K63) or through methionine.^9,10 Ubiquitin chains are distinguished by their unique binding domains, thereby mediating different biological reactions. Among them, K48, K63, and linear ubiquitin chains are the focus of extensive research.^11,12 The K48-linked ubiquitin chain is usually associated with proteasomal degradation, a process facilitated by the proteasome – a multisubunit complex capable of recognizing and breaking down substrates with ubiquitin modifications.^13,14 This process is not only crucial for signal transduction and transcriptional regulation but is also fundamental for maintaining protein homeostasis.¹⁵ In contrast, K63-linked polyubiquitination plays a role in DNA damage repair mechanisms, signal transduction pathways, and autophagy. Chains linked by K63, whether linear or branched/mixed, provide a scaffold for additional protein recruitment and signal transduction.^16,17 In addition to polyubiquitination, branched ubiquitination also substantially influences biological signaling, such as the mixed polyubiquitination of K11 and K63, which promotes insoluble aggregation of ATG14, thereby reducing autophagy.¹⁸ Ubiquitination is considered a crucial modification in cancer, where metabolic enzymes, transcription factors, and various signaling pathways are regulated, leading to its initiation and progression.^19,20

Tripartite motif (TRIM) family members, renowned for their ability to act as E3 ubiquitin ligases, are crucial for innate immunity, tumorigenesis, autophagy, and apoptosis, owing to their conserved N-terminal RING, B-box, and coiled-coil domains.²¹ Research on TRIM22 has focused mainly on its role in viral infections (such as HIV), autoimmune diseases (such as systemic lupus erythematosus), and autophagy.^22–26 However, the mechanism of TRIM22 in human cancers remains largely unexplored. Therefore, the precise function of TRIM22 and potential underlying mechanisms in glioblastoma remain unclear.

The B-cell lymphoma 2 (Bcl-2) family consists of genes that exhibit a high degree of homology with the Bcl-2 gene, all of which are intricately involved in the modulation of apoptosis. Within this family, the Bcl-2 protein is the most thoroughly researched member of the antiapoptotic family. In various malignancies, Bcl-2 family members help cells evade apoptosis and resist chemotherapy and targeted therapies, primarily through the upregulation of one or more antiapoptotic Bcl-2 family proteins. Numerous therapeutic agents directed at antiapoptotic proteins, such as Bcl-2, have progressed to clinical trials.²⁷ The present study investigated the function and potential mechanism of the TRIM22/Bcl-2 axis in the progression of GBM, aiming to identify novel targets for the treatment of GBM.

2. Materials and methods

2.1. Online cancer database analysis

TCGA GBM database from GEPIA2 and GEO (GDS1813) was used to conduct a comparative analysis of TRIM22 mRNA expression levels between GBM and healthy brain tissue. TCGA and Rembrandt (http://gliovis.bioinfo.cnio.es/) GBM databases were used to compare overall survival (OS). TRIM22 expression levels were evaluated across various tumor samples and their corresponding normal controls using the CPTAC (https://pdc.cancer.gov/pdc/).

2.2. Cell culture

The U251, U87, U118, and T98 human GBM cell lines and the 293T human embryonic kidney (HEK293T) cell line were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco 10,566,016) supplemented with 100 µg/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (FBS; Gibco, A5256701). The cells were grown in a steady-state incubator (Heal Force), with a controlled environment of 5% CO₂, and maintained at 37 °C.

2.3. Plasmid and lentivirus transfection

Flag and His tags were attached to the TRIM22 (Genechem, GOSE0415419) and Bcl-2 (Genechem, GOSE0387482) coding regions, respectively. Plasmids that carry HA-tagged ubiquitin (Genechem, GOSE0327411) and k63-ubiquitin (Genechem, GOSE0254146) were acquired from Genechem. SyngenTech developed a TRIM22-overexpressing lentiviral vector. The constructs were subjected to DNA sequencing to confirm their integrity. A short hairpin RNA (shRNA) targeting TRIM22, which was delivered via a lentiviral vector, was obtained from Genechem (GIEE0415417). The sequence of the shRNA designed to target the complementary DNA of human TRIM22 was 5’-CAATGAAATGAGAGTCATCTT-3’.

2.4. Western blot analysis

Cells and tissues were subjected to extraction with RIPA buffer (Cell Signaling Technology, 9806) to isolate the proteins. After quantification of the extracted protein, 25 μg of protein was separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The proteins were then transferred onto a PVDF membrane, which was incubated with an antibody solution overnight at 4 °C. GAPDH (Proteintech, 1:10000, 60004–1-Ig) served as a standard for protein normalization.

2.5. Immunoprecipitation (IP)

Proteins were extracted as described above from GBM and 293T cells. Following high-speed centrifugation, the protein supernatant was collected and subsequently subjected to immunoprecipitation with the specified antibodies at 4 °C overnight. The lysates were then collected and incubated with protein A/G magnetic beads (MedChemExpress, HY-K0202) at 4 °C overnight. After washing the immunocomplexes five times with RIPA lysis buffer, the bound proteins were eluted via boiling. The proteins were then separated by SDS‒PAGE and subjected to Western blot analysis. Details of the antibodies are provided in the Supplementary Material.

2.6. Reverse-transcription quantitative PCR (RT qPCR)

RNA was isolated from the cells utilizing TRIzol Reagent (Sigma – Aldrich, T9424) via a ventilator following the instructions provided by the manufacturer. The PrimeScript RT Reagent Kit (Takara, RR047) was used to convert RNA into cDNA through reverse transcription. Quantitative real-time PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711–02) in an ABI StepOnePlus system on a Lightcycler 480 II instrument (Roche Applied Science). Sangon Biotech Company supplied the primers, and the primer sequences are listed in Supplementary Material Table S1. The target gene expression levels were adjusted in relation to those of GAPDH. The fold changes were assessed using the comparative CT (ΔΔCT) method.

2.7. Cell counting Kit‑8 assay

A Cell Counting Kit-8 (CCK-8) assay (Biosharp, BS350C) was used to evaluate cell growth. Both the control and transfected cells were plated in 96-well plates (Corning, 3599) at a concentration of 1000 cells/well. CCK8 reagent (10 µl) was subsequently added to each well. Twenty-four hours after seeding, which was designated as Day 0), cell viability was measured using a microplate reader (Denovix). Cell viability was assessed at 24-hour intervals. The increase in cell proliferation was measured by comparing the change in fold growth from Day 0 to Day 4, and the results were plotted on a graph.

2.8. Colony‑forming assay

Treated cells were seeded into six-well dishes (Corning, 3516) at a concentration of 1000 cells/well, and they were incubated for two weeks, at which point a colonies emerged. The samples were fixed with 4% paraformaldehyde (Biosharp, BL538A) for 15 min and stained with crystal violet for 30 min (Biotime, C0121) at room temperature. Images were acquired, and the overall number of colonies was determined using ImageJ software.

2.9. EdU assay

Cellular proliferation in the control and treatment groups was assessed using an EdU assay. After the cells were incubated with 10 µM EdU for 1 hour at 37 °C, the growth medium was removed, and the remaining cells were fixed with 4% paraformaldehyde (Biosharp, BL538A) at ambient temperature for 30 min. The cells were then permeabilized with 0.5% Triton X-100 (Biosharp, BS084) for 20 minutes. The cells were then stained according to the manufacturer’s protocol, and images were acquired using an Invitrogen EVOS FL Auto (Life Technologies). ImageJ was utilized for both synthesizing images and counting cells. The ratio of EdU+ cells to DAPI+ cells served as an indicator of both the growth rate and the level of proliferation.

2.10. Transwell analysis

Cells (20,000 cells/insert) were seeded into the upper compartment of the Transwell inserts (pore size: 8 μm; Corning Costar; 3422), and 600 μL of medium supplemented with 20% FBS was added to the bottom compartment. The chambers were incubated at 37 °C for a period of 36 h, after which the cells were fixed with 4% paraformaldehyde (Biosharp, BL538A) and stained with crystal violet (Biotime, C0121). Images were acquired via the Invitrogen EVOS FL Auto (Life Technologies), and the total number of colonies was determined utilizing ImageJ software.

2.11. Cell apoptosis

Once the GBM cells reached 90% confluence, the cell suspensions were collected, centrifuged at 300 × g for three minutes, washed with cold PBS and resuspended in 1× binding buffer. Next, the cells were stained with Alexa Fluor 647-annexin V and propidium iodide (PI) in the dark for 15 minutes (YEASEN, 40304ES50). The cells were subjected to analysis via flow cytometry (FCM) within one hour. The resulting data were subsequently evaluated using FlowJo software.

2.12. Clinical samples and immunohistochemistry

The tissue microarray (HBraG160Su01), which consisted of 160 human GBM samples, was purchased from Shanghai Outdo Biotech Co. Ltd. All patient data were collected and utilized in line with the sanctioned guidelines set forth by the Institutional Review Boards of the involved organizations.

2.13. Ubiquitination screening assay

The proteins targeted by TRIM22-induced ubiquitination were identified through the R&D Systems Proteome Profiler Human Ubiquitin Array Kit (catalog ARY027), which consists of a diverse selection of 49 protein samples. The human ubiquitin array membrane, which was tagged with 49 antibodies, was incubated with cell lysates for one hour on a rocking shaker. The membrane was subsequently incubated with a cocktail of biotinylated pan-ubiquitin detection antibodies overnight at 4 °C. The membrane array was then incubated with streptavidin-HRP. The signal produced at each collection point indicated the amount of ubiquitinated protein that was attached.

2.14. Ubiquitination assays

The process of ubiquitination for exogenous Bcl-2 was investigated following the transfection of Flag-labeled TRIM22, His-labeled Bcl-2, and HA-labeled ubiquitin into the 293T and U251 cell lines. Proteins in the lysate were precipitated and analyzed through western blotting using targeted antibodies.

2.15. Cycloheximide (CHX) chase

U251 and U118 cells were infected with adenoviruses targeting TRIM22 (Genechem). After selection, CHX (MedChemExpress, HY-12320) was added to the culture medium to prevent protein synthesis, and the cell extracts were collected at specified intervals. The protein samples (20 μg) were subsequently analyzed via Western blotting.

2.16. Animal models

Male BALB/c nude mice (4 weeks old) were acquired from the Changzhou Cavens Experimental Animal Center. The mice were acclimated to their new surroundings for one week prior to the experiments. All the mice were assigned randomly into groups. Control and sh-TRIM22 U87 cells, which stably express the firefly luciferase gene, were stereotactically introduced into the brains of each mouse, utilizing 500,000 cells dispersed in 5 μL of PBS. Tumor progression was tracked via bioluminescence imaging at 7, 14, and 21 days postimplantation, and the IVIS Spectrum system was used to capture visual data. The data were then analyzed with Living Image Software. All animal care and experimental procedures strictly adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Animal Ethics Committee of Nanjing Medical University (Approval No. 2024234) prior to experimentation.

2.17. Statistical analysis

The data were statistically analyzed via GraphPad Prism software (version 8). The data are presented as the means ± SDs. An unpaired, two-tailed Student’s t test was used to compare two separate groups, while one-way or two-way ANOVA was used to conduct comparisons across more than two groups. A p value less than 0.05 was considered statistically significant.

3. Results

3.1. TRIM22 is upregulated in GBM

TRIM22 mRNA expression was first examined in TCGA GBM database from GEPIA2, which revealed a marked increase in TRIM22 mRNA levels in tumor samples (T) compared with healthy tissues (N) (Figure 1 (a,b)). These findings were confirmed in the GEO database (GDS1813) (Figure 1(c)).In addition, the expression levels of TRIM22 differed when various types of tumors were compared to their respective normal tissues (Figure 1(d)). The increased TRIM22 protein expression level was validated via immunohistochemistry (IHC). TRIM22 protein expression was elevated in gliomas and was positively correlated with tumor grade (Figure 1(e,f)). Moreover, lower expression of TRIM22 was correlated with increased OS in TCGA database, which aligns with the data from the Rembrandt database (Figure 1(g,h)). Collectively, these findings demonstrated a correlation between increased TRIM22 expression levels and the increasing grade of gliomas, suggesting that TRIM22 may play a role in the progression of gliomas.

Figure 1.

Investigation and verification of TRIM22 expression in glioma tissues.

(a) Comparison of TRIM22 mRNA levels in GBM versus normal tissues using TCGA data analyzed from GEPIA2. (b) Analysis of TRIM22 mRNA levels in different classes of GBM in TCGA GBM database from GEPIA2. (c) Comparative analysis of TRIM22 mRNA levels between GBM and healthy tissue samples, as recorded in the GEO database. The x-axis denotes sample groups, and the y-axis shows gene expression. The colors signify different groups. (d) TRIM22 expression data across normal and various cancerous tissues in CPTAC. (e) Representative TRIM22 IHC images of WHO grade I-IV glioma tissue arrays (n = 160); scale bar: 500 μm. (f) Quantification of the results of immunohistochemistry in tissue microarrays. (g, h) TRIM22 overexpression in glioma patient tumors from TCGA and Rembrandt databases was correlated with reduced OS. (*p < 0.05, **p < 0.01, and ***p < 0.001)

3.2. TRIM22 promotes the proliferation and migration of glioma cells

To validate the increased expression of TRIM22 in GBM (Figure 1), the presence of TRIM22 was validated in four different cell lines, and U251 and U87 cells were selected for use in subsequent experiments (Figure 2(a)). To investigate the impact of TRIM22 on gliomas, shRNA was used to decrease the expression of TRIM22 in human glioma cell lines (KD), and TRIM22 was overexpressed via a lentivirus (OE). Both Western blot analysis and RT‒qPCR revealed significant changes in the protein and mRNA expression levels of TRIM22, respectively (Figure 2(b)). The CCK-8 assays revealed that TRIM22 overexpression significantly increased the proliferation of U251 and U87 cells, whereas TRIM22 KD significantly inhibited the proliferation of GBM cells (Figure 2(c)). Colony‑forming assays revealed that GBM U251 and U87-shTRIM22 cells exhibited a marked decrease in their capacity to form single-cell colonies compared with that of the control group (con). The positive impact of TRIM22 was noted in GBM cells with elevated TRIM22 expression (Figure 2(d)). The Transwell migration assay confirmed the stimulatory impact of TRIM22 (Figure 2(e)). EdU assays revealed a reduction in tumor proliferation following the knockdown of TRIM22 (KD) (Figure 2(f)). To investigate the in vivo tumor formation-enhancing capabilities of TRIM22, an allograft tumor proliferation study was performed in immunodeficient mice. Compared with those derived from control cells, tumors derived from TRIM22-deficient U87 cells presented a slower growth rate (Figure 2(g)). Taken together, these findings suggested that TRIM22 plays a pivotal role in the proliferation and progression of glioma cells.

Figure 2.

TRIM22 enhances the growth and migration of glioma cells.

(a) Verification of TRIM22 knockdown and overexpression in U87 and U251 cells via Western blotting and RT‒qPCR. (b) TRIM22 expression in different glioma cell lines was analyzed via Western blotting, and GAPDH served as a loading control. (c) A CCK-8 assay was conducted using lentiviral transduced U87 and U251 cells. (d) Colony formation experiments were performed using U87 and U251 cells transduced with lentiviruses. (e) Visual depiction of the migratory cell counts in the cell migration assays. (f) EdU assays were conducted using U87 and U251 cells transduced with lentiviral vectors. Scale bar, 200 μm. (g) Images and quantification of in vivo bioluminescence imaging. (*p < 0.05, **p < 0.01, and ***p < 0.001)

3.3. Effect of TRIM22 on apoptosis in GBM

Because TRIM22 may play a significant role in glioma biological processes, we investigated the potential correlation between TRIM22 and neoplastic cell apoptosis. FCM was employed to evaluate the influence of TRIM22 overexpression and knockdown on GBM apoptosis. The U87 control group (con) exhibited an apoptosis rate of 11.03%, with 7.54% early apoptosis and 3.49% late apoptosis. In contrast, the TRIM22-deficient U87 group presented a significantly higher percentage of apoptotic cells (34.6%), with 21.8% early apoptotic cells and 12.8% late apoptotic cells. Similar findings were noted in the U251 cell line. These findings suggested that TRIM22-deficient GBM cells had a greater proportion of both early and late apoptotic cells than control GBM cells. The rate of apoptosis increased considerably when TRIM22 was knocked down, far surpassing that of the control group (Figure 3(a,b)). These findings suggested that TRIM22 knockdown promotes GBM cell apoptosis.

Figure 3.

Effect of TRIM22 knockdown and overexpression on GBM apoptosis.

(a) Flow cytometry employing dual staining with annexin V-647 and propidium iodide (PI) was utilized to assess apoptosis in U87 cells with either TRIM22 knockdown or overexpression. (b) Flow cytometry employing dual staining with annexin V-647 and propidium iodide (PI) was utilized to assess apoptosis in U251 cells with either TRIM22 knockdown or overexpression. (*p < 0.05 and **p < 0.01).

3.4. TRIM22 modulates the protein level of Bcl-2 in GBM

Recent studies have indicated that TRIM22, a member of the RING-type E3 ubiquitin ligases, is a crucial player in the progression of various cancers, including GBM.^28,29 To explore the core process behind this result, we conducted an impartial analysis using a ubiquitin microarray to evaluate the ubiquitination status of 49 distinct proteins. The proteins exhibiting notable differences encompass Bcl-2, HSP70, ErbB3, IRF3 and Insulin Receptor. In light of the established relationship between TRIM22 and apoptosis in glioma cells, our analysis will primarily concentrate on Bcl-2.^30–33 TRIM22 was identified as a likely catalyst for Bcl-2 activation (Figure 4(a)). The Bcl-2 protein serves as a crucial moderator of apoptosis and a potential therapeutic target for cancer.^34,35 Given the association of TRIM22 with apoptosis in GBM (Figure 3), we focused on Bcl-2. The impact of TRIM22 overexpression on Bcl-2 expression levels in GBM was examined. Western blot analysis revealed increased Bcl-2 protein levels in two cell lines infected with TRIM22-overexpression adenovirus (AD TRIM22) relative to those in the controls. We also examined the protein expression levels of phosphorylated Bcl-2 and Bax. The results showed that compared with the phosphorylated BCL-2 protein, the effect of TRIM22 on BAX was not significant. Evidence indicates that anti-apoptotic functions of BcI-2 can be regulated by its phosphorylation.^36,37 Although the Bcl-2 protein expression levels were increased, the Bcl-2 mRNA levels remained unchanged (Figure 4(b,c)). These findings suggested that TRIM22 influences Bcl-2 at the protein level rather than affecting Bcl-2 mRNA expression.

Figure 4.

TRIM22 modulates the protein level of Bcl-2 in glioblastoma.

(a) TRIM22 overexpression enhanced the ubiquitination of Bcl-2. A human ubiquitin array kit was used to identify the TRIM22 ubiquitination targets. Cell lysates obtained from either control or U251 cells transduced with adenoviruses expressing TRIM22 were utilized for each array. Each dot corresponds to the degree of ubiquitination detected in a target protein, as identified by an antibody targeting ubiquitin. RS =reference spot. (b) Western blotting was used to identify the expression levels of Bcl-2 and proteins associated with Bcl-2. (c) The mRNA levels of TRIM22 and Bcl-2 in GBM cells were examined (ns= no significance, *p < 0.05, **p < 0.01, and ****p < 0.0001).

3.5. TRIM22 promotes the stability of Bcl-2 via nondegradative ubiquitination

Because increased levels of TRIM22 increased Bcl-2 protein expression but did not affect Bcl-2 mRNA expression in U251 and U118 cells (Figure 4), Bcl-2 protein may be more stable within cells that have an abundance of TRIM22. Thus, we assessed the half-life of Bcl-2 by treated U251 and U118 cells with cycloheximide, which revealed an extended half-life of Bcl-2 in the AD-TRIM22 U251 and U118 lines (Figure 5(a,b)). Previous studies have indicated that the ubiquitin proteasome system (UPS) is capable of degrading Bcl-2.^38,39 TRIM22 overexpression induced the endogenous ubiquitination of Bcl-2 in U251 and 293T cells (Figure 5(c)), and the ubiquitination trend associated with K63 in Bcl-2 aligned with that in TRIM22 (Figure 5(d)). Thus, these findings suggested that TRIM22 enhances Bcl-2 stability through nondegradative ubiquitination.

Figure 5.

Effects of TRIM22 on the ubiquitination of Bcl-2.

(a) The Bcl-2 protein levels in AD-TRIM22 U251 cells subjected to cycloheximide (CHX) treatment for 0, 12, 24, and 36 h were evaluated via Western blotting. (b) The Bcl-2 protein levels in AD-TRIM22 U118 cells subjected to cycloheximide (CHX) treatment for 0, 12, 24, and 36 h were investigated via Western blotting. (c) Ubiquitination assay of Bcl-2 in U251 and 293T cells. (d) Anti-K63-linkage-specific polyubiquitin assay of Bcl-2 in U251 and 293T cells.

3.6. TRIM22 binds to Bcl-2

To explore the underlying process by which TRIM22 influences the growth and metastasis of GBM, IP was performed to determine whether there is an endogenous interaction between TRIM22 and Bcl-2. Coimmunoprecipitation (co-IP) analyses were conducted using the U251 and U87 cell lines. TRIM22 precipitated Bcl-2, and reverse co-IP experiments indicated that Bcl-2 also precipitated TRIM22 in glioblastoma cells  (Figure 6(a,b)) . To further confirm their interaction, Flag-TRIM22 and His-Bcl-2 were cotransfected into 293T cells, and a reciprocal IP was performed, which revealed efficient exogenous binding (Figure 6(c)) . In conclusion, these results demonstrated that TRIM22 promotes the stability of the Bcl-2 apoptosis-related protein through nondegradative ubiquitination, thereby inhibiting apoptosis in GBM and facilitating the occurrence and progression of tumors.

Figure 6.

TRIM22 binds to Bcl-2.

(a, b) Western blot analysis of coimmunoprecipitates utilizing either the anti-Bcl-2 or the anti-TRIM22 antibodies in U87 and U251 cell lysates. (c) Western blotting of coimmunoprecipitates was conducted using lysates derived from 293T cells cotransfected with Flag-TRIM22 and His-Bcl-2. (d) A visual representation illustrating the role of TRIM22 in activating Bcl-2 and promoting the growth of glioblastoma.

4. Discussion

The attributes of neoplastic cells, specifically their resistance to apoptosis and uncontrolled proliferative potential, present significant obstacles in the field of cancer treatment research.^40,41 Previous research has indicated that TRIM22 primarily serves to identify viral agents, impede viral replication processes, stimulate immune cell activity, and oversee ubiquitination mechanisms.^42,43 Recent research has highlighted the function of TRIM22 as an E3 ubiquitin ligase in tumors.^44,45 In this study, analysis of expression data from TCGA and GEO databases revealed that TRIM22 mRNA levels were elevated in GBM patients. In an independent group of primary human gliomas, elevated TRIM22 protein levels were associated with increased tumor grade. Cell function assays demonstrated that TRIM22 upregulation enhanced cell growth, invasion, and metastasis both in vitro and in vivo. These findings align with the previously reported results on TRIM22 in GBM, indicating that TRIM22 is instrumental in advancing the progression and malignancy of cancer cells.^28,29

TRIM22 is an E3 ubiquitin ligase that is pivotal in the development of tumors.⁴⁶ Ubiquitination can take place in multiple forms, and each type of polyubiquitination plays a distinct and essential role. The ubiquitination of K6 is involved in DNA repair, mitochondrial quality maintenance, tumor development, and Parkinson’s disease pathogenesis.⁴⁷ The ubiquitination of K11 is a key player in the cell cycle and transport events,⁴⁸ and the process of K27 ubiquitination is essential in the regulation of mitophagy and is also involved in the transmission of immune signals within T cells.⁴⁹ Moreover, the regulation of protein degradation and stress response through ubiquitination is a key focus in the field of K29 ubiquitination.⁵⁰ K33 polyubiquitination is involved in the interaction between proteins and mitochondrial autophagy.⁵¹ In addition, the Met1 enzyme is commonly associated with facilitating the linkage between the N-terminal methionine (M1) residue found on substrates and the C-terminal glycine residue of Ub, resulting in the formation of a peptide bond; the resulting signaling scaffold is instrumental in the modulation of inflammatory reactions, cellular apoptosis, and xenophagy.⁵² K48-linked or K63-linked chains are the most prevalent and best studied cases.⁵³ K48-linked chains serve as markers that guide proteins toward the proteasome for degradation, whereas K63-linked chains primarily function in processes that do not involve proteolysis, including inflammatory signaling and endocytosis. The present study revealed TRIM22 overexpression increased Bcl-2 expression, Bcl-2 phosphorylation, and K63-linked ubiquitination of Bcl-2.

The diverse proteins within the Bcl-2 family each play unique and vital roles throughout organismal development and in organismal functions. Diverse cancer cells depend on the activation of various prosurvival Bcl-2 proteins to maintain their longevity; for example, high expression of MCL-1 in neuroblastoma is associated with drug resistance.⁵⁴ Elevated amounts of Bcl-2 have been observed in various hematological malignancies and in specific solid tumors, such as colorectal cancer (CRC).^55,56 The role of Bcl-2 in preventing apoptosis is accomplished through its direct interaction with and binding to proapoptotic BH3-only proteins, which either directly or indirectly trigger the activation of Bax/BAK.⁵⁷ Bcl-2 is additionally implicated in the modulation of the autophagic process, and Bcl-2 phosphorylation inhibits Beclin1-dependent autophagic death.⁵⁸ Nevertheless, the impact of Bcl-2 phosphorylation on the enhancement or reduction of antiapoptotic capabilities remains unclear. The present findings indicated that activated TRIM22 facilitates the phosphorylation of Bcl-2 at threonine 56 and serine 70. Future research should consider the relationship between these site characteristics and the ubiquitination sites of TRIM22 to deepen the understanding of the function and mechanisms of TRIM22 in gliomas.

In summary, the present study revealed a novel regulatory mechanism for TRIM22. TRIM22 is significantly upregulated in glioblastoma multiforme (GBM), and it modulates the activation of Bcl-2 through its function as an E3 ubiquitin ligase. TRIM22 enhances the proliferation of glioblastoma multiforme (GBM) by facilitating the phosphorylation or stabilization of Bcl-2 via K63-mediated ubiquitination. The present findings lay foundation for developing diagnostic and treatment strategies aimed at addressing gliomas in patients by focusing on TRIM22.

The present study had several limitations. The present study explored the processes that control the regulation of Bcl-2 protein levels. Although the present findings demonstrated that TRIM22 regulates the stability of Bcl-2 through nondegradative polyubiquitination, it remains unclear whether TRIM22 induces K48/K63 branched or unbranched mixed-linkage ubiquitin chains on Bcl-2.^59,60 Additional forms of posttranslational modifications, including methylation and SUMOylation, may serve as regulatory mechanisms in the modulation of Bcl-2. Moreover, additional members of the Bcl-2 family may be subject to similar regulatory mechanisms; however, this phenomenon has yet to be explored.

Funding Statement

This work was supported by the Wuxi Taihu Lake Talent Plan, Supports for Leading Talents in Medical and Health Profession Grant No. [DJYX-2020]; the Scientific Research Project of Wuxi Health Commission Grant No. [2020ZHYB16]; the National Natural Science Foundation of China Grant No. [82172955]; and the Youth project of Wuxi commission of Health [Q202133]

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/23723556.2025.2518679.