Journal: Molecular Therapy. Nucleic Acids

Article Title: METTL3-mediated m 6 A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung

doi: 10.1016/j.omtn.2020.12.001

Figure Lengend Snippet: METTL3 is vital for the EMT and malignancy of T-HBE cells T-HBE cells were transfected with METTL3 siRNA or siRNA con. (A) The levels of global mRNA m 6 A were determined by RNA m 6 A colorimetric analysis (mean ± SD, n = 3). (B) Western blots were performed, and (C) relative protein levels (mean ± SD, n = 3) of METTL3, E-cadherin, N-cadherin, and vimentin were determined. (D) Colony-formation assays and Transwell assays (scale bars, 100 μm) were performed. (E) Relative colony numbers and (F) relative levels of cell invasion and migration were determined (mean ± SD, n = 3). (G) T-HBE cells (1 × 10 7 ) transfected with METTL3 siRNA or siRNA con were injected into nude mice (n = 5). (H) Weights of tumors in the three groups were measured (mean ± SD, n = 5). (I) The sizes of tumors formed in the mice were monitored every 7 days (mean ± SD, n = 5). ∗p < 0.05, different from T-HBE cells in the absence of METTL3 siRNA.

Article Snippet: Mice in the AAV-ShMETTL3 and AAV-NC shRNA (GeneChem, China) groups were dosed by intranasal instillation after CS exposure for 4 weeks.

Techniques: Transfection, Western Blot, Migration, Injection

Journal: Molecular Therapy. Nucleic Acids

Article Title: METTL3-mediated m 6 A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung

doi: 10.1016/j.omtn.2020.12.001

Figure Lengend Snippet: Silencing of ZBTB4 by a METTL3-m 6 A-YTHDF2-dependent mechanism T-HBE cells were transfected with METTL3 siRNA or siRNA con, pcD METTL3, or pcD con and YTHDF2 siRNA or siRNA con. (A) Methylated RNA immunoprecipitation (meRIP)-qPCR was applied to assess the m 6 A levels for ZBTB4 in CSE-HBE cells (mean ± SD, n = 3). (B) meRIP-qPCR was applied to assess the m 6 A levels for ZBTB4 in T-HBE cells (mean ± SD, n = 3). (C) Western blots were performed, and (D) relative protein levels (mean ± SD, n = 3) of METTL3 and ZBTB4 were determined. (E) Peaks indicating the relative abundance of m 6 A sites in ZBTB4 mRNA. (F) WT or m 6 A consensus sequence mutant ZBTB4 3′ UTR was fused with a firefly luciferase reporter. Mutations of m 6 A consensus sequences were generated by replacing A with G. (G) Relative activities of the WT and Mut luciferase reporters in T-HBE cells (mean ± SD, n = 3). (H) western blots were performed, and (I) relative protein levels (mean ± SD, n = 3) of YTHDF2 and ZBTB4 were determined. (J) RIP assays were performed in T-HBE cells to detect the direct binding between the ZBTB4 mRNA and YTHDF2 protein (mean ± SD, n = 3). (K) Relative activities of the WT and Mut luciferase reporters in T-HBE cells (mean ± SD, n = 3). ∗p < 0.05, different from control group.

Article Snippet: Mice in the AAV-ShMETTL3 and AAV-NC shRNA (GeneChem, China) groups were dosed by intranasal instillation after CS exposure for 4 weeks.

Techniques: Transfection, Methylation, RNA Immunoprecipitation, Western Blot, Sequencing, Mutagenesis, Luciferase, Generated, Binding Assay, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: METTL3-mediated m 6 A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung

doi: 10.1016/j.omtn.2020.12.001

Figure Lengend Snippet: METTL3 regulation of ZBTB4 is involved in the EMT and malignancy of T-HBE cells T-HBE cells were transfected with METTL3 siRNA and/or co-transfected with a ZBTB4 siRNA. (A) Western blots were performed, and (B) relative protein levels (mean ± SD, n = 3) of METTL3, ZBTB4, EZH2, E-cadherin, N-cadherin, vimentin, and H3K27me3 were determined. (C) Colony-formation assays and Transwell assays (scale bars, 100 μm) were performed, and (D) relative colony numbers and (E) relative levels of cell invasion and migration were determined (mean ± SD, n = 3). ∗p < 0.05, different from T-HBE cells transfected with METTL3 siRNA alone.

Article Snippet: Mice in the AAV-ShMETTL3 and AAV-NC shRNA (GeneChem, China) groups were dosed by intranasal instillation after CS exposure for 4 weeks.

Techniques: Transfection, Western Blot, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: METTL3-mediated m 6 A modification of ZBTB4 mRNA is involved in the smoking-induced EMT in cancer of the lung

doi: 10.1016/j.omtn.2020.12.001

Figure Lengend Snippet: Downregulation of METTL3 reverses the CS-induced EMT in the lungs of mice Male BALB/c mice at 6–8 weeks of age were divided into four groups: normal control (n = 6), CS (n = 6), CS+sh METTL3 (n = 6), and CS+NC shRNA (n = 6). (A) The levels of global mRNA m 6 A in the lung tissues of mice were determined by RNA m 6 A colorimetric analysis (mean ± SD, n = 6). (B) Western blots were performed, and (C) relative protein levels (mean ± SD, n = 3) of METTL3, ZBTB4, EZH2, E-cadherin, N-cadherin, vimentin, and H3K27me3 in the lung tissues of mice were determined. (D) Representative immunostaining images and (E) the levels (mean ± SD, n = 6) of METTL3, E-cadherin, and vimentin in the lung tissues of mice were determined by IRS. # p < 0.05, different from control group mice. ∗p < 0.05, different from normal CS group mice.

Article Snippet: Mice in the AAV-ShMETTL3 and AAV-NC shRNA (GeneChem, China) groups were dosed by intranasal instillation after CS exposure for 4 weeks.

Techniques: Control, shRNA, Western Blot, Immunostaining