Figure 4.

TrkC-miR2 expression alteration effect on the Wnt signaling pathway. A, Wnt signaling up-regulation after TrkC-miR2 overexpression in the SW480 cells. Luciferase activity was significantly increased in the cells overexpressing TrkC-premir2 compared with the controls. B, increased expression level of the genes involved in both canonical and non-canonical Wnt signaling pathway after TrkC-miR2 overexpression. C, Wnt inhibitor small molecules and TrkC-miR2 co-treatment effect on the Wnt signaling is shown through Top/flash assay. Although Wnt signaling is down-regulated after the XAV932 and IWP-2 small molecules application, this down-regulation has been compensated by TrkC-miR2 overexpression compared with the controls. When the cells were treated with the PNU74654 small molecule, significant Wnt signaling alteration was not detected with or without TrkC-miR2 overexpression. Error bars indicate S.D. of three experiments. D, successful down-regulation of TrkC-miR2 using anti-TrkC-miR2 sequence in SW480 cells (compared with the mock control). Also, APC2-3′-UTR overexpression as a scavenger, resulted in down-regulation of TrkC-miR2 (compared with the non-scavenger HBEGF-3′-UTR sequence), detected by RT-qPCR. Expression data were normalized against U48 as an internal control. E, RT-qPCR results showed significant up-regulation of APC2 expression after the down-regulation of TrkC-miR2 (using anti-TrkC-miR2) or after the scavenging of TrkC-miR2 (by APC2-3′-UTR overexpression) in SW480 cells. Expression data were normalized against GAPDH as an internal control. F, top/Fop flash assay after transfection of anti-TrkC-miR2 in SW480 cells showing non-significant Wnt signaling reduction compared with the mock transfected control. Also, overexpression of APC2-3′-UTR as a TrkC-miR2 scavenger again resulted in non-significant Wnt signaling attenuation compared with the off-target HBEGF-3′-UTR overexpression. ns, not significant. G, RT-qPCR analysis of TrkC-miR2 expression in HUH7 cell lines after TrkC-premiR2, anti-TrkC-miR2, and APC2-3′-UTR overexpression compared with mock control. TrkC-miR2 level was significantly increased in the cells overexpressing TrkC-premiR2; however, anti-TrkC-miR2 and APC2-3′-UTR scavenger constructs were not capable of significant reduction in TrkC-miR2 level. H, shows APC2 expression alteration after TrkC-premiR2, anti-TrkC-miR2, and APC2-3′-UTR overexpression. APC2-3′-UTR-specific primers were used for detection of APC2 expression alteration. Only successful overexpression of TrkC-miR2 (G) has resulted in significant reduction of APC2 transcripts. I, RT-qPCR against c-Myc and CCND1 genes (as the Wnt signaling target genes) after the overexpression of interested constructs. Data indicate no significant Wnt signaling alteration after the TrkC-miR2 expression alteration in Huh7 cells. J, shown is the Top/flash assay in the HUH7 cell line before and after TrkC-miR2 expression alteration. Similar to I, no significant Wnt signaling alteration was detected. *, p < 0.05; **, p < 0.01; ***, p < 0.001.