GUGAUAAUGGAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGCUUGCGAGGUAUGA GAAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA

Transcription

1 Precursor mmu-mir-8- GUGAUAAUGGAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGCUUGCGAGGUAUGA GAAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA Precursor mmu-mir-8- GACCAGUUGCCGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUGUGGUGGAACGAUGGAA ACGGAACAUGGUUCUGUCAAGCACCGCGGAAAGCAUCGCUCUCUCCUGCA Mature mir 8 UUGUGCUUGAUCUAACCAUGU Supplemental Figure S: The RNA sequence of primary and mature mir-8- and - are illustrated. Both mir-8- and - yield a single species of mature microrna, mir-8. The seed sequence is marked by a box.

2 Supplemental Figure. S A. MC3T3 Dkk elative transcript levels Days Tob Relative transcript levels Days Sfrp Sost B. BMSCs Rela7ve transcript levels.5.5 Days Dkk Tob Sfrp Supplemental Figure. S: Expression of mir- 8 target genes during osteoblast differen7a7on. Total RNA was analyzed for expression of Dkk, Tob, Sfrp and Sost by real Dme PCR at the indicated Dme points using MC3T3 cells (A) and BMSCs (B). Sost is not detected in BMSCs. Expression was normalized with GAPDH RNA.

3

4 Supplemental Figure S4: Axin CyclinD Fig S4. Stimulated Wnt signaling by mir-8 MC3T3 cells were treated by Control, mir-8 or anti-mir-8 Lentivirus. Expression of Wnt target genes, Axin and CyclinD, was analyzed by qpcr.

5 Supplemental Table S: Nucleotide sequences of primers and probes used for qrt-pcr and northern blot Bone Marker Genes - Murine Real Time qpcr Primers (5-3 ) Runx F: CGGCCCTCCCTGAACTCT R: TGCCTGCCTGGGATCTGTA Alp F: CCAACTCTTTTGTGCCAGAGA R: GGCTACATTGGTGTTGAGCTTTT Ocn F: CTGACAAAGCCTTCATGTCCAA R: GCGCCGGAGTCTGTTCACTA Tob F: ATGGCGTCCTCTCTGCTTG R: TGAAAGGTCAGCGTATGGCTT Sost F: AGCCTTCAGGAATGATGCCAC R: CTTTGGCGTCATAGGGATGGT Dkk F: TCAGTCAGCCAACCGATCTG R: TCTCTGTGGCATCGTTTCTTTT Sfrp F: CGTGGGCTCTTCCTCTTG R: ATGTTCTGGTACTCGATCCG Tcf F: AGCTTTCTCCACTCTACGAACA R: AATCCAGAGAGATCGGGGGTC Lef F: TGTTTATCCCATCACGGGTGG R: CATGGAAGTGTCGCCTGACAG Axin F: TGACTCTCCTTCCAGATCCCA R: TGCCCACACTAGGCTGACA CyclinD F: GCGTACCCTGACACCAATCTC R: ACTTGAAGTAAGATACGGAGGGC mir-8- F: CCATGGAACGTCACGCAGC mir-8- F: GCGGAAAGCACCGTGCTC Mature mir-8 F: TTGTGCTTGATCTAACCATGT hsost F: ACTTCAGAGGAGGCAGAAATGG R: CAAGGGGGAATCTTATCCAACTTTC hdkk F: AGGAGTGTGAAGTTGGGAGGTA R: GGTTTGAGTAATGACCGTGGTT hsfrp F: ATGACAACGACATAATGGAA R: GGAGATGCGCTTGAACTCTC htcf4 F: CCTTAGGGACGGACAAAGAG R: GAAGAGTTGCCCAACATTCC hlef F: CTCTACAACAAGGGACCCTC R: CTTGTTTGGAGTTGACATCTG Control Control Primers (5 3 ) GAPDH F: AGGTCGGTGTGAACGGATTTG R: TGTAGACCATGTAGTTGAGGTCA U6 F: CGCTTCGGCAGCACATATAC R: AAAATATGGAACGCTTCACGA Northern Probes Primers (5-3 ) Mouse mir-8 GGAAATCCCTGGCAATGTGAT

6 SUPPLEMENTAL TABLE S Target UTR Fragment Sequences Cloned in pmir-report-luc Vector (Ambion Inc) DKK WT 5 CTAGTCGTGAAAATGCTATTATTAAAAGAAAGCACACCATGGTTAATCATGTGCAATACAAGCACAGGGCAGCA 3 DKK MT 5 CGATGTG CGATGTG 3 SFRP WT 5 CTAGTCCGCGACCTCATTTCCGGTTTCCCAAGCACAGTCCCTACAGAACTTCAGTTTATAAGCACACAGTAACCCA3 SFRP MT 5 CGATGTG CGATGTG 3 TOB WT 5 CTAGTCGCATCATTTTTCATTTGCCAACCAAGCACAAAGCGCATCATTTTTCATTTGCCAACCAAGCACAAAGA3 TOB MT 5 GATGTG CGATGTG 3 SOST WT 5 CTAGTGGCAGTGTTAATATCGCTTTGTGAAGCACAAGTGGGCAGTGTTAATATCGCTTTGTGAAGCACAAGTGA3 SOST MT 5 CGATGTG CGATGTG 3 Supplemental Table S: Schematic illustration of the 3 UTR of TOB, DKK, SFRP and SOST mrna target sequences with mir-8 participating nucleotides. Three putative target sites of different targets bound to mir-8 predicted by TargetScan ( MICRORNA.ORG ( and MICROCOSM ( programs. Last 7 nucleotides of the respective mrna targets (AAGCACA) were the seed sequence of mir-8. In the mutant reporter, these sequences were mutated (CGATGTG).

7 Supplemental Table S3: Pathway Ontology for mir-8 Targets Enrichment Score :.86 Gene count P value Retinol metabolism 3 4.3E- Enrichment Score :.85 Gene count P value Dilated cardiomypathy 6 4.7E- Arrhythmogenic right ventricular cardiomyopathy (ARVC) 4.E- Hypertrophic cardiomyopathy (HCM) 4.7E- Enrichment Score :.8 Gene count P value Gap junction 7.E- Vascular smooth muscle contraction 8.4E- Long-term depression 6.9E- Purine metabolism 6.5E- Enrichment Score :.78 Gene count P value Insulin signaling pathway 7 7.5E- Glioma 4.6E- Natural killer cell mediated cytotoxicity 5.7E- Chemokine signaling pathway 6 3.5E- Chronic myeloid leukemia 3 4.9E- ErbB signaling pathway 3 5.6E- Neurotrophin signaling pathway 3 7.7E- Enrichment Score :.74 Gene count P value Vascular smooth muscle contraction 8.4E- Enrichment Score :.7 Gene count P value ECM-receptor interaction 6 3.E- Enrichment Score :.46 Gene count P value Wnt signaling pathway 5 4.E- Supplemental Table S3: Pathway ontology for mir-8 targets. Using TargetScan (ver 6.), the top 5 (from approximately 8 putative targets with conserved sequences) were examined for the enrichment of gene ontology categories. The results show signaling pathways related to either cell differentiation pathways of different tissues or various cancers. Notable categories related to osteogenesis and bone metastasis include insulin, gap junctions, ECM-receptor interaction and ErbB and chemokine pathways.