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. Author manuscript; available in PMC: 2015 Oct 15.
Published in final edited form as: Nat Commun. 2015 Apr 15;6:6879. doi: 10.1038/ncomms7879

Figure 5. HOXD10 and Smad7 are direct targets of miR-1269a.

Figure 5

(a,b) RT-qPCR of Smad7 (a) and HOXD10 (b) mRNA levels in SW480 and HT29 cells with a control vector (NC) or with an ectopic miR-1269a expression vector (miR-1269a). (c,d) Western blot of Smad7 (c) and HOXD10 (d) protein levels in SW480 and HT29 cells with a control vector (NC) or with an ectopic miR-1269a expression vector (miR-1269a). (e) Luciferase reporter assays confirming the miR-1269a binding sites in Smad7 and HOXD10 3′UTRs. 3′UTRs of Smad7 (left) and HOXD10 (right) containing wild-type (Wt) or mutated (Mut) putative miR-1269a binding sites were cloned into the 3′UTR of firefly luciferase (Fluc). Ectopic miR-1269a expression in SW480 cells downregulated luciferase in Wt cells, but not in Mut cells. Fluc signals were normalized by a simultaneously delivered Renillar luciferase (Rluc) expression plasmid. (f) Transwell migration assay (left) and Matrigel invasion assay (right) of SW480 cells carrying control (NC) or miR-1269a expression (miR-1269a) vectors. Transient expression of HOXD10 abrogated miR-1269a induced migration and invasion. Error bars denote the s.d. between triplicates. (g,h) Bright field and fluorescent (mCherry) images of livers isolated from mice orthotopically injected with SW620-NC and SW620-HOXD10 cells (j), and number of liver metastatic nodules (h). Ectopic HOXD10 expression reduced liver metastasis of SW620 cells. Error bars in (h) denote s.d. of each group (8 mice). Scale bar, 8mm. In remaining cases, error bars denote s.d. of triplicates. ***, p<0.001; **, p<0.01; ***, p<0.001, Student’s t-test.