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. 2020 Aug 27;11:955. doi: 10.3389/fgene.2020.00955

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Copyright © 2020 Cheng, Fu, Wang and Wang.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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METTL3 knockdown down-regulates PEDF expression and m6A methylation in PEDF mRNAs, as well as Wnt signaling activities. (A) PEDF expression and correlation of the expression of METTL3 with PEDF expression in TCGA database between DLBCL tissues and normal counterparts. ^∗P < 0.05. (B) mRNA expression of PEDF in 18 DLBCL tissues and 18 inflammatory lymph gland specimens was determined using qRT-PCR. ^∗∗P < 0.01. A positive correlation between mRNA expression of METTL3 and PEDF was detected by linear regression analysis. qRT-PCR (C) and western blotting (D) were used to analyze mRNA and protein expression of PEDF in SU-DHL4 and HBL1 cells with silenced expression of METTL3, respectively. ^∗∗P < 0.01. TOP/FOP-Flash reporter (E) was employed to determine Wnt signaling activity in SU-DHL4 and HBL1 cells with silenced expression of METTL3. (F) Western blotting assay of total and nuclear β-catenin proteins in DLBCL cells with silenced expression of METTL3. GAPDH and Lamin B1 were used as internal control and endogenous control of cell nuclear fraction, respectively. (G) Accumulation of β-catenin in the nucleus of the DLBCL cells with silenced expression of METTL3 according to confocal microscope images. (H) Western blotting assay of total and phosphorylated LRP6 proteins in DLBCL cells with silenced expression of METTL3. GAPDH was used as internal control. Me-RIP (I) assay was conducted to determine m6A methylation in PEDF transcripts in SU-DHL4 and HBL1 cells with silenced expression of METTL3. ^∗∗P < 0.01. (J) The half-life (T1/2) of PEDF mRNAs in SU-DHL4 and HBL1 cells transfected with Lv-shMETTL3 or Lv-NC (the control lentivirus).