'Supplementary Methods Scoring of immunohistochemical detections Supplementary Figure S1 Related to Figure 1 Supplementary Figure S2 Related to Figure 3 Supplementary Figure S3 Gene Ontology and KEGG pathway analysis of up-regulated mRNAs Supplementary Figure S4 Gene Ontology and KEGG pathway analysis of down-regulated mRNAs Supplementary Figure S5 Effects of miR-34a/b/c deletion on barrier protein expression in adenomas of ApcMin/+ mice Supplementary Figure S6 Related to Figure 7 Supplementary Table 1 List of antibodies and reagents Supplementary Table 2 Primers used for cloning and sequencing of the 3''-UTR of murine Wasf1 Supplementary Table 3 List of primers used for qPCR Supplementary Table 4 Subset of selected, up-regulated mRNAs with miR-34 seed-matching sequences Supplementary Table 5 Univariate and age/gender/tumor grade-adjusted Cox proportional hazard analysis of selected up-regulated mRNAs with miR-34 seed-matching sequences and overall survival in the TCGA COAD cohort Supplementary Reference Reference in Supplementary Methods'
ARTICLE ABSTRACT
The p53-inducible miR-34a and miR-34b/c genes are frequently silenced in colorectal cancer. To address the in vivo relevance of miR-34a/b/c function for suppression of intestinal tumor formation, we generated ApcMin/+ mice with deletions of the miR-34a and/or miR-34b/c genes separately or in combination. Combined deletion of miR-34a/b/c increased the number of intestinal stem cells as well as Paneth and Goblet cells, resulting in enlarged intestinal crypts. miR-34a/b/c-deficient ApcMin/+ mice displayed an increased tumor burden and grade and decreased survival. miR-34a/b/c-deficient adenomas showed elevated proliferation and decreased apoptosis and displayed pronounced bacterial infiltration, which may be due to an observed decrease in infiltrating immune cells and downregulation of barrier proteins. mRNA induction in miR-34a/b/c-deficient tumors was enriched for miR-34a/b/c seed-matching sites and for mRNAs encoding proteins related to epithelial–mesenchymal transition, stemness, and Wnt signaling. Accordingly, cells explanted from miR-34a/b/c-deficient adenomas formed tumor organoids at an increased rate. Several upregulated miR-34 targets displayed elevated expression in primary human colorectal cancers that was associated with lymph-node metastases (INHBB, AXL, FGFR1, and PDFGRB) and upregulation of INHBB and AXL in primary colorectal cancer was associated with poor patient survival. In conclusion, our results show that miR-34a/b/c suppress tumor formation caused by loss of Apc and control intestinal stem cell and secretory cell homeostasis by downregulation of multiple target mRNAs. Cancer Res; 77(10); 2746–58. ©2017 AACR.
