Fig. 1.

Alternative splicing of MKNK2 in CAC tissues is correlated with oncogenic characteristics. a The schematic model of Mnk2 isoforms. MKNK2a and MKNK2b possess different exon 13. The C-terminus of Mnk2a is translated from exon13a and exon13b, therefore contains a MAPK binding motif and can bind with MAPK members. In contrast, Mnk2b is generated by an alternative 3′ splicing site between exon13a and exon13b, leading to the absence of MAPK binding motif. b RNA levels of MKNK2a and MKNK2b were tested via RT-qPCR in 32 pairs of CAC tumor tissues and adjacent nontumorous tissues. P value was based on paired Student’s t-test. c Clinical significance of MKNK2 alternative splicing was evaluated by analyzing its correlation with KRAS and TP53 mutations. d Expressing of MKNK2a and MKNK2b were tested in distinct CAC cell lines via RT-PCR. Caco-2 and HT-29 cells are documented with wild type KRAS, while HCT-116 and SW480 cells with mutated-KRAS (G13D and G12V mutation, respectively). e RAS-G12V transfecting resulted in decreased MKNK2a and increased MKNK2b in both Caco-2 and HT-29 cells. f Actinomycin D (20 nM, 80 nM, 200 nM) was used to assess whether the RAS-G12V-induced upregulation of MKNK2b was caused by enhanced RNA synthesis or attenuated RNA-damaging. Accordingly, Ras-G12V can enhance MKNK2a-MKNK2b switch while actinomycin D inhibited RNA synthesis. g Colony formation assays revealed that RAS-G12V-induced oncogenic transformation was inhibited by Mnk2a while enhanced by Mnk2b transfection. P value was based on unpaired Student’s t-test comparing with RAS-G12V group. (H) A lower MKNK2a level and a higher MKNK2b level were found in tumors with larger size (n = 20) comparing with the smaller ones (n = 12). P value was based on unpaired Student’s t-test. (I) MTT proliferation assays showed the oncogenic effect of Mnk2b on promoting tumor proliferation in both HCT-116 and SW480 cells. P value was based on unpaired Student’s t-test comparing with cells transfected with pcDNA-vector. (J) Overexpressing Mnk2b enhanced colony formation of the CAC cell line HCT-116 and SW480 cells. Colonies were counted after incubation for 10 days in DMEM. P value was based on unpaired Student’s t-test comparing with cells transfected with pcDNA-vector