Cyanidin-3-O-glucoside inhibits the β-catenin/MGMT pathway by upregulating miR-214-5p to reverse chemotherapy resistance in glioma cells

Overcoming resistance to alkylating agents has important clinical significance in glioma. Cyanidin-3-O-glucoside (C3G) has a tumor-suppressive effect on tumor cells. However, whether it plays a role in temozolomide resistance in glioma is still unclear. We constructed a TMZ-resistant LN-18/TR glioma cell line, observed the effect of C3G on TMZ resistance in this cell line, and explored the role of miR-214-5p in chemoresistance. Results showed that β-catenin and MGMT were significantly upregulated in LN-18/TR cells. C3G upregulated miR-214-5p and enhanced the cytotoxic effect of temozolomide on LN-18/TR cells. Contrarily, C3G downregulated β-catenin and MGMT. Moreover, the miR-214-5p mimic downregulated β-catenin and MGMT in LN-18/TR cells, whereas the miR-214-5p inhibitor had the opposite effect; the miR-214-5p inhibitor significantly blocked the C3G-induced downregulation of β-catenin and MGMT. C3G or the miR-214-5p mimic enhanced temozolomide-induced apoptosis in LN-18/TR cells, whereas the miR-214-5p inhibitor blocked this effect. Furthermore, C3G or miR-214-5p agomir combined with TMZ significantly inhibited the growth of LN-18/TR tumors. Collectively, our research discovered the potential signaling mechanism associated with C3G-mediated suppression of TMZ resistance in LN-18/TR cells through miR-214-5p, which can facilitate the treatment of MGMT-induced resistance in glioma cells.

www.nature.com/scientificreports/ and other malignant tumors, highlighting the relatively high development value of C3G. However, the effect of C3G on chemotherapy resistance in glioma is still unclear, and further research is needed. In recent years, microRNAs (miRNAs) have been found to play important roles in the occurrence and development of malignant tumors. miRNAs are generally 20-25 nt in length and play physiological roles through the post-transcriptional regulation of mRNAs 14 . Many miRNAs cause mRNA degradation through specific base pairing with target mRNAs, ultimately affecting the expression of the encoded proteins 15,16 . Some miRNAs are abnormally expressed and play a role in the resistance of tumor cells to chemotherapeutics [17][18][19] . miR-214-5p has a tumor-suppressive effect in a variety of malignant tumors mediated by inhibiting tumor cell proliferation, migration, invasion, and other malignant biological behaviors 20,21 . However, whether miR-214-5p plays a role in TMZ resistance in glioma is still unclear. This study aimed to construct a TMZ-resistant glioma cell line, observe the effect of C3G on TMZ resistance in these cells, and explore the role of miR-214-5p in chemoresistance. This will provide theoretical support for the clinical treatment of glioma.

Results β-Catenin and O-6-methylguanine-DNA methyltransferase (MGMT) are upregulated in LN-18/ TR cells.
To study the role of C3G in TMZ resistance in glioma cells, we used LN-18 cells to construct the TMZ-resistant glioma cell line LN-18/TR. LN-18/TR cells had significantly enhanced resistance to TMZ ( Fig. 2A, B). The resistance index of LN-18/TR cells to TMZ was 4.51. The drug resistance protein MGMT plays an important role in glioma cells; therefore, we examined the expression changes of MGMT and its upstream signal protein β-catenin. The expression of β-catenin and MGMT were significantly higher in LN-18/TR cells than in LN-18 cells (Fig. 2C, D).
CTNNB1 has miR-214-5p-binding sequences. To confirm whether miR-214-5p can target CTNNB1 (the gene encoding β-catenin), we conducted bioinformatics analysis. The results of the miRcode analysis showed that CTNNB1 had a miR-214-5p-binding site, and the sequence of the site was relatively conserved among species (Fig. 4A). RNA22 v2 analysis also revealed that CTNNB1 mRNA and miR-214-5p had potential interaction sites (Fig. 4B). Additionally, we found that the 3′-untranslated region (3′-UTR) of CTNNB1 mRNA has a complementary pairing sequence with the seed region of miR-214-5p (Fig. 4C). A dual luciferase reporter gene experiment revealed that the luciferase activity of the cells transfected with plasmid containing miR-214-5p mimic and 3′-UTR of the wild-type CTNNB1 mRNA was significantly reduced (Fig. 4D).
C3G inhibits resistance to TMZ through miR-214-5p. We further observed the effect of C3G/miR-214-5p on TMZ-induced apoptosis. TMZ could weakly induce apoptosis in LN-18/TR cells, although treatment with 30, 60, and 90 μM C3G enhanced TMZ-induced apoptosis (Fig. 6a, b). miR-214-5p mimic transfection also enhanced TMZ-induced apoptosis in LN-18/TR cells (Fig. 6a, b). To observe the role of miR-214-5p in C3Genhanced TMZ-induced apoptosis in LN-18/TR cells, we transfected the miR-214-5p inhibitor into LN-18/TR cells and assessed apoptosis after treatment with C3G and TMZ. The results showed that the miR-214-5p inhibitor blocked the effect of C3G on TMZ-induced apoptosis in LN-18/TR cells (Fig. 7a, b). . However, at the end of the experiment, the body weights of the mice in the TMZ group were significantly lower than those in the control group, whereas the body weights of the mice in the C3G combined with TMZ or miR-214-5p agomir combined with TMZ group were significantly lower than those in the TMZ group (Fig. 8D).

Discussion
In this study, we successfully constructed a TMZ-resistant glioma cell line (LN-18/TR) through intermittent administration of TMZ and a gradual increase in its concentration. The resistance index of LN-18/TR cells to TMZ was 4.51. We found that levels of β-catenin and MGMT were significantly upregulated in LN-18/TR cells compared with those in the parental (non-resistant) cells. β-catenin is a functional protein encoded by CTNNB1 www.nature.com/scientificreports/ that can form a protein complex with the adhesion protein cadherin to participate in cell adhesion 22,23 . β-catenin is also an important signaling molecule in the classic Wnt signaling pathway that participates in the regulation of cell proliferation, differentiation, and apoptosis 24,25 . β-catenin plays an important role in the resistance of various malignant tumors to chemotherapy drugs, immunosuppressants, and targeted drugs [26][27][28] . MGMT is a unique DNA repair protein found in bacteria and mammals that can repair DNA damage caused by alkylation 29 . Under normal circumstances, MGMT protects normal tissues from the damage of alkylating agents, whereas in tumor tissues, it increases resistance to alkylating chemotherapeutics 30,31 . Previous studies have shown that MGMT is a downstream target of Wnt/β-catenin and can mediate Wnt/β-catenin-induced TMZ resistance 32 . Bi et al. 33 found that β-catenin can upregulate MGMT to maintain TMZ resistance in glioma cells, whereas cordycepin can inhibit the β-catenin/MGMT pathway to alleviate TMZ resistance. These findings suggest that the resistance of the LN-18/TR cells to TMZ is related to β-catenin/MGMT signaling. C3G, a flavonoid widely found in plants, has been proven to exert a tumor-suppressive effect in malignant tumors. Our study found that C3G can sensitize LN-18/TR cells to TMZ. We also showed that C3G can upregulate miR-214-5p in LN-18/TR cells. Bioinformatics analysis showed that CTNNB1 (the gene encoding β-catenin) and miR-214-5p have multiple potential interaction sites, indicating that CTNNB1 could be a target of miR-214-5p. Therefore, we speculated that C3G might downregulate β-catenin through miR-214-5p, thereby sensitizing LN-18/TR cells to TMZ.
For further verification, we analyzed the molecular mechanism underlying the relationship among C3G, miR-214-5p, β-catenin, and MGMT. We found that C3G decreased β-catenin and MGMT expression in LN-18/ TR cells. A miR-214-5p mimic and inhibitor exerted negative and positive regulatory effects, respectively, on the expression of β-catenin and MGMT in LN-18/TR cells. In addition, a miR-214-5p inhibitor blocked the C3Ginduced downregulation of β-catenin and MGMT in LN-18/TR cells. These results suggest that C3G targets β-catenin through miR-214-5p, thereby inhibiting the expression of MGMT. We then assessed the effect of C3G and miR-214-5p on TMZ-induced apoptosis in LN-18/TR cells. Both C3G and the miR-214-5p mimic enhanced TMZ-induced apoptosis in LN-18/TR cells. In contrast, the miR-214-5p inhibitor blocked the effect of C3G on TMZ-induced apoptosis in LN-18/TR cells. Therefore, C3G enhances TMZ-induced apoptosis in LN-18/TR cells through miR-214-5p. In vivo results showed that C3G or miR-214-5p agomir enhanced the response of LN-18/ www.nature.com/scientificreports/ TR xenograft tumors to TMZ. However, C3G or miR-214-5p agomir combined with TMZ caused severe weight loss in nude mice with xenograft tumors. It is suggested that the toxic and side effects of the two might be relatively strong, and corresponding chemical modification or preparation methods need to be improved. Based on these results, we hypothesized that C3G might upregulate miR-214-5p and inhibit β-catenin, thereby inhibiting the expression of MGMT, which leads to TMZ sensitization in LN-18/TR cells. Our research has discovered the potential signaling mechanism associated with C3G-mediated suppression of TMZ resistance in LN-18/TR cells through miR-214-5p, which can facilitate the treatment of MGMT-induced resistance in glioma cells.
In conclusion, our study confirmed that C3G has a TMZ-chemosensitization effect in glioma cells, which is related to the upregulation of miR-214-5p. miR-214-5p downregulates β-catenin, potentially via direct binding to CTTNB1, which downregulates MGMT and ultimately sensitizes cells to TMZ. Therefore, C3G might be a potential novel therapeutic drug for TMZ-resistant gliomas.  The total apoptosis rate after each treatment. **P < 0.01 compared to the control; ## P < 0.01 compared to control + TMZ; △ P < 0.05; && P < 0.01 compared to NC + TMZ. Western blotting. After drug (C3G, miR-214-5p mimic, or miR-214-5p inhibitor) treatment, cells were collected in a 1.5 mL centrifuge tube, and 0.12 mL radio-immunoprecipitation assay lysis buffer (Solarbio) was added to lyse the cells for 10 min. The lysates were centrifuged at 11,000×g for 20 min at 4 °C, and the supernatant (total protein) was transferred to a 0.5 mL tube. The total protein was quantified using a bicinchoninic acid protein quantification kit (Solarbio), and 10 µL protein samples (3.5 mg/mL) were prepared in the loading buffer. Samples (total protein) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred from the gel to a polyvinylidene difluoride (PVDF) membrane (Absin, China). The PVDF membrane was blocked with 5% skimmed milk for 3 h and then incubated with pri- Immunofluorescence analysis. Sterilized coverslips were placed in a six-well plate, and 5 × 10 5 cells per well were added. Cells were cultured for an appropriate time and fixed with 4% paraformaldehyde (Beyotime Biotechnology, China). The wells were rinsed with PBS, and 1 mL 0.1% Triton X-100 (Sigma-Aldrich) was added to each well for 10 min. The wells were rinsed again with PBS, and goat serum (Tianhang, China) was added dropwise to the coverslips and incubated for 1 h. The serum was blotted with absorbent paper, and diluted β-catenin (1:100) or MGMT (1:50) antibodies were added dropwise onto the coverslip. Coverslips were incubated overnight at 4 °C. Slides were washed with PBS, and diluted fluorescent secondary antibody (Invitrogen, Carlsbad, CA, USA) was added dropwise to the coverslip. Coverslips were incubated for 1 h, washed with PBS, and added with Prolong Gold Antifade Reagent (Beyotime Biotechnology) and 4′,6-diamidino-2-phenylindole (Solarbio) dropwise. Slides were incubated for 5 min in the dark and mounted overnight at room temperature. The next day, the slides were imaged using an ECLIPSE Ts2R-FL fluorescence microscope (Nikon, Japan).

Real-time fluorescence quantitative polymerase chain reaction (RT-FqPCR).
Cells were collected in a 1.5 mL tube and the total RNA was extracted with a Trizol kit (Sigma-Aldrich) following the manufacturer's protocol. The miScript II RT Kit (Qiagen, Duesseldorf, Germany) was used to reverse transcribe total RNA into cDNA. cDNA was amplified using miR-214-5p primers and SYBR Premix Ex Taq   www.nature.com/scientificreports/ Transfection. LN-18/TR cells were seeded in 10 cm dishes at 1 × 10 6 cells/dish. The following day, DMEM containing miRNA mimic (50 nM; Thermo Fisher Scientific, Waltham, MA, USA) or miRNA inhibitor (120 nM; Thermo Fisher Scientific) and the corresponding negative control (NC) were prepared. Transfection was performed following the manufacturer's instructions. Cells were cultured for 24 h, and the medium was replaced. After 24 h, cells were harvested for RT-FqPCR to verify the level of miRNA.
Dual luciferase reporter gene experiment. The 3′UTR of CTNNB1 mRNA was linked downstream to the firefly luciferase reporter gene in the pmirGLO vector plasmid (Ke Lei Biotechnology Co., Ltd, China) to obtain a wild-type (wt) plasmid. In addition, the complementary paired sequences of the 3′UTR of CTNNB1 mRNA and miR-214-5p seed region were mutated and linked downstream to the firefly luciferase reporter gene of the pmirGLO vector plasmid to obtain a mutant (mut) plasmid. LN-18 cells were seeded in a 24-well plate, and the experiment was divided into the control group (transfected with pmirGLO vector plasmid and pmir-GLO vector plasmid + miR-214-5p mimic), wt group (transfected with wt plasmid and wt plasmid + miR-214-5p mimic), and mut group (transfected with mut plasmid and mut plasmid + miR-214-5p mimic). The plasmid transfection kit (Ke Lei Biotechnology Co., Ltd) and luciferase reporter test kit (Ke Lei Biotechnology Co., Ltd) were used for plasmid transfection and luciferase activity detection, respectively, according to the manufacturer's instructions.
Flow cytometry. After drug treatment, cells were collected in a 10 mL tube and resuspended in 1 × Binding Buffer (Solarbio) at a concentration of 1 × 10 6 cells/mL. The cells were mixed quickly and thoroughly. The cell suspension (100 µL) was transferred to a flow tube. To this, fluorescein isothiocyanate Annexin V and propidium iodide (Solarbio) were added according to the manufacturer's instructions and incubated for 20 min. Apoptosis was analyzed with an Accuri C6 Plus flow cytometer (BD Bioscience, San Jose, CA, USA).
Animal experiments. The experiment was approved by the ethics committee of Hubei University of Chinese Medicine. Animal experiments were conducted in accordance with the Declaration of Helsinki and the ARRIVE guidelines. LN-18/TR cells were inoculated into the right armpit of BALB/c nude mice (HFK Bioscience Co., Ltd, China) at 5 × 10 6 per mouse. When tumors grew to approximately 100-150 mm 3 , animals were divided into groups as follows, with six animals in each group: control, TMZ (20 mg/kg/d, ip), C3G (10 mg/ kg/d, ip) + TMZ (20 mg/kg/d, ip), miR-214-5p agomir (1 mg/kg/3d, iv; Thermo Fisher Scientific) + TMZ group (20 mg/kg/d, ip). C3G or miR-214-5p agomir treatment was performed 4 h before TMZ administration. The experiment was carried out for 26 days, and the tumor long diameter, tumor short diameter, and nude mouse body weight were recorded three times per week. At the end of the experiment, all nude mice were euthanized, and the tumor was stripped and weighed. Tumor volume was calculated as follows: tumor volume (mm 3 ) = long diameter × short diameter 2 /2.

Statistical analyses.
The GraphPad Prism software (Version 8.4.0, GraphPad Software Inc., San Diego, CA, USA) for Windows was used to analyze the data. All results are shown as means ± standard deviations. Comparisons between two groups were performed by a t-test; comparisons between multiple groups were performed by one-way analysis of variance followed by the least significant difference-t test for multiple comparisons. P < 0.05 was considered significant.
Ethics declarations. We confirm the study was carried out in compliance with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.

Date availability
All data generated or analyzed during this study are included in this published article.