Figure 4.

Snail mediates M3-induced malignancy in CRC. m⁶A RIP-qPCR analysis of Snai1 mRNA in shNC- and shM3-infected (A) HCT-116 and (B) SW480 cells. (C) Snail protein expression levels (right, quantitative analysis) in stable with/without M3 knockdown HCT-116 and SW480 cells. (D) Snail mRNA expression levels in stably infected HCT-116 and SW480 cells with/without M3 knockdown. (E) Wild-type cells were treated with S-adenosylhomocysteine at a final concentration of 100 nM for 48 h, followed by detection of Snail protein expression (right, quantitative analysis) using western blot analysis. shNC and shM3 cells were included for analysis. (F) Snail mRNA expression levels in shNC and shM3 cells treated with Act-D (5 µg/ml) for the indicated time points were evaluated by qPCR. (G) Snail mRNA expression levels in CRC and adjacent normal tissue cells treated with Act-D (5 µg/ml) for the indicated time points were evaluated by qPCR. The proliferation rate of (H) HCT-116 and (I) SW480 cells transfected with shNC and shM3 and/or overexpressing Snail was evaluated using a Cell Counting Kit-8 assay at the indicated time points. (J) shNC or shM3 cells transiently overexpressing Snail or pcDNA3 vector were subjected to invasion assays for 24 h and subsequently analyzed with CytoSelect™ 24-well Cell Invasion assay kits (magnification, ×100; 8 µm; left). The invaded cells were then quantitatively analyzed (right). All experiments were performed in triplicate. Error bars represent standard deviation. *P<0.05, **P<0.01, ***P<0.001. M3, methyltransferase-like 3; CRC, colorectal cancer; m⁶A, nitrogen 6-methyladenosine; RIP, RNA immunoprecipitation; qPCR, quantitative PCR; sh, small hairpin RNA; NC, negative control; Act-D, actinomycin D.