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. 2023 Jul 10;43(7):354–369. doi: 10.1080/10985549.2023.2210032

CircTTLL13 Promotes TMZ Resistance in Glioma via Modulating OLR1-Mediated Activation of the Wnt/β-Catenin Pathway

Jun Li 1,✉, Junfeng Ma 1, Shan Huang 1, Jun Li 1, Liang Zhou 1, Jiahua Sun 1, Lin Chen 1
PMCID: PMC10348032  PMID: 37427890

Abstract

Glioma, originating from neuroglial progenitor cells, is a type of intrinsic brain tumor with poor prognosis. temozolomide (TMZ) is the first-line chemotherapeutic agent for glioma. Exploring the mechanisms of circTTLL13 underlying TMZ resistance in glioma is of great significance to improve glioma treatment. Bioinformatics was adopted to identify target genes. The circular structure of circTTLL13 and its high expression in glioma cells were disclosed by quantitative real time-PCR (qRT-PCR) and PCR-agarose gel electrophoresis. Functional experiments proved that oxidized LDL receptor 1 (OLR1) promotes TMZ resistance of glioma cells. CircTTLL13 enhances TMZ resistance of glioma cells via modulating OLR1. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pulldown, mRNA stability, N6-methyladenosine (m6A) dot blot and RNA total m6A quantification assays were implemented, indicating that circTTLL13 stabilizes OLR1 mRNA via recruiting YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and promotes m6A methylation of OLR1 pre-mRNA through recruiting methyltransferase-like 3 (METTL3). TOP/FOP-flash reporter assay and western blot verified that circTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1. CircTTLL13 promotes TMZ resistance in glioma through regulating OLR1-mediated Wnt/β-catenin pathway activation. This study offers an insight into the efficacy improvement of TMZ for glioma treatment.

Keywords: circTTLL13, OLR1, Wnt/β-catenin pathway, TMZ resistance, glioma

Graphical Abstract

graphic file with name TMCB_A_2210032_UF0001_C.jpg

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INTRODUCTION

Being the most prevalent primary brain tumor, glioma originates from neuroglial progenitor cells, accounting for about 81% of central nervous system (CNS) malignancies.1 The major risk factors for glioma include family history, germline mutation, and ionizing radiation exposures. In addition, the incidence of glioma increases with age, and men have a higher incidence of glioma than women.2 As an aggressive brain tumor, glioma features invasive growth and difficulty in complete tumor resection, which limits its 5-year survival rate.3 Temozolomide (TMZ) is considered the first-line treatment for glioma, which prolongs the overall survival of patients with glioma.4,5 However, TMZ resistance undermines the efficacy of this drug. Hence, elucidating the mechanisms underlying TMZ resistance in glioma is crucial to improving its efficacy for glioma patients.

Circular RNA (circRNA) is a category of endogenous noncoding RNA featuring a closed circular structure.6 Despite the inability to code proteins, circRNAs play crucial roles in modulating various malignancies, including glioma. For instance, circPTN has been reported to sequester miR-145-5p and miR-330-5p to facilitate cell proliferation and stemness in glioma;6 circ0014359 propels glioma development by targeting miR-153/PI3K pathway; and circNFIX interference suppresses glioma progression both in vitro and in vivo by increasing miR-378e expression to downregulate RPN2 expression.7 Furthermore, the former studies revealed that circRNAs can regulate TMZ resistance of glioma. For example, circNFIX, transferred by exosomes, increases TMZ resistance in glioma;8 circ0005198 promotes TMZ resistance of glioma cells by targeting miR-198/TRIM14 axis;9 circHIPK3 inhibits TMZ sensitivity in glioma by targeting miR-524-5p/PI3K/AKT pathway;10 and circ0110757 overexpresses ITGA1 to promote TMZ resistance in glioma through downregulating miR-1298-5p.11 Herein, we used the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) to screen out circTTLL13, the focus of our study. It is a novel circRNA, which has never been reported. Its host gene, TTLL13 has been found to be a polyglutamylase.12

Oxidized LDL receptor 1 (OLR1), as reported before, can modulate multiple cancers. For instance, OLR1 facilitates the metastasis of pancreatic cancer via enhancing c-Myc expression and HMGA2 transcription.13 Moreover, it has been reported that OLR1 is closely linked to survival of head and neck squamous cell carcinoma patients.14 However, the correlation of OLR1 with glioma has never been investigated.

Wnt/β-catenin pathway, as a highly conserved and tightly controlled molecular mechanism, modulates cell proliferation and differentiation.15 It functions by modulating β-catenin to regulate the expressions of key genes.16 Moreover, as reported by previous studies, this pathway participates in the regulation of glioma. For instance, thioridazine reinforces P62-mediated autophagy and apoptosis in glioma cells through targeting Wnt/β-catenin pathway;17 miR‑301a modulates glioma progression via targeting Wnt/β-catenin pathway;18 CASC7 suppresses the development of glioma through modulating Wnt/β-catenin pathway;19 and Coronin 3 propels the progression in glioma via targeting the Wnt/β-catenin pathway.20 Nevertheless, the correlations of circRNAs with this signaling pathway in glioma have rarely been reported.

In our study, we intended to probe into the role of circTTLL13 in glioma and figure out the underlying mechanisms concerning TMZ resistance. As TMZ plays an important role in glioma treatment, we urgently need to clarify the mechanisms underlying the regulation of TMZ resistance. The findings in our study may contribute to improving glioma treatment.

RESULTS

OLR1 facilitates TMZ resistance of glioma cells

GSE100736 and GSE113510 datasets were adopted to analyze the mRNAs highly expressed in TMZ-resistant glioma tissues under the certain conditions (P value ≤ 0.05, logFC ≤ –5). As shown in the Venn diagram, six common high-expressed mRNAs in TMZ-resistant glioma tissues were obtained, namely, KYNU, BASP1, CDKN2B, OLR1, PLAC8, and BASP1P1. Among them, KYNU and CDKN2B have been studied thoroughly in glioma;21–23 and UALCAN (http://ualcan.path.uab.edu/index.html) predicted that BASP1 is significantly low-expressed in glioma tissues (Fig. 1A). Therefore, OLR1, PLAC8, and BASP1P1 were selected as candidate genes. After that, qRT-PCR was implemented to examine OLR1, PLAC8, and BASP1P1 expression levels in U343, U251, U343/TMZ, U251/TMZ, and HA. The results showed that only OLR1 was evidently overexpressed in U343/TMZ and U251/TMZ cells relative to the other cells (Fig. 1B). Hence, OLR1 was selected for the ensuing experiments. MTT assay was used to evaluate IC50 values of U343, U251, U343/TMZ and U251/TMZ cells. It was unveiled that the IC50 value of TMZ in U343/TMZ and U251/TMZ cells was much higher than that in U343 and U251 cells (Fig. 1C). According to the references, the IC50 value of TMZ in U343/TMZ is about 280 μM,24 and the IC50 value of TMZ in U251/TMZ is about 830 μM.25 Subsequently, we detected OLR1 mRNA and protein levels in U343/TMZ and U251/TMZ cells after the transfection of si-OLR1-1/2/3. It was uncovered that OLR1 levels were downregulated after the transfection (Fig. 1D). Owing to the higher efficiency, we chose si-OLR1-1 and si-OLR1-2 for the ensuing experiments. Next, CCK-8 assay was performed to determine the IC50 value of TMZ in U343, U343/TMZ, U251, and U251/TMZ cells under the treatment of different concentrations of TMZ. It was unmasked that the IC50 value of TMZ-resistant and parental glioma cells was decreased evidently after interference with OLR1 (Fig. 1E). We used TMZ (the concentrations are based on the IC50 value of TMZ in difference cells) to treat U343, U343/TMZ, U251, and U251/TMZ cells for 48 h. Next, the proliferation of these TMZ-treated cells was uncovered by EdU assay. The outcomes showcased that cell proliferation was decreased after OLR1 depletion (Fig. 1F). Furthermore, flow cytometry analysis was applied to investigate the apoptosis of TMZ-treated cells, showing that cell apoptosis was promoted after OLR1 silencing (Fig. 1G). Taken together, OLR1 promotes the resistance of glioma cells to TMZ.

FIG 1.

FIG 1

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OLR1 promotes TMZ resistance of glioma cells. (A) The Venn diagram showed six common mRNAs jointly screened out by GSE100736 and GSE113510 datasets. As shown in box plot, UALCAN (https://ualcan.path.uab.edu/index.html) predicted that BASP1 is low-expressed in glioma tissue compared with normal tissue. (B) PLAC8, OLR1, and BASP1P1 expression levels were detected by qRT-PCR in HA, U343, U251, U343/TMZ and U251/TMZ cells. (C) MTT assay detected IC50 value of TMZ in U343/TMZ and U251/TMZ cells relative to U343 and U251 cells. (D) The efficiency of si-OLR1-1/2/3 was detected by qRT-PCR and Western blot in U343/TMZ and U251/TMZ cells. (E and F) CCK-8 and EdU assays were implemented to detect IC50 value and cell proliferation in TMZ-treated U343, U251, U343/TMZ, and U251/TMZ cells subsequent to OLR1 depletion. (G) Flow cytometry analysis was implemented to detect cell apoptosis in TMZ-treated U343, U251, U343/TMZ and U251/TMZ cells after the knockdown of OLR1. Student’s t test and one-way/two-way ANOVA were adopted for difference comparison. **P < 0.01.

CircTTLL13 is highly expressed in TMZ-resistant glioma cells and located in their cytoplasm and nucleus

The results in Fig. 1 showed that OLR1 improves TMZ resistance of glioma cells, but the upstream molecular mechanism underlying OLR1 regulation remained unclear. It has been reported that circRNAs play key roles in the modulation of cancer drug resistance.26 Hence, we used the GEO database to screen out the circRNAs with significantly high expression in drug-resistant tissues. According to the results of GSE139826 dataset, the top 10 significantly high-expressed circRNAs in drug-resistant glioma tissues were selected as candidates under the certain condition (P value ≤ 0.05). Afterward, we used GSE139826 and GSE100736 datasets to analyze the correlation of OLR1 with these 10 candidates. The results showed that the correlations of circ0036784, circ0131661, circ0139332 and circ0124252 with OLR1 expression were regarded to be statistically significant, as their P values are < 0.05 (Fig. 2A). Therefore, we selected circ0036784, circ0131661, circ0139332 and circ0124252 as the candidates. Based on their host genes, circ0036784, circ0131661, and circ0139332 were termed as circTTLL13, circSLC26A8, and circROR2 respectively. Next, we used qRT-PCR to examine circTTLL13, circSLC26A8, circROR2, and circ0124252 expression levels in TMZ-resistant glioma cells subsequent to the respective transfection of si-circTTLL13-1/2/3, si-circSLC26A8-1/2/3, si-circROR2-1/2/3, and si-circ0124252-1/2/3. It was shown that their expressions were downregulated after the transfection. Moreover, we found that si-circTTLL13-1/2/3 could not affect the host gene of circTTLL13, TTLL13 mRNA (Fig. 2B to E). Because of the higher efficiencies, we selected si-circTTLL13-1/2, si-circSLC26A8-1/2, si-circROR2-1/2 and si-circ0124252-1/2 for the ensuing experiments. Using these plasmids, we implemented qRT-PCR and Western blot to probe the OLR1 mRNA and protein levels after silencing circTTLL13, circSLC26A8, circROR2, and circ0124252, respectively in U251/TMZ cells. The results uncovered that OLR1 levels were decreased most significantly relative to the control group after interference with circTTLL13 (Fig. 2F), so circTTLL13 was selected for the follow-up assays. We then implemented qRT-PCR to investigate the expression of circTTLL13 in U343, U251, U343/TMZ, U251/TMZ and HA cells. The results showed that circTTLL13 was significantly overexpressed in U343/TMZ and U251/TMZ cells compared with that in U343, U251, and HA cells (Fig. 2G). Next, we verified whether circTTLL13 is circRNA and its localization in TMZ-resistant glioma cells. We performed PCR-agarose gel electrophoresis in U343/TMZ and U251/TMZ cells and found that circTTLL13 was amplified by convergent primer and divergent primer in complementary DNA (cDNA), and was only amplified by convergent primer in genomic DNA (gDNA). This verified the circular structure of circTTLL13 (Fig. 2H). As circRNA features stable structure, we then evaluated the stability of circTTLL13. We performed qRT-RCR in U343/TMZ and U251/TMZ cells to test the expressions of circTTLL13 and TTLL13 mRNA after the treatment of RNase R. The results showcased that TTLL13 mRNA was almost digested by rNase R, while circTTLL13 remained almost unchanged (Fig. 2I). Furthermore, qRT-PCR was conducted to unveil circTTLL13 and TTLL13 expression levels every 6 h in U343/TMZ and U251/TMZ cells subsequent to the treatment of actinomycin D (ActD), an inhibitor of RNA synthesis. The results showed that TTLL13 mRNA degraded at a faster rate than circTTLL13 (Fig. 2J). As indicated in Fig. 2H to J, circTTLL13 has a circular structure. FISH and subcellular fractionation assays were implemented to unclose the subcellular localization of circTTLL13 in U343/TMZ and U251/TMZ cells. As indicated in Fig. 2K, circTTLL13 was located in both cytoplasm and nucleus. The results of subcellular fractionation assay further showed that circTTLL13 was located in both cytoplasm and nucleus (Fig. 2L). Taken together, circTTLL13 is overexpressed in TMZ-resistant glioma cells and located in their cytoplasm and nucleus.

FIG 2.

FIG 2

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CircTTLL13 is highly expressed in TMZ-resistant glioma cells and located in their cytoplasm and nucleus. (A) GSE139826 and GSE100736 datasets were selected to detect the correlation of 10 potential circRNAs with OLR1 expression. (B) The efficiency of si-circTTLL13-1/2/3 was detected by qRT-PCR in U343/TMZ and U251/TMZ cells. (C to E) The efficiency of si-circSLC26A8-1/2/3, si-circROR2-1/2/3 and si-circ0124252-1/2/3 was detected by qRT-PCR in U251/TMZ cells. (F) OLR1 mRNA and protein levels were detected by qRT-PCR and Western blot after the depletion of circTTLL13, circSLC26A8, circROR2, or circ0124252 in U251/TMZ cells. (G) The expression of circTTLL13 was detected by qRT-PCR in U343, U251, U343/TMZ, U251/TMZ, and HA cells. (H) PCR-agarose gel electrophoresis was implemented in U343/TMZ and U251/TMZ cells to explore the circular structure of circTTLL13. (I and J) The stability of circTTLL13 and TTLL13 mRNA was detected by qRT-PCR in U343/TMZ and U251/TMZ cells using RNase R and ActD treatments. (K and L) FISH and subcellular fractionation assays were implemented to detect the subcellular localization of circTTLL13 in U343/TMZ and U251/TMZ cells. Student’s t test and one-way ANOVA were used for comparing differences. *P < 0.05, **P < 0.01.

CircTTLL13 facilitates TMZ resistance of glioma cells via modulating OLR1

Next, we verified whether circTTLL13 can promote TMZ resistance of glioma cells through regulating OLR1. First, we detected OLR1 mRNA and protein levels in U343/TMZ and U251/TMZ cells after the transfection of pcDNA3.1-OLR1. It was shown that both OLR1 mRNA and protein were overexpressed after the transfection (Fig. 3A). Next, we carried out EdU assay to investigate the influence of OLR1 on IC50 of TMZ. It was disclosed that OLR1 overexpression caused an increase of IC50 in U343 and U251 cells (Fig. 3B). Rescue assays were then implemented for the exploration of mechanism. We performed functional experiments in TMZ-treated U343/TMZ and U251/TMZ cells subsequent to si-NC, si-circTTLL13, si-circTTLL13+pcDNA3.1 or si-circTTLL13+pcDNA3.1-OLR1 transfection. The results of CCK-8 showed that IC50 value was decreased after interference with circTTLL13, but partially rescued by the overexpression of OLR1 (Fig. 3C). The outcomes of EdU assay disclosed that cell proliferation was hampered after the interference with circTTLL13, but was then partially rescued by the overexpression of OLR1 (Fig. 3D). The results of flow cytometry analysis showed that cell apoptosis was promoted after the knockdown of circTTLL13, but was then partially rescued by the enhancement of OLR1 (Fig. 3E). Taken together, circTTLL13 promotes TMZ resistance of glioma cells via modulating OLR1.

FIG 3.

FIG 3

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CircTTLL13 promotes TMZ resistance of glioma cells via modulating OLR1. (A) The efficiency of pcDNA3.1-OLR1 was detected by qRT-PCR and Western blot in U343/TMZ and U251/TMZ cells. (B) CCK-8 assays examined IC50 value in U343 and U251 cells after OLR1 overexpression. (C and D) CCK-8 and EdU assays were performed to detect IC50 value and cell proliferation in TMZ-treated U343/TMZ and U251/TMZ cells after the transfection of si-NC, si-circTTLL13, si-circTTLL13+pcDNA3.1 or si-circTTLL13+pcDNA3.1-OLR1. (E) Flow cytometry analysis was implemented to detect cell apoptosis in TMZ-treated U343/TMZ and U251/TMZ cells after the transfection of si-NC, si-circTTLL13, si-circTTLL13+pcDNA3.1 or si-circTTLL13+pcDNA3.1-OLR1. One-way ANOVA was adopted for comparing differences. **P < 0.01.

CircTTLL13 stabilizes OLR1 mRNA via recruiting YTHDF1

We next probed into the molecular mechanisms by which circTTLL13 regulates OLR1. First, the expression of circTTLL13 was examined by qRT-PCR in U343/TMZ and U251/TMZ cells subsequent to the transfection of pcDNA3.1-circTTLL13. It was showcased that its expression was upregulated after the transfection (Fig. 4A). We then performed luciferase reporter assay to evaluate the interaction of circTTLL13 with OLR1 promoter or OLR1 3’ UTR. It was shown that after the cotransfection of pcDNA3.1-circTTLL13, the luciferase activity in pGL3+OLR1-promoter group remained unchanged, while that in pmirGLO+OLR1-3’ UTR was markedly decreased (Fig. 4B and C). The results indicated the interaction between OLR1 3’ UTR and circTTLL13. It has been reported that circRNA can recruit RNA-binding protein (RBP) to bind to the 3’ UTR region of downstream mRNA, thereby stabilizing mRNA.27 Subsequently, we utilized starBase database (http://rna.sysu.edu.cn/encori/index.php) to identify the RBPs that can be combined with circTTLL13 under the certain condition (CLIP-Data ≥ 5); and screen out the RBPs that can be combined with OLR1 under the certain condition (CLIP-Data ≥ 2). As indicated in a Venn diagram, seven candidates were identified, namely, U2AF2, TAF15, FUS, PTBP1, UPF1, EIF4A3, and YTHDF1 (Fig. 4D). Afterward, we performed RIP assay in U251/TMZ cells to explore the binding of circTTLL13 with these candidates. The results uncovered that circTTLL13 was most enriched in the anti-YTHDF1 group (Fig. 4E). Moreover, we performed RNA pulldown assay in TMZ-resistant glioma cells and found that YTHDF1 was enriched in bio-circTTLL13, further proving the interaction (Fig. 4F). Subsequently, RIP assay was conducted to investigate OLR1 3’ UTR enrichment in the anti-YTHDF1 group. The results indicated the interaction between YTHDF1 and OLR1 3’ UTR. (Fig. 4G). It has been reported that YTHDF1, as an m6A reader, can stabilize mRNA.28 We then used SRAMP database (http://www.cuilab.cn/sramp) to predict m6A sites of OLR1. As indicated in Fig. 4H, OLR1 has a highly possible m6A site in the 3’ UTR (the 3’ UTR of OLR1 is located after the 601st base of the gene sequence). We performed Me-RIP in TMZ-resistant glioma cells to detect the enrichment of OLR1 3’ UTR. The results unveiled that OLR1 3’ UTR was enriched in the complex pulled down by m6A antibody (Fig. 4I). Afterward, RNA pulldown assay was conducted in TMZ-resistant glioma cells to detect the enrichment of YTHDF1 under different conditions.YTHDF1 was abundant in the OLR1 3’ UTR sense group, while YTHDF1 could not be pulled down after the mutation of m6A site in OLR1 3’ UTR, indicating that YTHDF1 can recognize the m6A site of OLR1 3’ UTR (Fig. 4J). Next, YTHDF1 mRNA and protein levels after interference with circTTLL13 were probed by qRT-PCR and Western blot in TMZ-resistant glioma cells. After the depletion of circTTLL13, YTHDF1 levels remained unchanged, indicating that circTTLL13 cannot affect YTHDF1 mRNA and protein (Fig. 4K). We then detected the expression of YTHDF1 in U343/TMZ and U251/TMZ cells after the transfection of si-YTHDF1-1/2/3 or pcDNA3.1-YTHDF1. It was shown that YTHDF1 expression was downregulated after the transfection of siRNAs and upregulated after the transfection of overexpression vectors (Fig. 4L). Owing to the higher efficiency, we chose si-YTHDF1-1 and si-YTHDF1-2 for the follow-up experiments. Afterward, we detected OLR1 mRNA and protein levels using qRT-PCR and Western blot subsequent to si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-YTHDF1 transfection. The results showed that OLR1 levels were decreased after interference with circTTLL13, but could not be rescued by the overexpression of YTHDF1 (Fig. 4M). The results indicated that circTTLL13 cannot promote OLR1 levels through regulating YTHDF1 expression. We performed mRNA stability assay to detect the effect of YTHDF1 on OLR1 mRNA stability. The results of qRT-PCR indicated that YTHDF1 can enhance the stability of OLR1 mRNA (Fig. 4N). To sum up, circTTLL13 stabilizes OLR1 mRNA by recruiting YTHDF1.

FIG 4.

FIG 4

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CircTTLL13 stabilizes OLR1 mRNA via recruiting YTHDF1. (A) The efficiency of pcDNA3.1-circTTLL13 was uncovered by qRT-PCR in U343/TMZ and U251/TMZ cells. (B and C) Luciferase reporter assays were conducted in U251/TMZ cells to detect the interaction between circTTLL13 and OLR1 promoter and 3’ UTR. (D) The Venn diagram showed the potential RBPs that can both interact with circTTLL13 and OLR1, which are screened out by starBase database (https://rna.sysu.edu.cn/encori/index.php). (E) RIP assay was performed in U251/TMZ cells to detect the interaction of circTTLL13 with U2AF2, TAF15, FUS, PTBP1, UPF1, EIF4A3, and YTHDF1. (F and G) RNA pulldown and RIP assays detected the interaction of YTHDF1 with circTTLL13 and OLR1 3’ UTR in U251/TMZ and U343/TMZ cells. (H) SRAMP database (http://www.cuilab.cn/sramp) forecasted the m6A site of OLR1. (I) Me-RIP was performed to detect m6A modification level of OLR1 3’ UTR in U251/TMZ and U343/TMZ cells. (J) RNA pulldown assay proved that YTHDF1 can recognize the m6A site of OLR1 3’ UTR in U251/TMZ and U343/TMZ cells. (K) YTHDF1 mRNA and protein levels were disclosed by qRT-PCR and Western blot in U251/TMZ and U343/TMZ cells after the knockdown of circTTLL13. (L) The efficiencies of si-YTHDF1-1/2/3 and pcDNA3.1-YTHDF1 were investigated by qRT-PCR in U343/TMZ and U251/TMZ cells. (M) The mRNA and protein levels of YTHDF1 were detected by qRT-PCR and Western blot in U251/TMZ and U343/TMZ cells after the transfection of si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-YTHDF1. (N) The stability of OLR1 was detected by mRNA stability assay in α-amanitin-treated U251/TMZ and U343/TMZ cells after the knockdown of YTHDF1. Student’s t test and one-way/two-way ANOVA were applied for comparing differences. **P < 0.01.

CircTTLL13 promotes m6A methylation of OLR1 pre-mRNA by recruiting METTL3

The results in Fig. 4 indicated that circTTLL13 recruits YTHDF1 to recognize m6A site in OLR1 3’ UTR, thereby stabilizing its mRNA expression. As shown in FISH and subcellular fractionation assays, circTTLL13 is located in both cytoplasm and nucleus. YTHDF1 generally recognizes the m6A site after methyltransferases (such as METTL3 and METTL14) in nucleus produce the effect. Therefore, we speculated that circTTLL13 can recruit methyltransferase in the nucleus to promote m6A methylation of OLR1. We performed Me-RIP assay in U251/TMZ cells to investigate OLR1 enrichment, finding that OLR1 3’ UTR was enriched in the complex pulled down by m6A antibody, and its enrichment was decreased significantly by circTTLL13 downregulation. The results further confirmed that circTTLL13 can recruit methyltransferase in the nucleus to promote m6A methylation of OLR1 (Fig. 5A). We then used SRAMP database to predict the m6A site of OLR1 pre-mRNA. The prediction showed that the m6A site of OLR1 pre-mRNA coincides with the m6A site of OLR1 mature mRNA recognized by YTHDF1 in Fig. 4 (Fig. 5B). Subsequently, we conducted RIP and RNA pulldown assay in TMZ-resistant glioma cells to detect the relationship with METTL3, circTTLL13 and OLR1 pre-mRNA. As shown in Fig. 5C, circTTLL13 was enriched in the anti-METTL3 group relative to the anti-IgG group, proving the interaction between METTL3 and circTTLL13. The results in Fig. 5D revealed that OLR1 pre-mRNA could interact with METTL3, and the interaction was suppressed after circTTLL13 knockdown. The results of RNA pulldown assay showed that both METTL3 and OLR1 pre-mRNA were enriched in the bio-circTTLL13 group (Fig. 5E). The results in Fig. 5C to E and Fig. S3A to C proved the co-existence of METTL3, circTTLL13, and OLR1 pre-mRNA in the same complex. We then detected the expression of METTL3 in TMZ-resistant glioma cells subsequent to the transfection of si-METTL3-1/2/3 or pcDNA3.1-METTL3. The results verified the efficiencies of these plasmids (Fig. 5F). Owing to the higher efficiency, we selected si-METTL3-1 and si-METTL3-2 for the follow-up experiments. Subsequently, m6A Dot Blot assay was used to detect the m6A level of different total RNA concentrations (50, 100, 200 and 400 ng) in TMZ-resistant glioma cells. The results showed that when the RNA concentration changed, the level of total m6A was decreased after interference with METTL3 (Fig. 5G). Next, RNA total m6A quantification was used to detect the m6A methylation level of Poly(A)+ RNAs in TMZ-resistant glioma cells subsequent to interference with METTL3. After the silencing of METTL3, the level of total m6A was decreased (Fig. 5H). We performed Me-RIP in TMZ-resistant glioma cells to detect the enrichment of OLR1 pre-mRNA. The results showed that OLR1 pre-mRNA was enriched in the complex pulled down by m6A antibody, and the enrichment was significantly suppressed by METTL3 depletion (Fig. 5I). Afterward, RNA pulldown assay was conducted in TMZ-resistant glioma cells to detect the enrichment of METTL3 under different conditions. The results disclosed that METTL3 was abundant in OLR1 pre-mRNA sense group, while YTHDF1 could not be pulled down after the mutation of m6A site in OLR1 pre-mRNA, indicating that METTL3 can promote m6A methylation of OLR1 pre-mRNA (Fig. 5J). Next, METTL3 expression level was examined by qRT-PCR in TMZ-resistant glioma cells after the knockdown of circTTLL13. The results showed that METTL3 expression remained unchanged, indicating that circTTLL13 cannot influence METTL3 expression (Fig. 5K). We performed qRT-PCR and Western blot in TMZ-resistant glioma cells to detect OLR1 mRNA and protein levels after the transfection of si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-METTL3. From the results, OLR1 mRNA and protein were decreased after interference with circTTLL13, but was then partially rescued by the overexpression of METTL3 (Fig. 5L). Taken together, circTTLL13 promotes m6A methylation of OLR1 pre-mRNA by recruiting METTL3.

FIG 5.

FIG 5

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CircTTLL13 promotes m6A methylation of OLR1 pre-mRNA by recruiting METTL3. (A) Me-RIP was performed to detect m6A modification level of OLR1 3’ UTR in U251/TMZ cells subsequent to circTTLL13 silencing. (B) SRAMP database predicted the m6A site of OLR1 pre-mRNA. (C) RIP assay was used to detect the interaction of METTL3 with circTTLL13 in U251/TMZ and U343/TMZ cells. (D) RIP assay disclosed the interaction between METTL3 and OLR1 pre-mRNA and the influence of circTTLL13 interference on the interaction in U251/TMZ and U343/TMZ cells. (E) RNA pulldown assay probed the interaction of circTTLL13 with METTL3 or OLR1 pre-mRNA in U251/TMZ and U343/TMZ cells. (F) The efficiencies of si-METTL3-1/2/3 and pcDNA3.1-METTL3 were detected by qRT-PCR in U343/TMZ and U251/TMZ cells. (G) m6A Dot Blot assay was performed in U251/TMZ and U343/TMZ cells to detect the m6A level of different total RNA concentrations (50, 100, 200, and 400 ng) after METTL3 depletion. (H) RNA total m6A quantification was used to detect the m6A methylation level of poly(A)+ RNAs in U251/TMZ and U343/TMZ cells after METTL3 depletion. (I) Me-RIP was performed to detect m6A modification level of OLR1 pre-mRNA in U251/TMZ and U343/TMZ cells subsequent to METTL3 knockdown. (J) RNA pulldown assay proved that METTL3 can promote m6A methylation of OLR1 pre-mRNA in U251/TMZ and U343/TMZ cells. (K) The expression of METTL3 was detected by qRT-PCR in U251/TMZ and U343/TMZ cells after the knockdown of circTTLL13. (L) The mRNA and protein levels of OLR1 were disclosed by qRT-PCR and Western blot in U251/TMZ and U343/TMZ cells after the transfection of si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-METTL3. Student’s t test and one-way ANOVA were applied for comparing differences. **P < 0.01.

CircTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1

The results in Fig. 1 showed that OLR1 promotes the TMZ resistance of glioma cells, but the underlying mechanisms remained unclear. It has been reported that OLR1 can activate Wnt/β-catenin pathway,29 and NF-κB pathway.30 Growing studies indicated that Wnt/β-catenin and NF-κB pathways can facilitate the drug resistance of cancer chemotherapy.31 Hence, we next explored whether circTTLL13 activates Wnt/β-catenin pathway or NF-κB pathway to promote the TMZ resistance of glioma cells. We conducted western blot to detect the levels of β-catenin and p65 in cytoplasm and nucleus of U251/TMZ cells subsequent to si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-OLR1 transfection. The results showed that after the knockdown of circTTLL13, β-catenin level in nucleus was decreased while that in cytoplasm was increased; and OLR1 overexpression partially reversed the effects. Comparatively, p65 level remained unchanged after the transfection (Fig. 6A). The results indicated that circTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1. Subsequently, TOP/FOP-flash reporter assay was performed in U251/TMZ cells to detect TOP/FOP luciferase activity after the transfection of si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-OLR1. The results disclosed that Wnt/β-catenin pathway was inhibited by circTTLL13 depletion, and was then partially rescued by OLR1 overexpression (Fig. 6B). Western blot was implemented to explore the levels of Cyclin D1 and c-Myc (the downstream proteins of Wnt/β-catenin pathway) after the transfection. The results revealed that cyclin D1 and c-Myc levels were decreased after the silencing of circTTLL13, and were then partially reversed by OLR1 overexpression (Fig. 6C). Taken together, circTTLL13 activates Wnt/β-catenin pathway by regulating OLR1.

FIG 6.

FIG 6

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CircTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1. (A) Western blot detected the levels of nuclear β-catenin, cytoplasmic β-catenin, nuclear p65, cytoplasmic p65 after the transfection of si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-OLR1 in U251/TMZ cells. (B) TOP/FOP-flash reporter assay was performed in U251/TMZ cells to detect TOP/FOP luciferase activity after the transfection of si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-OLR1. (C) Western blot detected the levels of cyclin D1 and c-Myc after the transfection of si-NC, si-circTTLL13-1, si-circTTLL13-1 + pcDNA3.1 or si-circTTLL13-1 + pcDNA3.1-OLR1 in U251/TMZ cells. One-way ANOVA was used to compare differences. **P < 0.01.

DISCUSSION

Glioma accounts for about 81% of CNS malignancies, and it is the most frequent primary brain tumor.1 As TMZ is considered as the first-line treatment for glioma, it is important to research the underlying mechanisms about TMZ resistance. In our study, we explored by which mechanisms circTTLL13 and OLR1 regulate TMZ resistance in glioma.

We utilized GEO database and qRT-PCR to identify OLR1 as the focus of our study. According to the literature review, GSTM3TV2 upregulates OLR1 expression by competitively binding to let-7 to enhance gemcitabine resistance in pancreatic cancer.32 In line with the findings, OLR1 was found to promote TMZ resistance in glioma. To validate this conclusion, we performed MTT, CCK-8, EdU and flow cytometry assays in TMZ-treated TMZ-resistant glioma cells. Subsequently, to explore the upstream mechanisms of OLR1, we adopted bioinformatic tools and qRT-PCR to identify circTTLL13. The previous studies disclosed that circRNAs have the ability to modulate the resistance of TMZ in glioma via the downstream mRNA. It has been reported that circ0072083 enhances NANGO expression to promote TMZ resistance in glioma.33 Furthermore, circ-HIPK3 propels TMZ resistance through targeting miR-421/ZIC5 axis in glioma.34 In the current study, we performed rescue experiments to detect the underlying mechanisms. The results of CCK-8, EdU and flow cytometry assays indicated that circTTLL13 facilitates TMZ resistance of glioma cells via modulating OLR1.

Luciferase reporter assays were performed to verify the interaction between circTTLL13 and OLR1 3’ UTR. Base on the literature review, we found that some circRNAs can recruit RBP to bind to the downstream mRNAs, thereby stabilizing mRNA.27 Herein, the potential RBPs interacting with circTTLL13 and OLR1 were screened out. RIP and RNA pulldown assays were performed to identify YTHDF1. As reported in the previous studies, METTL3 enhances the stability of c-Myc by YTHDF1-mediated m6A modification, thus promoting oral squamous cell carcinoma tumorigenesis;28 m6A-modified PDK4 positively modulates mRNA stability via interacting with YTHDF1/eEF-2 complex;35 and METTL3 recruits YTHDF1 to reinforce HK2 stability to facilitate the development of cervical cancer.36 In our study, we implemented mechanism experiments and mRNA stability assay, validating that circTTLL13 stabilizes OLR1 mRNA via recruiting a RBP YTHDF1.

YTHDF1 and other m6A readers exerts their functions after the formation of m6A catalyzed by methyltransferases.37 In this study, Me-RIP assay was performed to confirm that circTTLL13 can recruit methyltransferase in the nucleus to promote m6A methylation of OLR1. We used a bioinformatic website to predict the potential methyltransferase and obtained METTL3. The former studies reported that METTL3 can downregulate COL3A1 expression by enhancing its m6A methylation, thereby suppressing the metastasis of triple-negative breast cancer cells;38 and METTL3 promotes c-Myc expression by increasing m6A methylation levels of MYC mRNA, thereby facilitating the development of prostate carcinoma.39 In the current study, we confirmed that METTL3, recruited by circTTLL13, can facilitate m6A methylation of OLR1 pre-mRNA.

The former report indicated that OLR1 can activate Wnt/β-catenin pathway.29 Moreover, Wnt/β-catenin pathway has been reported to promote the drug resistance of cancer chemotherapy.31 We performed western blot, indicating that circTTLL13 activates Wnt/β-catenin signaling pathway instead of NF-κB signaling pathway by regulating OLR1. TOP/FOP-flash reporter assay and Western blot were performed, further verifying that circTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1.

However, due to the lack of materials and fund, we did not conduct in vivo experiments, which undermine the stringency of our report. Moreover, the investigation of Wnt/β-catenin pathway required more evidence to elucidate its participation in OLR1 regulation. In the future, we will research the functions of OLR1 in vivo and the specific mechanisms underlying Wnt/β-catenin pathway activation in our further study.

In conclusion, circTTLL13 promotes TMZ resistance in glioma via regulating OLR1-mediated Wnt/β-catenin pathway activation. Our study probed into the roles of circTTLL13, OLR1 and Wnt/β-catenin pathway in regulating TMZ resistance of glioma cells, and the underlying mechanisms. The findings in our study contributed to understanding of the mechanisms underlying TMZ resistance in glioma, which may improve the overall survival of glioma patients.

METHODS

Cell culture and vector construction

Human glioma cell lines (U343 and U251) were attained from ATCC (USA) and CRCPUMC (China) respectively. Human TMZ-resistant glioma cell lines (U343/TMZ and U251/TMZ) were acquired from MEXN (MXC861, China). Human astrocytes (HA) were acquired from Otwo Biotech (HTX1910, China). HA, U343 and U343/TMZ cells were cultivated in 89% RPMI 1640 added with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. U251 and U251/TMZ cells were kept in MEM-EBSS with 10% FBS. Full sequences of OLR1 and circTTLL13 were inserted into pcDNA3.1 vector for establishment of the overexpression vector, with empty pcDNA3.1 vector as the negative control (NC). In addition, small interfering RNAs (siRNAs) targeting OLR1, circTTLL13, circSLC26A8, circROR2, circ0124252, YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and methyltransferase-like 3 (METTL3) were built as knockdown vectors.

Quantitative real time- PCR

The total RNA was isolated from glioma cells utilizing TRIzol. The extracted RNAs were subjected to reverse transcription into cDNA and the samples were subjected to qRT-PCR. The outcomes were calculated based on the approach of 2−ΔΔCt. Bio-repeats were implemented in triplicate.

Western blot

The total protein was subjected to extraction from glioma cells. The protein samples were then treated with SDS-PAGE for protein separation. Subsequently, the samples were transferred onto PVDF membranes, followed by being blocked for 1 h in 5% defatted milk. After that, the blocked membranes were incubated with the primary antibodies at 4 °C overnight. Subsequently, secondary antibodies were incubated with the membranes. The blots were then developed. The primary antibodies encompassed anti-YTHDF1, anti-METTL3, anti-p65, anti-β-catenin, anti-β-actin, anti-histone H3, anti-cyclin D1, and anti-c-Myc. β-actin was utilized as the internal reference. Bio-repeats were conducted in triplicate.

Flow cytometry analysis

The, glioma cells were subjected to lysis and centrifugation. Cell samples were subjected to re-suspension in PBS with rNase. Next, the samples were subjected to staining with annexin V/propidium iodide (PI) and the fluorescence was examined by a flow cytometer. Bio-repeats were conducted in triplicate.

Cell counting kit-8 (CCK-8) assay

CCK-8 assay was carried out by use of CCK-8 kit following the supplier’s protocol. The glioma cells underwent different concentrations of TMZ respectively (0, 100, 200, 300, 400 μM), followed by the supplementation of 100 μL CCK-8 solution to every well. The absorbance at 450 nm was evaluated by utilizing a spectrophotometer.

5-Ethynyl-2’-deoxyuridine (EdU) assay

Glioma cells were put into 12-well plates. The transfected cells were treated with 50 μmol/L EdU reagent for 2 h, and then washed in PBS. The samples were cultured in 4% phosphate-buffered paraformaldehyde for fixation. Next, the cells were stained, followed by viability determination with a fluorescence microscope. Bio-repeats were conducted in triplicate.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay

Glioma cells were kept in 96-well plates. Twenty-four hours after seeding, 10 μL of 5 mg/mL MTT was added to every well, followed by culturing in an incubator for 4 h. The supernatant was discarded and 150 μL DMSO was supplemented for dissolution of the crystal. Optical density (OD) was determined at 490 nm.

RNA-binding protein immunoprecipitation (RIP) assay

The cells were lysed using RIPA lysis buffer. In addition, the magnetic beads were respectively conjugated with anti-m6A, anti-YTHDF1, anti-METTL3 and anti-IgG and the cell lysate was incubated with conjugated magnetic beads. The precipitated RNAs by the above antibodies were determined by qRT-PCR. Bio-repeats were performed in triplicate.

RNA pulldown assay

CircTTLL13, OLR1 pre-mRNA sense, OLR1 pre-mRNA anti-sense, OLR1 pre-mRNA sense (Mut), OLR1 3’ UTR sense, OLR1 3’ UTR anti-sense and OLR1 3’ UTR sense (Mut) were subjected to biotinylation. Afterward, the structure buffer was supplemented to biotinylated RNAs for secondary structure formation. The samples were denatured by heating and ice-bathing, followed by 2 h of incubation with streptavidin beads at 4 °C. After that, the cell lysate was prepared for setup of three groups (input, bio-NC, and bio-RNA). The lysates in three groups were subjected to overnight incubation with the beads at 4 °C. The pulldown products were analyzed by Western blot. Bio-repeats were conducted in triplicate.

Luciferase reporter and TOP/FOP-flash reporter assays

The sequences of OLR1 3’ UTR was subcloned into pmirGLO vector to create pmirGLO+OLR1-3’ UTR, with empty vector as NC. In addition, pmirGLO + IL-10-3’ UTR was cotransfected with pcDNA3.1-circTTLL13 or empty vector. The sequences of OLR1 promoter was inserted into pGL3 vector to build pGL3+OLR1-promoter. The empty pGL3 vector was applied as NC. The relative luciferase activity was uncovered after 36 h of incubation. To conduct TOP/FOP-flash assay, TOP/FOP-flash was subjected to cotransfection into transfected glioma cells. The TOP/FOP ratio was calculated to unclose pathway activities. Bio-repeats were performed in triplicate.

Fluorescence in situ hybridization (FISH) assay

Glioma cells were plated in 12-well plates for FISH assay. FISH kit was used to perform the assay. , The cells were fixed in 4% paraformaldehyde and next permeabilized in 0.5% Triton X-100. Thereafter, the cells were incubated with FISH probe labeled with digoxigenin (DIG), followed by treatment with anti-DIG for signal detection. A laser confocal microscope was used for imaging. The nucleus of cells was counterstained by DAPI solution. Bio-repeats were operated in triplicate.

Subcellular fractionation assay

Nucleus and cytoplasm of cells were separated and purified by use of PARIS kit based on the protocols of manufacturer. The internal references for cytoplasm and nucleus referred to β-actin and U6 respectively. Bio-repeats were implemented in triplicate.

RNA N6-methyladenosine (m6A) quantification

The total RNA was extracted by use of TRIzol. The m6A modification level of poly(A)+ RNAs was examined by the employment of m6A RNA Methylation Quantification Kit as per the instruction of supplier. Briefly, the RNAs accompanied with m6A standard were coated on wells. Detection antibody solution and capture antibody solution were supplemented. The m6A levels were quantified through the reading of absorbance of each well at 450 nm wavelength.

M6A dot blot assay

TRIzol was utilized to extract total RNA. Then, GenElute™ mRNA Miniprep Kit was used to purify poly real (A) + RNAs. After that, 50, 100, 200, and 400 ng double diluent were added into the RNAs and methylene blue.

mRNA stability assay

Glioma cells were treated with 50 mM α-amanitin for blocking RNA transcription. Afterward, qRT-PCR was utilized to probe OLR1 and β-actin expression levels in α-amanitin-treated cells subsequent to YTHDF1 interference at 0, 6, 12, 18, 24 h, respectively. Bio-repeats were implemented in triplicate.

Statistical analysis

The experimental data were displayed as mean ± standard deviation (SD). Data analysis was implemented using SPSS software. Student’s t test, and one-way/two-way analysis of variance (ANOVA) were used for the analysis of differences between groups. P value < 0.05 was regarded to indicate statistical significance.

ACKNOWLEDGEMENTS

We thank all our experimenters for their efforts.

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