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

Abstract

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.

Figure 1

Molecular structure of cyanidin-3-O-glucoside (C3G).

Figure 2

Expression of β-catenin and MGMT in LN-18/TR cells. (A) LN-18 and LN-18/TR cells were treated with temozolomide (TMZ; 80, 160, 320, 640, 1280, and 2560 μM) for 72 h, and the cell proliferation inhibition rate was detected by the MTT method. (B) IC₅₀ of TMZ in cells. (C) Western blotting was performed to detect β-catenin and MGMT protein expression in cells. (D) Immunofluorescence was performed to detect β-catenin and MGMT protein expression in LN-18 and LN-18/TR cells. **P < 0.01 compared to LN-18 cells.

Figure 3

Cyanidin-3-O-glucoside (C3G) induces sensitization to temozolomide (TMZ) and the upregulation of miR-214-5p in LN-18/TR cells. (A) LN-18 and LN-18/TR cells were treated with C3G (5, 10, 30, 60, 90, and 120 μM) for 72 h, and the cell proliferation inhibition rate was detected by the MTT method. (B) LN-18 and LN-18/TR cells were treated with C3G (30, 60, and 90 μM) combined with TMZ (80, 160, 320, 640, 1280, and 2560 μM), and the cell proliferation inhibition rate was detected by the MTT method. (C) The IC₅₀ of TMZ in LN-18/TR cells treated with 30, 60, and 90 μM C3G. (D) LN-18/TR cells were transfected with mimics of miR-132-3p, miR-28-3p, miR-502-3p, miR-214-5p, miR-151a-3p, and miR-1304-3p, or inhibitors of miR-2682-5p, miR-216-5p, miR-6087, miR-28-5p, miR-196b-5p, and miR-3687, and corresponding negative controls. The expression levels of each miRNA were detected using RT-FqPCR. **P < 0.01. (E) IC50s of TMZ in LN-18/TR cells transfected with each miRNA mimic. **P < 0.01 compared to NC4. (F) IC50s of TMZ in LN-18/TR cells transfected with each miRNA inhibitor. (G) LN-18/TR cells were treated with 30, 60, and 90 μM C3G, and the expression of miR-214-5p was assessed by RT-FqPCR. **P < 0.01 compared to control.

Figure 4

Bioinformatics prediction of the binding of CTNNB1 to miR-214-5p. (A) miRcode predicted miR-214-5p-binding sites in CTNNB1 mRNA and the conservation of the binding sites among species. (B) RNA22 v2 predicted the sequence of the miR-214-5p-binding site in CTNNB1 mRNA. (C) 3′-UTR of CTNNB1 mRNA and miR-214-5p seed region complementary paired sequence. (D) The dual luciferase reporter gene experiment verifies the interaction between miR-214-5p and 3′-UTR of CTNNB1 mRNA and the functions. **P < 0.01 compared to NC. CTTNB1: gene encoding the β-catenin protein.

Figure 5

Cyanidin-3-O-glucoside (C3G) inhibits the β-catenin/MGMT signaling pathway through miR-214-5p. (A) LN-18/TR cells were treated with C3G (30, 60, and 90 μM) for 48 h, and β-catenin and MGMT protein expression was detected in the cell protein extracts. **P < 0.01 compared to the control. (B) RT-FqPCR was performed to detect miR-214-5p levels in miR-214-5p mimic or negative control (NC) groups. **P < 0.01 compared to NC. (C) RT-FqPCR was conducted to detect miR-214-5p levels in miR-214-5p inhibitor or NC groups. **P < 0.01 compared to NC. (D) Following promotion or inhibition of the miR-214-5p expression/activity in LN-18/TR cells, β-catenin and MGMT expression was detected in the cell protein extracts. **P < 0.01 and ^△△P < 0.01 compared to NC1 and NC2, respectively. (E) LN-18/TR cells were treated with 30, 60, or 90 μM C3G for 48 h after transfection of NC or miR-214-5p mimic, and β-catenin and MGMT expression was detected in the cell protein extracts. **P < 0.01, ^##P < 0.01, ^△△P < 0.01 compared to 30, 60, and 90 μM C3G, respectively.

Figure 6

Cyanidin-3-O-glucoside (C3G) and miR-214-5p enhances temozolomide (TMZ)-induced apoptosis in LN-18/TR cells. (a) LN-18/TR cells were treated with 1000 µM TMZ alone or TMZ plus 30, 60, or 90 μM C3G, NC, or miR-214-5p mimic for 72 h. Flow cytometry was performed to detect apoptosis after each treatment. (b) 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.

Figure 7

Cyanidin-3-O-glucoside (C3G) enhances the sensitivity of LN-18/TR cells to temozolomide (TMZ) by upregulating miR-214-5p. (a) LN-18/TR cells transfected with NC or miR-214-5p inhibitor were treated with 1000 μM TMZ or TMZ plus 30, 60, or 90 μM C3G for 72 h. Flow cytometry was performed to detect apoptosis after each treatment. (b) The total apoptosis rate after each treatment. *P < 0.05 compared to the control; ^##P < 0.01 compared to NC + TMZ; ^△△P < 0.01 compared to 30 μM C3G + TMZ; ^&&P < 0.01 compared to 60 μM C3G + TMZ; ^$$P < 0.01 compared to 90 μM C3G + TMZ.

Figure 8

Cyanidin-3-O-glucoside (C3G) and miR-214-5p promote temozolomide (TMZ) sensitivity in vivo. (A) Representative images of control, TMZ, C3G + TMZ, miR-214-5p agomir + TMZ group tumors. (B) Tumor weights of each group of animals. **P < 0.01. (C) The tumor volume changes in each group of animals. Compared with control, **P < 0.01. (D) Changes in body weights of animals in each group. Compared with control, **P < 0.01. Compared with TMZ, ^##P < 0.01.