Fig 2: miR-193a-3p functions as a tumor-suppressor in colorectal cancer cells and its expression was decreased by overexpression of a BRAF protein. a Transfection of precursors of miR-193a-3p increased the expression of mature miR-193a-3p (top), and inhibited cell viability of the three cell lines irrespective of their KRAS/BRAF mutational status (bottom). b, c Transfection of the precursors of miR-193a-3p inhibited cell invasion ability in RKO and HCT116 cells. d miR-193a-3p overexpression reduced the mRNA levels of EMT-related genes ZEB1, SNAI1, and SNAI2 in RKO cells. e The mutant BRAF (V600E) overexpression activates its downstream pathway. Western blot analysis shows that the overexpression of mutant BRAF protein caused an increase in phosphorylated levels of BRAF and its downstream MEK and ERK, in the HCT8, LIM2405, SW48 and DiFi colorectal cancer cells compared to those transfected with a control vector. f miR-193a-3p expression was decreased in the KRAS/BRAF-wild-type SW48 and DiFi cells, but not in the KRAS-mutant HCT8 cells and the BRAF-mutant LIM2405 cells 72 h after overexpression of mutant BRAF protein. The Student’s t test was used to analyze statistical differences

Fig 3: A model highlighting the resistance mechanism in GTL16 GTL16R1 and GTL16R3.Highly expressed SND1-BRAF fusion protein signals through the MEK/ERK pathway and short-circuits c-Met inhibition.

Fig 4: Propranolol inhibited MAPK pathwayA-D. BRAF, MEK1/2, ERK1/2 and AKT phosphorylation following treatment with propranolol 100µM for 24h in A375, P-3 and P-6 cell lines. Aa-Df, Quantification of A-D. Results are presented as mean±SEM, *P<0.05, **P<0.01,***P<0.001, ****P<0.0001. All experiments were repeated for at least three times independently.

Fig 5: Higher ERK activity coincides with highly expressed SND1-BRAF fusion proteins in GTL16R1 and GTL16R3.(A) Western immunoblot of GTL16, GTL16R1 and GTL16R3 treated with inhibitors as single agent or combinations for 4 hr. (B) Densitometry measurement of the relative intensity of phospho ERK normalized to total ERK within each sample. (C) Densitometry measurement of the relative intensity of phospho AKT S473 normalized to total AKT within each sample.