Supplementary Table 1

Transcription

1

2

3

4

5 Supplementary Table 1 Table S1. Individual measurements of tumor volume (cm 3 ) for the indicated times and injected cell type. Mouse LTR LTR LTR LTR LTR LTR LTR LTR LTR LTR week 1 week 2 week 3 week 4 week 5 1 n. m. n. m n. m n. m n. m. n. m. n. m. n. m n. m n. m n. m n. m. n. m. n. m. 4 n. m. n. m. n. m. n. m n. m n. m n. m n. m n. m n. m n. m. n. m. n. m. n. m n. m n. m. n. m. 9 n. m. n. m. n. m. n. m. n. m. n. m. n. m. n. m. n. m. n. m n. m n. m n. m n. m n. m n. m n. m n. m n. m n. m.: not measurable

6 Supplementary Table 2 Table S2. Summary of mir-143 and mir-145 target sites in the 3 UTR/CDS of KRAS, KLF5, CD44 and BRAF. The seed sequence is underlined. Gene Position Genbank Acc. N KRAS NM_ KRAS NM_ KLF NM_ KLF NM_ CD NM_ CD NM_ BRAF NM_ Predicted consequential pairing of target region (top) and mirna (bottom) 5'...UCAUGUUAAAAGAAGUCAUCUCA... (UTR) 3' CUCGAUGUCACGAAGUAGAGU (mir-143) 5'...ACAGUUUGCACAAGU--UCAUCUCA... (UTR) 3' CUCGAUGUCACGAAGUAGAGU (mir-143) 5'...GAAAACCACAACUAAAACUGGAA... (UTR) 3' UCCCUAAGGACCCUUUUGACCUG (mir-145) 5'...UUACUCAAGCAGAUC-UCAUCUCA... (UTR) 3' CUCGAUGUCACGAAGUAGAGU (mir-143) 5'...CUUCUA-AGU-CUUCAUCUCA... (UTR) 3' CUCGAUGUCACGAAGUAGAGU (mir-143) 5'... UUUUCAACUUGAAAGAAACUGGAC... (UTR) 3' UCCCUAAGGACCCUUUUGACCUG (mir-145) 5'... CUUUCAGUGCUACCUUCAUCUCU... (CDS) 3' CUCGAUGUCACGAAGUAGAGU (mir-143)

7 Supplementary Table 3 Table S3. Sense Oligo sequence for the constructs bearing the wt and mutated target regions used in luciferase assay. The italic nucleotides are Xho-5 -protruding ends of the double strand oligo. Underlined nucleotides identified the seed wt and mutated sequences. Name KLF5 mir-143 KLF5 mir-145 Ras mir-143(1) Ras mir-143(2) CD44 mir-143 CD44 mir-145 Raf mir-143 XhoI 3 UTR oligos wt (top) and mutated (bottom) target regions TCGATAATGTATATGGCTTTACTCAAGCAGATCTCATCTCATGACAGGCAGCCACGTCTCAACATGGGT TCGATAATGTATATGGCTTTACTCAAGCAGATCGCTAGCCATGACAGGCAGCCACGTCTCAACATGGGT TCGAAACAAACAAAAGCAAGAAAACCACAACTAAAACTGGAAATGTATATTTTGTATATTTGAGAAAAC TCGAAACAAACAAAAGCAAGAAAACCACAACTAATGTACAAAATGTATATTTTGTATATTTGAGAAAAC TCGATTACTATCCATTTCTTCATGTTAAAAGAAGTCATCTCAAACTCTTAGTTTTTTTTTTTTACAACT TCGATTACTATCCATTTCTTCATGTTAAAAGAAGGCTAGCCAAACTCTTAGTTTTTTTTTTTTACAACT TCGATGTTAAGTTACAGTTTGCACAAGTTCATCTCATTTGTATTCCATTGATTTTTTT TCGATGTTAAGTTACAGTTTGCACAAGTGATATCCATTTGTATTCCATTGATTTTTTT TCGAAATGGTAAGGAGACTCTTCTAAGTCTTCATCTCAGAGACCCTGAGTTCCCACTCAGACC TCGAAATGGTAAGGAGACTCTTCTAAGTCTATGCATCAGAGACCCTGAGTTCCCACTCAGACC TCGATTCTTTTTATTTTCTTTTCAACTTGAAAGAAACTGGACATTAGGCCACTATGTGTTGTTACTGCC TCGATTCTTTTTATTTTCTTTTCAACTTGAAAGATCTAGAACATTAGGCCACTATGTGTTGTTACTGCC TCGACTAGCCTTTCAGTGCTACCTTCATCTCTTTCAGTTTTTCAAAATCC TCGACTAGCCTTTCAGTGCTACCTGATATCCTTTCAGTTTTTCAAAATCC

8 Supplementary Table 4 Table S4. DAVID platform analysis of the enrichment of dysregulated genes in mir-143 and mir-145 expressing HCT-116 cells in functional categories described in the KEGG database. Term Count % PValue List Total Pop Hits Pop Total Fold Enrichment Bonferroni Benjamini FDR hsa03030:dna replication E E E hsa04110:cell cycle E hsa03410:base excision repair

9 Supplemetary Table 5 Table S5. Detail of the genes belonging to the Kegg pathways described in table 3, with the ratio of differential expression in HCT-LTR143-5 relative to HCT-LTR, parametric p-value and FDR. Symbol mir vs ctrl hsa03030:dna replication Description Parametric p-value MCM minichromosome maintenance complex component 4 (MCM4) transcript variant 1, mrna 9.59E PRIM primase, DNA, polypeptide 2 (58kDa) (PRIM2) 1.20E DNA DNA replication helicase 2 homolog (yeast) (DNA2) 3.78E RFC replication factor C (activator 1) 5, 365kDa (RFC5) transcript variant E PRIM primase, DNA, polypeptide 1 (49kDa) (PRIM1) 4.01E MCM minichromosome maintenance complex component 5 (MCM5) 2.20E MCM minichromosome maintenance complex component 6 (MCM6) 3.53E MCM minichromosome maintenance complex component 2 (MCM2) 2.92E MCM minichromosome maintenance complex component 7 (MCM7) transcript variant E POLE 0.67 polymerase (DNA directed) epsilon (POLE) 1.86E LIG ligase I, DNA, ATP-dependent (LIG1) 1.32E RFC replication factor C (activator 1) 4, 37kDa (RFC4) transcript variant E POLE polymerase (DNA directed) epsilon 2 (p59 subunit) (POLE2) 1.92E POLA polymerase (DNA directed) alpha 1 (POLA1) 1.72E POLD polymerase (DNA-directed) delta 3, accessory subunit (POLD3) 2.83E MCM minichromosome maintenance complex component 3 (MCM3) 6.02E PCNA 0.69 proliferating cell nuclear antigen (PCNA) transcript variant E FDR

10 Symbol mir vs ctrl hsa04110:cell cycle Description Parametric p-value TFDP transcription factor Dp-1 (TFDP1) 7.16E MAD2L MAD2 mitotic arrest deficient-like 1 (yeast) (MAD2L1) 2.38E SMC1A 0.66 structural maintenance of chromosomes 1A (SMC1A) 2.97E CCNE cyclin E2 (CCNE2) 1.01E ORC6L 0.52 origin recognition complex, subunit 6 like (yeast) (ORC6L) 4.56E CDC25C 0.61 cell division cycle 25 homolog C (S pombe) (CDC25C) transcript variant E RBL retinoblastoma-like 1 (p107) (RBL1) transcript variant E E2F E2F transcription factor 3 (E2F3) 7.84E MCM minichromosome maintenance complex component 5 (MCM5) 2.20E MCM minichromosome maintenance complex component 6 (MCM6) 3.53E CDK cyclin-dependent kinase E MCM minichromosome maintenance complex component 2 (MCM2) 2.92E ANAPC anaphase promoting complex subunit 4 (ANAPC4) 1.38E BUB1B 0.62 BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) (BUB1B) 3.98E TTK 0.62 TTK protein kinase (TTK) 9.69E ANAPC anaphase promoting complex subunit 1 (ANAPC1) 1.17E PCNA 0.69 proliferating cell nuclear antigen (PCNA) transcript variant E MCM minichromosome maintenance complex component 4 (MCM4) transcript variant E CCND cyclin D2 (CCND2) 9.91E ORC3L 0.58 origin recognition complex, subunit 3-like (yeast) (ORC3L) transcript variant E DBF DBF4 homolog (S cerevisiae) (DBF4) 2.58E MCM minichromosome maintenance complex component 7 (MCM7) transcript variant E CDC cell division cycle 7 homolog (S cerevisiae) (CDC7) 1.93E BUB BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) (BUB1) 4.96E SKP S-phase kinase-associated protein 2 (p45) (SKP2) transcript variant E SKP S-phase kinase-associated protein 1 (SKP1) transcript variant E MCM minichromosome maintenance complex component 3 (MCM3) 6.02E FDR

11 Symbol mir vs ctrl hsa03410:base excision repair Description Parametric p-value APEX APEX nuclease (apurinic/apyrimidinic endonuclease) 2 (APEX2) nuclear gene encoding mitochondrial protein 4.17E POLB 0.69 polymerase (DNA directed) beta (POLB) 6.72E LIG ligase III, DNA, ATP-dependent (LIG3) nuclear gene encoding mitochondrial protein, transcript variant alpha 4.38E POLE 0.67 polymerase (DNA directed) epsilon (POLE) 1.86E LIG ligase I, DNA, ATP-dependent (LIG1) 1.32E XRCC X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) 2.16E POLE polymerase (DNA directed) epsilon 2 (p59 subunit) (POLE2) 1.92E POLD polymerase (DNA-directed) delta 3, accessory subunit (POLD3) 2.83E NEIL nei endonuclease VIII-like 3 (E coli) (NEIL3) 2.23E UNG 0.65 uracil-dna glycosylase (UNG) nuclear gene encoding mitochondrial protein, transcript variant E PCNA 0.69 proliferating cell nuclear antigen (PCNA) transcript variant E FDR

12 Supplementary Table 6 Table S6. Additional genes differentially expressed in HCT-LTR143-5 relative to HCT- LTR, but not belonging to statistically enriched Kegg functional categories. Symbol mir vs ctrl GFR-PI3K Description Parametric p-value GH GH1 growth hormone PIK3CB PIK3R Symbol mir vs ctrl phosphoinositide-3-kinase,pik3cb catalytic, beta polypeptide phosphoinositide-3-kinase, Pik3r1 regulatory subunit 1 (alpha) p27 degradation Description FDR Parametric p-value SKP S-phase kinase-associated SKP1 protein SKP S-phase kinase-associated Skp2 protein 2 (p45) NEDD neural precursor cell expressed, developmentally down-regulated 8 nedd8 FDR TFDP TFDP1 transcription factor Dp