Research Paper Volume 13, Issue 8 pp 12224—12238

Figure 4. miR-338-5p targets the m6A methylase METTL3. (A) Venn map of the downstream genes of miR-338-5p predicted by the websites miRDB (http://www.mirdb.org/, Target Score ≥ 50), TargetScan (http://www.targetscan.org/vert_71/, cumulative weighted context ++ score < -0.15), DIANA TOOLS (http://diana.imis.athena-innovation.gr/DianaTools, miTG score > 0.45) and miRWalk (http://mirwalk.umm.uni-heidelberg.de/; energy < -23, accessibility < 0.01, au > 0.45). (B) The PPI network of seven miR-338 downstream genes and their related genes constructed by GeneMANIA (http://genemania.org/). Larger circle of the related genes in the figure reflects higher core level, otherwise reflects lower core level. (C) Absolute METTL3 expression in GC obtained by UALCAN analysis. The blue box on the left represents the expression of normal samples, and the red box on the right represents the expression of GC samples. (D) TargetScan to predict the binding sites of miR-338-5p to METTL3. (E) RT-qPCR determination of miR-338-5p expression in MGC-803 and HGC-27 cells in response to miR-338-5p mimic, with U6 as internal control. (F) Dual-luciferase reporter gene assay to detect luciferase activity in the presence of miR-338-5p mimic. (G) RT-qPCR and Western blot assay to examine METTL3 expression after transfection with miR-338-5p mimic, with β-actin as internal control. Measurement data are expressed as mean ± standard deviation. * p < 0.05. Unpaired t test was employed for data comparison between two groups, and cell experiments were repeated 3 times independently.