METTL3 knockdown promotes temozolomide sensitivity of glioma stem cells via decreasing MGMT and APNG mRNA stability

Abstract

Chemo-resistance hinders the therapeutic efficacy of temozolomide (TMZ) in treating glioblastoma multiforme (GBM). Recurrence of GBM even after combination of maximal tumor resection, concurrent radio-chemotherapy, and systemic TMZ applocation is inevitable and attributed to the high therapeutic resistance of glioma stem cells (GSCs), which can survive, evolve, and initiate tumor tissue remodeling, the underlying mechanisms of GSCs chemo-resistance, have not been fully elucidated up-to-now. Emerging evidence showed that METTL3-mediated N6-methyladenosine (m6A) modification contributed to the self-renew and radio-resistance in GSCs, however, its role on maintenance of TMZ resistance of GSCs has not been clarified and need further investigations. We found that the cell viability and half-maximal inhibitory concentration (IC50) of GSCs against TMZ significantly decreased after GSCs underwent serum-induced differentiation to adherent growth of tumor cells. Besides, METTL3 expression and total m6A modification declined dramatically in consistence with GSCs differentiation. Knockdown of METTL3 weakened self-renew, proliferation and TMZ IC50 of GSCs, whereas enhanced TMZ induced γH2AX level, indicating upregulation of double-strand DNA damage. We also found that mRNA stability of two critical DNA repair genes (MGMT and APNG) was regulated by METTL3-mediated m6A modification. In conclusion, we speculated that METTL3-mediated m6A modification of MGMT and APNG mRNAs played crucial roles on suppression of TMZ sensitivity of GSCs, which suggest a potential new therapeutic target of METTL3 against GBM.

Fig. 1. Both total m6A RNA modification and METTL3 expression of GSCs were significantly higher than those of the corresponding differentiated cells.

A The colorimetric m6A quantification assay was conducted to examine the total m6A levels of two GSCs cell lines and the corresponding differentiated GSCs. B Dot Blot was performed to confirm the total m6A levels of two GSCs cell lines and differentiated GSCs. C The mRNA expression level of major m6A methyltransferases (METTL3, METTL14, RBM15, WTAP, and VIRMA) and demethylase (FTO and ALKBH5) of both two GSCs cell lines and their differentiated cells were analyzed by real-time PCR. D The dys-regulated level of METTL3 and FTO was confirmed by Western Blotting. *P ＜ 0.05, **P < 0.01 and ***P < 0.001 versus indicated groups.

Fig. 2. METTL3 knockdown impaired self-renew and stemness maintenance of GSCs.

A METTL3 knockdown significantly decreased the number of tumorspheres formed by GSC-11 and GSC-23 cells in vitro. B IF analysis was performed to evaluate the effect of METTL3 knockdown on CD133 expression of GSC-11 cells. C Real-time PCR was performed to detect the effect of METTL3 knockdown on the mRNA levels of several stemness markers. D Me-RIP-PCR was applied to detect the m6A methylation levels of SOX2, CD44, CD133, Nestin and Oct4 in GSCs after METTL3 knockdown. E The effects of METTL3 knockdown on the protein levels of several GSCs markers were detected by Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 versus indicated groups.

Fig. 3. METTL3 knockdown in GSCs not only improved TMZ sensitivity of GSCs, but also elevated TMZ-induced DNA damage.

A The effect of METTL3 knockdown (shMETTL3-1 and shMETTL3-2) on TMZ IC50 value of GSCs (GSC-11 and GSC-23) was analyzed by CCK-8 assay. B Edu assay was applied to evaluate the effect of METTL3 knockdown (shMETTL3-1 and shMETTL3-2) on proliferation ability of GSCs (GSC-11 and GSC-23). C The level of γH2AX content was analyzed with ELISA to evaluate DNA damage extent of GSCs (GSC-11 and GSC-23) after TMZ administration. ***P < 0.001 versus indicated groups.

Fig. 4. METTL3 knockdown downregulated mRNA stability of two critical DNA repair genes (MGMT and APNG) in GSCs.

A The effect of METTL3 knockdown (shMETTL3-1 and shMETTL3-2) in GSCs (GSC-11 and GSC-23) on the level of major repair enzymes, including APNG, CBX5, MGMT, MSH2, MSH6, MLH1, XRCC3, and XPC, was analyzed by real-time PCR. B The effect of METTL3 knockdown (shMETTL3-1 and shMETTL3-2) on m6A methylated level of APNG and MGMT was analyzed by Me-RIP-qPCR. C The effect of METTL3 knockdown (shMETTL3-1 and shMETTL3-2) on mRNA stability of APNG and MGMT was evaluated by transcription assay *P < 0.05 and ***P < 0.001 versus indicated groups.

Fig. 5. In vivo tumorigenicity assay disclosed METTL3 knockdown improved TMZ sensitivity of GSCs.

A The xenograft tumor size of NC or METTL3 knockdown GSC-11 and GSC23 cells with or without TMZ treatment. B Tumor growth curve. C Weight of the implanted tumors from GSCs in subcutaneous xenograft model of BALB/c nude mice with or without TMZ treatment. D IHC analysis of METTL3, MGMT, APNG, and cleaved caspase-3 in subcutaneous model. *P ＜ 0.05, **P ＜ 0.01, ***P < 0.001 versus indicated groups.