Fig. 4: Metformin upregulated the expression of p21 by reducing METTL3-associated m⁶A modification.

A The breast cancer tissue with the high staining of METTL3 expressed a low level of p21 while the tissue with the low staining of METTL3 expressed a high level of p21. B, C The mRNA and protein expression of p21 was upregulated after METTL3 knockdown in SUM-1315 and MCF-7 cells. The relative RNA level was calculated by the 2^−ΔΔCt method and normalized based on β-actin. The average mRNA level of p21 in shNC group was set as 1. D, E Knockdown of METTL3 prolonged the half-life of p21 transcript in SUM-1315 and MCF-7 cells. F–H Overexpression of METTL3 attenuated the regulation of METTL3 and p21 by metformin both in mRNA and protein level in SUM-1315 and MCF-7 cells. The relative RNA level was calculated by the 2^−ΔΔCt method and normalized based on β-actin. The average mRNA level of METTL3 and p21 in oeNC group was set as 1. **p < 0.01, ***p < 0.001 vs. oeNC-Met group. I MeRIP followed by qRT-PCR showed that METTL3 knockdown could decrease the amount of p21 mRNA modified by m⁶A. The relative enrichment level of p21was calculated by the 2^−ΔCt method and normalized based on Input. **p < 0.01 vs. shNC group. J, K After METTL3 knockdown, luciferase activity of wild-type 3′-UTR of p21 was increased in SUM-1315 and MCF-7 cells while mutant abolished this induction. **p < 0.01 vs. shNC group, ns means not significant. shNC, scramble control; shMETTL3 1/2, METTL3 knockdown; oeNC, negative control; oeNC-Met, oeNC-Met negative control group treated with metformin; oeMETTL3-Met, METTL3 overexpression group treated with metformin; WT-p21, wild-type 3′-UTR of p21, Mut-p21, mutant-type 3′-UTR of p21. Data represented the mean ± SD.