Home Exosomal circular RNA NT5E driven by heterogeneous nuclear ribonucleoprotein A1 induces temozolomide resistance by targeting microRNA-153 in glioma cells
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Exosomal circular RNA NT5E driven by heterogeneous nuclear ribonucleoprotein A1 induces temozolomide resistance by targeting microRNA-153 in glioma cells

  • Renjie Wang , Ruichao Jia , Junqiang Dong , Nan Li and Haiqian Liang ORCID logo EMAIL logo
Published/Copyright: November 6, 2023
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Oncologie
From the journal Oncologie Volume 26 Issue 1

Abstract

Objectives

Exosomally transferred circular RNAs (circRNAs) are critical in cancer. However, the study of exosomal circRNAs in glioma resistance remains limited. Here, we further investigated the function and mechanism of exosomal circular RNA NT5E (circNT5E) in temozolomide-resistant glioma cells (TMZ-GCs).

Methods

Exosomes were isolated from TMZ-GCs and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. CircNT5E, microRNA-153 (miR-153), and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) levels were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in TMZ-sensitive and TMZ-resistant GCs and in treated TMZ-GCs. In addition, the colocalization of circNT5E and miR-153 was confirmed by fluorescence in situ hybridization (FISH) and dual-luciferase reporter assays. Internalization of exosomes was observed by immunofluorescence staining. TMZ resistance, proliferation, and pAKTser473 protein levels were evaluated by a Cell Counting Kit-8 (CCK-8) assay, an EdU incorporation assay, and Western blotting, respectively. In addition, tumor growth was examined using a xenograft tumor model in nude mice.

Results

We first proved that circNT5E was highly abundant in exosomes derived from TMZ-GCs. Then, we discovered that circNT5E could serve as a miR-153 sponge. Finally, knockdown of circNT5E reduced TMZ resistance and cell proliferation and downregulated AKTser473 phosphorylation by targeting miR-153 in TMZ-GCs. Moreover, our data revealed that exosomes derived from TMZ-GCs also had obvious effects on inducing the TMZ resistance and proliferation of GCs. Moreover, we revealed that the packaging of circNT5E into exosomes can be driven by hnRNP A1.

Conclusions

Collectively, our findings proved that exosomal circNT5E transferred in a manner mediated by hnRNPA1 could accelerate TMZ resistance by targeting miR-153 in GCs, indicating that exosomal circNT5E is a therapeutic target for TMZ-resistant glioma.

Keywords: circular RNA NT5E; glioma; microRNA-153; exosome; temozolomide resistance; heterogeneous nuclear ribonucleoprotein A1

Introduction

Glioma is the most common malignant tumor. Patients with highly malignant gliomas have a survival time of less than 1 year. Temozolomide (TMZ) has a clear therapeutic effect on glioma [1]. TMZ combined with radiotherapy can increase the survival rate of glioma patients. Although TMZ is a first-line chemotherapy drug for glioma, the high recurrence and fatality rate of glioma remain threats to human health. Therefore, finding ways to prevent glioma growth and increase TMZ sensitivity to protect against the development of tolerance to chemotherapy is one of the hot research topics in glioma therapy.

Exosomes are a class of functional bilayer membrane structures produced in multivesicular bodies and released from various cells. Exosomes can carry multiple bioactive molecules, including lipids, proteins, circular RNAs (circRNAs), microRNAs (miRNAs), etc. Exosomes have been considered novel transmitters of intercellular signal communication [2]. A study confirmed that exosomes can participate in tumor angiogenesis, growth, metastasis, etc. [3]. Circular RNAs (circRNAs) are structurally stable and evolutionarily conserved in eukaryotes. Studies have proven that circRNAs play a key role in tumors and cardiovascular and neurodegenerative diseases [4, 5]. In recent years, researchers have identified a vast number of circRNAs in gliomas using high-throughput sequencing technologies [6]. Among these identified circRNAs, circRNAs highly expressed in glioma cells (GCs) have been verified to affect glioma cell apoptosis, proliferation, metastasis and drug resistance [7]. Exosomes, as transporters, play a biological role mostly dependent on the biomolecules they carry within them. Exosome-derived circRNAs utilize exosomes and their characteristics of stability and expression differences to cooperatively influence cancer progression [8]. Previous studies from our group and other groups showed that circular RNA NT5E (circNT5E) has prominent effects on inducing the progression of glioblastoma, lung cancer, and bladder cancer [9], [10], [11], [12]. However, the action and mechanism of exosomal circNT5E in mediating TMZ resistance in GCs are not clear.

Thus, we investigated the function and possible mechanisms of exosomal circNT5E in TMZ resistance in glioma. We first revealed circNT5E levels in exosomes derived from temozolomide-resistant glioma cells (TMZ-GCs) and explored the effects of circNT5 and exosomes derived from TMZ-GCs on cell proliferation and TMZ resistance. From a mechanistic perspective, we screened for and confirmed the downstream genes of circNT5E that regulate TMZ resistance in GCs and the possible mechanisms of circNT5E packaging into exosomes. Therefore, we might have uncovered a novel mechanism of TMZ resistance in glioma.

Materials and methods

Cell culture

U87 and U251 cells were obtained from the American Type Culture Collection (ATCC). U87 cells were grown in minimum essential medium (MEM; cat. no. 41500034, Gibco, Inc., NY, USA) supplemented with 10 % fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), and U251 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; cat. no. 12800017, Gibco, Inc., NY, USA) supplemented with 10 % FBS at 37 °C and 5 % CO2 [13].

Establishment of TMZ-resistant cells

Cells were treated with progressively increasing concentrations of TMZ (Cat# T2577, Sigma-Aldrich, St. Louis, MO, USA) in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) for 20 days at each concentration for a total of 5–6 months at 37 °C and 5 % CO2. TMZ concentration required to induce resistance in cells was 50 μg/mL [14, 15].

Exosome isolation

The supernatant was collected after 72 h of cell culture (TMZ-resistant U87 and U251 cells). Differential centrifugation was performed to isolate exosomes using a Total Exosome Isolation Kit (Invitrogen™, Carlsbad, CA, USA) [16].

Transmission electron microscopy (TEM)

Briefly, exosome samples (10 μL) and phosphotungstic acid (10 μL) were sequentially precipitated dropwise onto a copper grid. After drying for a few minutes, exosomes were observed by TEM (TEM-2100, JEOL, Akishima, Tokyo, Japan) [14].

Nanoparticle tracking analysis (NTA)

Exosome samples (5 μL) were diluted to 30 μL with PBS, and the particle size and concentration were tested using a Flow Nano Analyzer (NanoFCM, Co., Ltd., Xiamen, China) [14].

Cell treatment

Lentivirus (LV)-scramble and LV-si-circNT5E were purchased from GenePharma (Suzhou, China). The negative control siRNA (NC) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) siRNA (si-hnRNPA1), anti-NC, and anti-microRNA-153 (anti-miR-153) constructs were acquired from GenePharma (Suzhou, China). U251-R and U87-R cells (1 × 105 cells/well) in 6-well plates were transfected with LV-scramble, LV-si-circNT5E, anti-miR-153, anti-NC, si-hnRNPA1, or NC using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s specifications. U87 and U251 cells were also treated with 10, 20, and 40 μg/mL exosomes derived from TMZ-resistant U251 and U87 cells [17].

Western blot

Total protein was isolated with radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). After quantification, proteins (40 μg) were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After closure, the membranes were incubated with a diluted primary antibody (Abcam, Waltham, MA, ab134045, ab125011, ab8805, ab285034) and the corresponding diluted secondary antibody (ab6789, Abcam, Waltham, MA). Finally, the membranes were immersed in enhanced chemiluminescence (ECL) reagent (Millipore, Billerica, MA, USA), and the protein bands were visualized [18].

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was obtained with TRIzol (Invitrogen, MA, USA) and reverse transcribed into cDNA with a reverse transcription kit (Takara, Tokyo, Japan). Then, qRT-PCR was performed with SYBR Green qPCR Master Mix (Bio‐Rad Laboratories, Hercules, CA). The relative expression levels were determined by the 2−ΔΔCt method [18].

Dual-luciferase reporter assay

The pmirGLO vector was used to construct wild-type (WT) and mutant (MUT) circNT5E plasmids. Then, TMZ-GCs were cotransfected with WT-circNT5E, MUT-circNT5E, miR-153 mimics or NC. Luciferase activity was assayed based on the instructions of the Dual Luciferase Reporter Gene Assay Kit (Solarbio Science & Technology Co., Ltd. Beijing, China) [19].

Fluorescence in situ hybridization (FISH)

We first designed and synthesized a circNT5E probe labeled with fluorescein isothiocyanate (FITC) and a miR-153 probe labeled with Texas Red (obtained from RiboBio, Guangzhou, China). TMZ-GCs were seeded on 1 cm × 1 cm slides in a 24-well plate and fixed with 4 % paraformaldehyde. Experiments were performed with a FISH kit (RiboBio, Guangzhou, China), and the cells were finally fixed with 4′6-diamidino-2-phenylindole (DAPI; Roche, Shanghai, China). The stained slides were photographed with a laser confocal scanning microscope [20].

Cell Counting Kit-8 (CCK-8) assay

After 48 h of treatment as indicated, cells (2 × 103 cells/well) in 96-well plates were spiked with 10 μL CCK-8 reagent (Beyotime, Shanghai, China). After 2 h, the optical density (OD) was measured at 450 nm by a Spectrophotometer (Thermo Fisher Scientific Inc., Waltham MA, USA), and the IC50 was calculated [14].

EdU incorporation assay

Cells (4 × 104 cells/well) were seeded in a 96-well plate and cultured for 48 h. The cells were processed in several steps, including culture in medium containing EdU (1:1,000) for 2 h, fixation with 4 % paraformaldehyde, Apollo staining, and Hoechst 33342 staining. Then, the cells were examined using a fluorescence microscope (BX63; Olympus Corp., Tokyo, Japan) [21].

Immunofluorescence (IF)

Cells were seeded in 6-well plates and fixed. After washing, the cells were permeabilized with 0.5 % Triton X-100, blocked with 2 % bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), and incubated with exosome solutions labelled with PKH67 (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C and then with a fluorescent secondary antibody for 1 h protected from light. After 15 min of DAPI staining, the slides were mounted and observed [22].

In vivo xenograft modeling

Specific pathogen-free (SPF) grade male BALB/c nude mice were obtained from Beijing Huafukang Biotechnology Co., Ltd. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The animal study was approved by the animal management committee of the Characteristic Medical Center of the Chinese People’s Armed Police Force (No. 2021-0021). The animals in this study were all male, and there was no influence of sex on (or association with) the results of the study. U251 cells were treated with TMZ and/or exosomes derived from TMZ-resistant U251 cells. Then, the abovementioned U251 cells (4 × 106) were inoculated into the dorsal surface of mice. After 28 days, the mice were sacrificed, and the tumors were excised and weighed [14].

Statistical analysis

Data analysis and image generation were performed mainly using SPSS 24.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Software, SDG, USA). The measurement data are presented as the means±standard deviations (SDs). The t test and one-way ANOVA were applied for comparisons. p<0.05 was considered to indicate a significant difference [18].

Results

CircNT5E is more abundant in exosomes derived from TMZ-GCs

To investigate the influence of exosomal circNT5E on the functions of TMZ-GCs, we first isolated exosomes from TMZ-GCs. TMZ-resistant U87 and U251 cells were first generated (IC50 values for TMZ were shown in Figure 1A) based on a previous study [15]. We observed by TEM that exosome derived from TMZ-GCs had a circular vesicle structure (Figure 1A). NTA indicated that the vesicle size was mainly between 100 and 200 nm (Figure 1B). Then, we monitored CD63 and TSG101 expression on the isolated vesicles by Western blotting. The data revealed that these two proteins were expressed on exosomes from TMZ-sensitive and TMZ-resistant U87 and U251 cells, especially TMZ-GCs (Figure 1C). Thus, we confirmed that the isolated vesicles were exosomes derived from TMZ-GCs. Subsequently, we analyzed circNT5E expression in TMZ-sensitive and TMZ-resistant GCs and discovered that circNT5E expression was observably increased in TMZ-GCs relative to GCs (Figure 1D). In addition, we transduced LV-scramble and LV-si-circNT5E plasmids into TMZ-GCs. We demonstrated that the circNT5E level was lower in TMZ-GCs transduced with LV-si-circNT5E than in those transduced with LV-scramble (Figure 1E). However, circNT5E silencing did not significantly change NT5E mRNA expression (Figure 1F).

Figure 1: CircNT5E is more abundant in exosomes derived from TMZ-GCs. (A) Representative images of exosomes secreted by TMZ-resistant U87 cells (U87-R) and TMZ-resistant U251 cells (U251-R). IC50 values for TMZ in different cell lines were shown. Scale bar=100 nm. (B) The concentration and distribution of the exosomes was evaluated by NTA. (C) Western blot analysis showed the levels of CD63 and TSG101 in exosomes derived from U87 or U87-R and U251 or U251-R cells. (D) qRT-PCR was adopted to determine the circNT5E level in TMZ-sensitive and TMZ-resistant GCs. After circNT5E silencing, the changes in circNT5E (E) and NT5E mRNA (F) levels were confirmed with qRT-PCR in TMZ-sensitive and TMZ-resistant GCs. ns, no statistical significance; *p<0.05, **p<0.01, ***p<0.001.
Figure 1:

CircNT5E is more abundant in exosomes derived from TMZ-GCs. (A) Representative images of exosomes secreted by TMZ-resistant U87 cells (U87-R) and TMZ-resistant U251 cells (U251-R). IC50 values for TMZ in different cell lines were shown. Scale bar=100 nm. (B) The concentration and distribution of the exosomes was evaluated by NTA. (C) Western blot analysis showed the levels of CD63 and TSG101 in exosomes derived from U87 or U87-R and U251 or U251-R cells. (D) qRT-PCR was adopted to determine the circNT5E level in TMZ-sensitive and TMZ-resistant GCs. After circNT5E silencing, the changes in circNT5E (E) and NT5E mRNA (F) levels were confirmed with qRT-PCR in TMZ-sensitive and TMZ-resistant GCs. ns, no statistical significance; *p<0.05, **p<0.01, ***p<0.001.

CircNT5E silencing attenuates TMZ resistance and cell proliferation and reduces the pAKTser473 level in TMZ-GCs by targeting miR-153

Next, we further elucidated the mechanism of circNT5E in TMZ-GCs. In the circBank database, we screened for the binding sites of multiple miRNAs in circNT5E. Through literature analysis, we found that miR-153 is closely related to glioma. miR-153 also became the target of our subsequent research. First, the FISH results indicated that circNT5E and miR-153 were colocalized in TMZ-GCs (Figure 2A). Moreover, we determined the binding sites of miR-153 within WT or mutant circNT5E (Figure 2B). miR-153 overexpression observably reduced the luciferase activity of the WT circNT5E reporter but had no effect on the mutant circNT5E reporter, suggesting that circNT5E can directly bind to miR-153 (Figure 2C). Then, we transfected anti-miR-153 into TMZ-GCs to downregulate miR-153 expression. The qRT-PCR data showed that the miR-153 level was notably decreased in the anti-miR-153 group compared to the anti-NC group, indicating the successful downregulation of miR-153 in TMZ-GCs. Then, the CCK-8 assay data showed that the IC50 of TMZ in the circNT5E silencing group was markedly lower than that in the control (LV-scramble+anti-NC) group and that miR-153 inhibition dramatically increased the IC50 of TMZ in TMZ-GCs, while miR-153 inhibition also prominently reversed the effect of circNT5E silencing on decreasing the IC50 of TMZ in TMZ-GCs (Figure 2D). In addition, the EdU incorporation assay results also indicated that circNT5E silencing markedly diminished cell proliferation and that miR-153 inhibition significantly increased cell proliferation, while the attenuating effect of circNT5E silencing on cell proliferation was notably restored by miR-153 inhibition in TMZ-GCs (Figure 2E and F). Additionally, the Western blot results revealed that circNT5E silencing markedly decreased the pAKTser473 level, that miR-153 inhibition observably increased the pAKTser473 level, and that miR-153 inhibition prominently attenuated the decrease in the pAKTser473 level mediated by circNT5E silencing in TMZ-GCs (Figure 2G and H).

Figure 2: CircNT5E silencing attenuates TMZ resistance and cell proliferation and reduces the pAKTser473 level in TMZ-GCs by targeting miR-153. TMZ-GCs were transduced with LV-scramble or LV-si-circNT5E and anti-miR-153 or anti-NC. (A) The colocalization of circNT5E and miR-153 was analyzed using FISH in TMZ-GCs. (B) The possible binding sites of miR-153 within WT or mutant circNT5E. (C) TMZ-GCs were cotransfected with the WT circNT5E or mutant circNT5E plasmids and miR-153 mimic or mimic-NC, and luciferase activity was determined using a dual-luciferase reporter assay. (D) qRT-PCR analysis of miR-153 in TMZ-GCs transfected with anti-miR-153. The IC50 of TMZ was tested by a CCK-8 assay. (E and F) An EdU incorporation assay was adopted to assess the change in cell proliferation in each group. (G and H) Western blotting was applied to monitor the changes in the levels of pAKTser473 and AKT. *p<0.05, **p<0.01, ***p<0.001. Scale bar: 100 μm.
Figure 2:

CircNT5E silencing attenuates TMZ resistance and cell proliferation and reduces the pAKTser473 level in TMZ-GCs by targeting miR-153. TMZ-GCs were transduced with LV-scramble or LV-si-circNT5E and anti-miR-153 or anti-NC. (A) The colocalization of circNT5E and miR-153 was analyzed using FISH in TMZ-GCs. (B) The possible binding sites of miR-153 within WT or mutant circNT5E. (C) TMZ-GCs were cotransfected with the WT circNT5E or mutant circNT5E plasmids and miR-153 mimic or mimic-NC, and luciferase activity was determined using a dual-luciferase reporter assay. (D) qRT-PCR analysis of miR-153 in TMZ-GCs transfected with anti-miR-153. The IC50 of TMZ was tested by a CCK-8 assay. (E and F) An EdU incorporation assay was adopted to assess the change in cell proliferation in each group. (G and H) Western blotting was applied to monitor the changes in the levels of pAKTser473 and AKT. *p<0.05, **p<0.01, ***p<0.001. Scale bar: 100 μm.

Exosomes derived from TMZ-GCs promote the TMZ resistance and proliferation of GCs

To further elucidate the impact of exosomal circNT5E on GCs, we first cocultured U87 and U251 cells (TMZ-sensitive GCs) with exosomes derived from PKH67-labeled TMZ-GCs. Our results indicated that U87-R and U251-R cell-derived exosomes could be endocytosed by GCs (Figure 3A). Then, GCs were treated with exosomes derived from TMZ-GCs (10, 20, and 40 μg/mL), and the qRT-PCR data revealed that these exosomes dramatically upregulated circNT5E in GCs and that the upregulation of circNT5E increased with increasing exosome concentration (Figure 3B). Then, the EdU incorporation assay results revealed that TMZ markedly reduced the EdU/Hoechst ratio and that treatment with exosomes derived from TMZ-GCs significantly elevated the EdU/Hoechst ratio, while the reduction in the EdU/Hoechst ratio mediated by TMZ in GCs was notably reversed by exosomes derived from TMZ-GCs (Figure 3C and D). Moreover, U251 cells were treated with TMZ and/or exosomes derived from U251-R cells, which were then injected subcutaneously into nude mice. The in vivo results showed that TMZ treatment observably reduced the tumor size and weight and that exosomes derived from U251-R cells markedly increased the tumor size and weight, while the reductions in the tumor size and weight mediated by TMZ were significantly reversed by exosomes derived from U251-R cells (Figure 3E).

Figure 3: Exosomal circNT5E promotes the TMZ resistance and proliferation of GCs. (A) After labeling with PKH67, exosomes derived from TMZ-GCs were cocultured with U87 and U251 cells. The internalization of exosomes was observed by immunofluorescence staining. (B) The expression of circNT5E was analyzed through qRT-PCR in GCs after treatment with exosomes derived from TMZ-GCs. (C and D) An EdU incorporation assay was adopted to evaluate the proliferation of GCs that were treated with TMZ and/or exosomes derived from TMZ-GCs. (E) U251 cells were first treated with TMZ and/or exosomes derived from TMZ-resistant U251 cells and applied to establish a xenograft tumor model in nude mice (there were three mice in each group). Tumor weight was calculated after 28 days. *p<0.05, **p<0.01, ***p<0.001. Scale bar: 100 μm.
Figure 3:

Exosomal circNT5E promotes the TMZ resistance and proliferation of GCs. (A) After labeling with PKH67, exosomes derived from TMZ-GCs were cocultured with U87 and U251 cells. The internalization of exosomes was observed by immunofluorescence staining. (B) The expression of circNT5E was analyzed through qRT-PCR in GCs after treatment with exosomes derived from TMZ-GCs. (C and D) An EdU incorporation assay was adopted to evaluate the proliferation of GCs that were treated with TMZ and/or exosomes derived from TMZ-GCs. (E) U251 cells were first treated with TMZ and/or exosomes derived from TMZ-resistant U251 cells and applied to establish a xenograft tumor model in nude mice (there were three mice in each group). Tumor weight was calculated after 28 days. *p<0.05, **p<0.01, ***p<0.001. Scale bar: 100 μm.

HnRNPA1 mediates circNT5E packaging into exosomes

More importantly, we further investigated whether HnRNPA1 can drive the packaging of circNT5E into exosomes. We first adopted RBPDB analysis to predict the interactions between RBP motifs and circNT5E, and the data indicated that the relative score was 100 % or 97 %, the matching sequence was UAGGGA or UAGGGU, and the threshold was 0.7 (Figure 4A). We also identified the target sites with WebLogo 3 (Figure 4B). Then, by transfection, we silenced hnRNPA1 in the TMZ-GCs, and the qRT-PCR results verified that hnRNPA1 was markedly downregulated in the si-hnRNPA1 group compared with the si-NC group, indicating the successful silencing of hnRNPA1 (Figure 4C). In addition, we discovered that hnRNPA1 silencing prominently reduced the circNT5E abundance in exosomes derived from TMZ-GCs (Figure 4D). Finally, we also revealed that exosomes from hnRNPA1-silenced TMZ-GCs dramatically decrease the level of circNT5E in U87 and U251 cells (Figure 4E). These results suggested that circNT5E could be packaged into exosomes by hnRNPA1 and that exosomal circNT5E could also accelerate TMZ resistance by targeting miR-153 in GCs (Figure 5).

Figure 4: HnRNPA1 mediates circNT5E packaging into exosomes. (A) The interactions between RBP motifs and circNT5E were predicted through RBPDB analysis (threshold=0.7). (B) The target sites were also visualized with WebLogo 3. qRT-PCR for the confirmation of hnRNPA1 (C) and circNT5E (D) expression in TMZ-GCs transfected with si-hnRNPA1 or NC. (E) The circNT5E level was confirmed in GCs treated with exosomes derived from hnRNPA1-silenced TMZ-GCs. *p<0.05, **p<0.01, ***p<0.001. Scale bar: 100 μm.
Figure 4:

HnRNPA1 mediates circNT5E packaging into exosomes. (A) The interactions between RBP motifs and circNT5E were predicted through RBPDB analysis (threshold=0.7). (B) The target sites were also visualized with WebLogo 3. qRT-PCR for the confirmation of hnRNPA1 (C) and circNT5E (D) expression in TMZ-GCs transfected with si-hnRNPA1 or NC. (E) The circNT5E level was confirmed in GCs treated with exosomes derived from hnRNPA1-silenced TMZ-GCs. *p<0.05, **p<0.01, ***p<0.001. Scale bar: 100 μm.

Figure 5: CircNT5E could be packaged into exosomes by hnRNPA1 and that exosomal circNT5E could also accelerate TMZ resistance by targeting miR-153 in GCs.
Figure 5:

CircNT5E could be packaged into exosomes by hnRNPA1 and that exosomal circNT5E could also accelerate TMZ resistance by targeting miR-153 in GCs.

Discussion

Glioma is a common primary intracranial tumor with a poor prognosis [23]. Currently, chemoresistance has been a major obstacle in glioma therapy, and TMZ is a commonly applied chemotherapeutic agent for glioma, but most patients eventually become resistant to TMZ during the course of chemotherapy [24]. Therefore, it is extremely important to elucidate the mechanism of TMZ resistance to improve the effect of chemotherapy in patients with glioma.

Exosomes are a class of extracellular vesicles secreted by cancer cells and stromal cells in the tumor microenvironment [25]. Research has shown that exosomes can increase chemotherapeutic drug resistance by transferring their contents into cancer cells [26]. A recent study revealed that exosomes have a significant regulatory effect on glioma progression [27]. Currently, some circRNAs have been found to be able to be monitored in serum, urine, and tumor-derived exosomes, and these circRNAs can participate in cell growth, angiogenesis, tumor drug resistance, and the response to targeted therapies. A recent study suggested that exosomal circNFIX could also increase TMZ resistance in glioma [28]. These studies proved that exosomal circRNAs could alter TMZ resistance in glioma.

According to the circBase database, circNT5E (hsa_circ_0077232) is formed by the cyclization of the NT5E precursor RNA, located at chr6:86180954-86205509 in the genome. Research has shown that circNT5E can be upregulated in glioblastoma samples [29]. Our previous study also revealed that circNT5E could induce glioblastoma tumorigenesis by targeting miR-422a [11]. However, the impact of circNT5E on TMZ resistance in glioma has not been reported. In this study, we first proved that the circNT5E abundance can be increased in exosomes derived from TMZ-GCs, suggesting that exosomal circNT5E might be associated with TMZ resistance in glioma. Our data also showed that circNT5E silencing can reduce TMZ resistance, cell proliferation and the pAKTser473 level in TMZ-GCs, suggesting that circNT5E silencing has an obstructive role in the malignant progression of TMZ-GCs. We also revealed that exosomes derived from TMZ-GCs increased the TMZ resistance and proliferation of GCs, indicating that exosomes could accelerate glioma progression. Therefore, we preliminarily concluded that exosomal circNT5E has an obvious accelerating effect on TMZ resistance and malignant progression in glioma.

A study confirmed that circRNAs have roles as miRNA sponges, regulate selective splicing, bind RNA-binding proteins, and encode proteins. In our study, we discovered through bioinformatics analysis that miR-153 might be a downstream target gene of circNT5E. miR-153 has a significant attenuating role in the development of multiple cancers, such as gastric [30], prostate [31], and pancreatic cancers [32]. Studies have also demonstrated that miR-153 can also prevent the malignant progression of glioma [33, 34]. In our study, we further demonstrated that the role of exosomal circNT5E in inducing TMZ resistance in glioma cells can be achieved by targeting miR-153.

hnRNPA1, a member of the hnRNPA/B subfamily, is an essential RNA-binding protein in vivo. hnRNPA1 can shuttle freely between the nucleus and cytoplasm and participate in transcriptional and posttranscriptional regulation by regulating pre-mRNA and mRNA splicing, thus maintaining gene stability [35]. In addition, hnRNPA1 can activate different pathways by binding to different genes, thus affecting cell biological functions. In recent years, several studies have indicated that hnRNPA1 can mediate the packaging of biomolecules into exosomes. For example, hnRNPA1 can induce miR-27b-3p packaging into exosomes [36]; hnRNPA1 can also mediate lincROR packaging into exosomes [37]. In our study, we also proved that hnRNPA1 can mediate circNT5E packaging into exosomes.

There are several major limitations in this study that could be addressed in future research. First, we preliminarily investigated the effect of exosomes derived from U251-R cells on tumor growth in nude mice by an in vivo study. However, the small sample size in the in vivo study may have contributed to the variability of the results. In future studies, we will expand the sample size of nude mice to further explore the role and mechanism of exosomal circNT5E transferred in a manner mediated by hnRNPA1 in glioma xenograft models. Second, this study showed that exosomal circNT5E transferred in a manner mediated by hnRNPA1 may be a potential therapeutic target for TMZ-resistant glioma, mainly through its cellular and molecular mechanisms. In future studies, we will extend the experimental studies to explore its clinical application in TMZ-resistant glioma patients. Third, the U87 and U251 cell lines were mainly used to investigate the potential mechanism of exosomal circNT5E transferred in a manner mediated by hnRNPA1 in this study. Due to the heterogeneity of patients with glioma, it is not sufficient to simply perform cell line-related studies. The study of the tumor microenvironment and genetic heterogeneity is also an important direction for our future research. Fourth, the current study mainly explored the role of the circNT5E/miR-153 axis in TMZ-resistant glioma cells. Further investigation of other possible mechanisms is also of great interest in TMZ-resistant glioma. Fifth, the predictions of RBPDB have inherent biases. Due to the limited time given by the journal club, this concept was beyond the scope of the current study; however, we will further validate the biological mechanism by which hnRNPA1 binds circNT5E outside of cells in future studies.

Conclusions

In summary, our results suggested that circNT5E could be packaged into exosomes by hnRNPA1 and that exosomal circNT5E could also accelerate TMZ resistance by targeting miR-153 in GCs. Therefore, we revealed that inhibition of exosomal circNT5E might provide the possibility to combat TMZ resistance in glioma.

Corresponding author: Haiqian Liang, PhD, Department of Neurosurgery, Characteristic Medical Center of Chinese People’s Armed Police Force, Tianjin 300162, China, and Chinese Glioma Cooperative Group (CGCG), Beijing, China, E-mail:
Renjie Wang and Ruichao Jia contributed equally to this work.

Funding source: Natural Science Foundation of Tianjin Municipality

Award Identifier / Grant number: 21JCQNJC01400

  1. Research ethics: All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The animal study was approved by the animal management committee of the Characteristic Medical Center of the Chinese People's Armed Police Force (No. 2021-0021). The animals in this study were all male, and there was no influence of sex on (or association with) the results of the study.

  2. Informed consent: Informed consent was obtained from all individuals included in this study.

  3. Author contributions: The authors confirm contribution to the paper as follows: study conception and design: WRJ; data collection: WRJ, JRC, DJQ, LN and LHQ; analysis and interpretation of results: WRJ, LN and LHQ; draft manuscript preparation: WRJ. All authors reviewed the results and approved the final version of the manuscript.

  4. Competing interests: Authors state no conflict of interest.

  5. Research funding: This work was supported by the Natural Science Foundation of Tianjin, No. 21JCQNJC01400 (to WRJ).

  6. Data availability: Data available on request from the authors.

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Received: 2023-06-27
Accepted: 2023-10-17
Published Online: 2023-11-06

© 2023 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  2. Review Articles
  3. Advances in ferroptosis of cancer therapy
  4. Immunotherapy in hepatocellular carcinoma: an overview of immune checkpoint inhibitors, drug resistance, and adverse effects
  5. The role of matrix metalloproteinase-2 in the metastatic cascade: a review
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  12. AKAP12 inhibits the proliferation of ovarian cancer by activating the Hippo pathway
  13. AQP1 as a novel biomarker to predict prognosis and tumor immunity in glioma patients
  14. Exosomal circular RNA NT5E driven by heterogeneous nuclear ribonucleoprotein A1 induces temozolomide resistance by targeting microRNA-153 in glioma cells
  15. miR‐30a‐3p inhibits the proliferation of laryngeal cancer cells by targeting DNMT3a through regulating DNA methylation of PTEN
  16. Disulfidptosis-related long non-coding RNAs predict prognosis and indicate therapeutic response in non-small cell lung carcinoma
  17. Case Report
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https://doi.org/10.1515/oncologie-2023-0256
Keywords for this article
circular RNA NT5E; glioma; microRNA-153; exosome; temozolomide resistance; heterogeneous nuclear ribonucleoprotein A1
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Review Articles
  3. Advances in ferroptosis of cancer therapy
  4. Immunotherapy in hepatocellular carcinoma: an overview of immune checkpoint inhibitors, drug resistance, and adverse effects
  5. The role of matrix metalloproteinase-2 in the metastatic cascade: a review
  6. The tumor microenvironment: a key player in multidrug resistance in cancer
  7. Robotic vs. laparoscopic approach in obese patients with endometrial cancer: which is the best? A mini-review
  8. SLC25 family with energy metabolism and immunity in malignant tumors
  9. Research Articles
  10. Catalase expression is an independent prognostic marker in liver hepatocellular carcinoma
  11. A novel immune-associated prognostic signature based on the immune cell infiltration analysis for hepatocellular carcinoma
  12. AKAP12 inhibits the proliferation of ovarian cancer by activating the Hippo pathway
  13. AQP1 as a novel biomarker to predict prognosis and tumor immunity in glioma patients
  14. Exosomal circular RNA NT5E driven by heterogeneous nuclear ribonucleoprotein A1 induces temozolomide resistance by targeting microRNA-153 in glioma cells
  15. miR‐30a‐3p inhibits the proliferation of laryngeal cancer cells by targeting DNMT3a through regulating DNA methylation of PTEN
  16. Disulfidptosis-related long non-coding RNAs predict prognosis and indicate therapeutic response in non-small cell lung carcinoma
  17. Case Report
  18. Primary retroperitoneal choriocarcinoma with lung and liver metastasis in a male patient: case report
  19. Short Commentary
  20. Clinical pharmacy services in cancer patients with hypertension
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