Fig. 6

Mutant K-Ras inhibits miR-199b expression in NSCLC cells by stimulating DNA methylation of the miR-199b promoter. a Demethylation agent treatment increased miR-199b expression in K-Ras-mutated NSCLC cell lines. H2122 and A549 cells were treated with the indicated concentration of 5-azacytidine (5`-aza) for 12 days. Next, they were subjected to miR-199b expression analysis. Cell culture medium was changed every third day. b Demethylation agent treatment abolished mutant K-Ras overexpression-induced inhibition of miR-199b expression. H1975 cells were transfected with mutant K-Ras (G12D) expression plasmid or empty vectors. After 24 h of transfection, cells were treated with or without 5 μM 5`-aza for 6 days. Then, cells were subjected to qRT-PCR and Western blot analysis. c Mutant K-Ras increased DNA methylation of the miR-199b promoter in H1975 cells. Cells were transfected with K-Ras (G12D). After 96 h of transfection, cells were subjected to bisulfite sequencing analysis. d Silencing of K-Ras inhibits DNA methylation of the miR-199b promoter in the K-Ras mutated cell H2122. Cells were transfected with K-Ras shRNA expression vector or scramble vector. After 96 h of transfection, cells were subjected to bisulfite sequencing analysis. e A schematic model of lung tumorigenesis regulation and the progression of K-Ras-mutated NSCLC by the miR-199b regulatory axis