Abstract C36: miR-22 represses Wnt Signaling in human colon cancer cells

Abstract

Abstract Background: Wnt signaling is an evolutionarily conserved pathway that plays an important role in processes like development, differentiation and metastasis. Aberrant activation of Wnt signaling has been implicated in various tumors, predominantly of breast and colon. MicroRNAs are short non-coding RNAs that are frequently deregulated in cancer. Expression of the microRNA miR-22 was reported to be significantly lower in colorectal cancer tissues than that in normal adjacent mucosa. Based on bioinformatics predictions, we identified several Wnt signaling intermediates as potential targets for miR-22. As Wnt signalling is constitutively active in colon cancer cell lines SW480 and SW620, we hypothesize that miR-22 might be a potential regulator of Wnt signaling in colon cancer. Methods: Wnt signaling was stimulated in HEK293 cells by treating them with 25mM Lithium Chloride (LiCl). Wnt signaling activation was assayed using TOPFlash/FOPFlash luciferase activity in HEK293, SW480 and its metastatic derivative SW620, after overexpressing miR-22 using a plasmid construct. CyclinD1 promoter luciferase assay, Western blotting and quantitative RT-PCR were used to study the expression of downstream targets of Wnt signaling. Direct targets of miR-22 were validated by performing 3'-UTR luciferase assays. Plasmid constructs were used to overexpress or knockdown c-Myc. Results: Overexpression of miR-22 resulted in decreased TOP-Flash luciferase activity in LiCl-treated HEK293 cells and the colon cancer cells SW480 and SW620. miR-22 repressed Wnt signaling target genes CyclinD1, c-Myc and TCF7L2, at both mRNA and protein levels. It also decreased the mesenchymal markers (Snail and Vimentin) at the protein level. Overexpression of miR-22 down-regulated BCL9L protein levels and also targeted the BCL9L 3' UTR, as demonstrated by luciferase reporter assay. In HEK293 cells, overexpression of c-MYC repressed miR-22 levels whereas knockdown of endogenous c-Myc by shRNA up-regulated miR-22 levels. Conclusions: miR-22 inhibits Wnt signaling and potentially exerts this effect by targeting the Wnt activator BCL9L. The repression of mesenchymal markers by miR-22 suggests a role for it in Epithelial to Mesenchymal Transition. Further, the Wnt target gene c-Myc regulates the expression of miR-22, suggesting the existence of a positive feedback loop involving miR-22/Wnt Signaling/c-Myc by which miR-22 may indirectly regulate its own expression. Our findings therefore demonstrate a novel role for miR-22 in the regulation of Wnt signaling in colon cancer cells. Citation Format: Chetlangia Neha, Srinivasa Rao Rao, Devarajan Karunagaran. miR-22 represses Wnt Signaling in human colon cancer cells. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C36.