SUPPLEMENTARY FIGURES AND TABLES

Transcription

1 SUPPLEMENTARY FIGURES AND TABLES Suppl. Table 1 Oligonucleotides used in the study The table shows the structure and nucleotide position of the oligonucleotides used in the study along with their application (Exp.). The asterisk indicates that the nucleotide position is calculated relative to the mir-21 stem-loop sequence. ChIP1S/ChIP1AS and ChIP2S/ChIP2AS encompass the region of the MIR21 5 -flanking sequence containing the upstream RARE-1 and RARE-2 sites, respectively. Pri-miR-21 2S/Pri-miR-21 2AS encompass part of the transcribed region of MIR21 and were used as a negative control for the amplification procedure. The deletion mutant of the RARE-1 present in the 5 -flanking region of MIR21 was generated as follows: The circular plasmid, mir21 S, containing 1.5 Kb of MIR21 5 -flanking region upstream of the firefly luciferase reporter, was used as the template to amplify the entire DNA sequence between the two limiting amplimers, which were endowed with an artificially added restriction site at their 5 -end (italics in the oligonucleotide name). The resulting amplified bands were cut with the appropriate restriction endonunuclease and re-circularised using T4 DNA ligase. All the plasmids and deletions thereof were checked by sequencing. To prepare the constructs containing the 3 UTR of the potential mir-21 targets downstream of the renilla luciferase reporter gene, we proceeded as follows. The amplimers listed were used to amplify the entire 3 UTR region, which was subsequently cloned in the pcr2.1 plasmid (Invitrogen). Inserts were re-amplified with versions of the same oligonucleotides as above containing an artificial NotI site at their 5 -end. The PCR amplified products were cut with NotI and inserted in the same site of the phrl plasmid. The correct orientation of the 3 UTR relative to the luciferase reporter was verified by sequencing. Deletions of the 3-UTRs of IL1B, ICAM1 and PLAT were generated with the same strategy described for the RARE-1 deletion constructs. Suppl. Table 2 Genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells MCF-7 (MCF7) and MD-MB231 (MDA) cells were treated for 6 and 48 hours with ATRA (1 µm). Total RNA was extracted and used to perform whole-genome gene expression microarray studies. The table lists the 481 genes whose ATRA-dependent regulation is significantly different (p<0.01) in the two cell lines. When genes belong to the blue cluster (see Fig. 5A), gene symbols are marked in blue, when they belong to the orange cluster the same gene symbol column is marked in orange. When genes are predicted to be potential mir-21 targets according to the prediction algorithms, Miranda, TargetScan or PITA, GenBank accession numbers (GENBANK_ACC) are marked in light blue. In the blue cluster, gene titles are marked in yellow, when the corresponding genes are annotated as belonging to inflammatory (INF) and immune responses (IMM) or leukocyte migration (LEUK) pathways. Values are expressed as the Log 2 ratios of the ATRA vs. vehicle treatment. When the Log 2 ratio is < -0.5 or > 0.5 the corresponding cells are marked in green and red, respectively. Suppl. Table 3 mir-21 predicted seed sequences present in the 3 UTR present in the 3 UTRs of CCR1, TNFAIP3, PTX3, IL1B, ICAM1, PLAT and ITGB3 The table shows the sequence and position of the various mir-21 predicted seed sequences located in the 3 UTR of the indicated transcripts along with the corresponding accession number. ITGB3 is characterized by 3 seed sequences, while all the other mrnas are endowed with a single seed sequence. 1

2 Suppl. Table 1 Exp. gene oligonucleotide sequence position Acc.No. Cloning of MIR21 promoter MIR 21 MIR21 promoter S 5 -acaacttcacccctcactgatctc-3-4,693/-4,670* MIR21 promoter AS 5 -tagtcctcagagtaaggtcagctc -3-3,236/-3213* Real Time PCR Pri-miR-21 Pri-miR-21 1S 5 -aatggccttgcactcttcttatg-3 487/509 AY Pri-miR-21 1AS 5 -gttgaccaaacatgacatcagaaac-3 527/551 Pri-miR-21 2S 5 -tggtggcacttagagtcttttgtg-3 2,614/2,637 AY Pri-miR-21 2AS 5 -aggattttatggagaaatgggga-3 2,661/2,683 ChIP MIR21 ChIP 1S 5 -ccagaagttagggatatgttagca-3-4,396/-4,373* ChIP 1AS 5 -tacctccagggttcaagtgattct-3-4,094/-4,071* ChIP 2S 5 -actgtctaccataaaccatgaaagga /-3579* ChIP 2AS 5 -cccactagtcagaagtcagtgattaaca-3-3,547/-3,520* Deletion of MIR21 promoter MIR21 RARE1 S-StuI 5 -ctaggcctccagcctggcaaagatggtgaa-3-4,204/-4,183* RARE1 AS-StuI 5 -ctaggcctgatcccccttcctcggcctcc-3-4,245/-4,225* Point mutation of MIR21 promoter MIR21 Mut1 5 -gaagggggatcacgaggacaggagttcaagaccag-3-4,235/-4,201* Mut2 5 -accatgaaaggattcaaagtgcatagttccttctttgttcc-3-3,690/-3,550* Cloning of 3 UTR CCR1 3'-CCR1 S 5 -ctcagaccataggaggccaaccca-3 1,140/1,163 NM_ '-CCR1 AS 5 -cacctgggaaagtgatcacaacttg-3 1,631/1,655 PLAT 3'-PLAT S 5 -ccaggaacacccgactcctcaaaa-3 1,899/1,922 NM_ '-PLAT AS 5 -cagaagtcaattaagtccaaactcag-3 3,147/3,173 PTX3 3'-PTX3 S 5 -atgttgtgaaactccacttgaagc-3 1,290/1,313 NM_ '-PTX3 AS 5 -aatgacgtgagctagttttataaaat-3 1,885/1,910 TNFAIP3 3'-TNFAIP3 S 5 -taaccggaaacaggtgggtcacct-3 2,437/2,460 NM_ '-TNFAIP3 AS 5 -gaaatccaacaaagaataggtggctttc-3 4,336/4,363 ICAM1 3'-ICAM1 S 5 -tgaaaccgaacacacaagccacgc-3 1,887/1,910 NM_ '-ICAM1 AS 5 -tttggcagttgagaaagctttattaac-3 3,223/3,250 IL1B 3'-IL1B S 5 -agagagctgtacccagagagtcct-3 898/921 NM_ '-IL1B AS 5 -cagtgaagtttatttcagaaccattg-3 1,470/1,495 Maspin 3'-Maspin S 5'-gtggcatagcccatgttaagtcct-3' 1,196/1,219 NM_ '-Maspin AS 5'-gtagttcaataatattttattgtcaatagc-3' 2,528/2,557 Deletion of 3 -UTR PLAT PLAT dels-sali 5 -atgtcgacaatttagattatgggggctctg-3 3,117/3,138 NM_00093 PLAT delas-sal I 5 -atgtcgactcctcttcctgaagttcacttc-3 3,084/3,105 ICAM1 ICAM1 dels-sali 5 -atgtcgacaacaccacacctggcaaattt-3 3,141/3,161 NM_ ICAM1 delas-sali 5 -atgtcgactcccagctactcaggaggctga-3 3,107/3,128 IL 1B IL 1B dels-sali 5 -atgtcgacgattatttaaatgggaatattt-3 1,423/1,444 NM_ IL 1B del AS-SalI 5 -atgtcgacattttcagtcttaattaaagga-3 1,390/1,441 Cloning of cdna PLAT PLAT S 5'-gggacgctgtgaagcaatcatgga-3' 191/214 NM_00093 PLAT AS 5'-ttttgaggagtcgggtgttcctgg-3' 1,899/1,922 PLAT S/Kozac-NotI 5'-atgcggccgcgccaccatggatgcaatgaagagagggct-3' 209/232 NM_00093 PLAT AS-XhoI 5'-atctcgagtcacggtcgcatgttgtcacgaa-3' 1,876/1,898 ICAM1 ICAM1 S 5'-ttgcaacctcagcctcgctatgg-3' 301/323 NM_ ICAM1 AS 5'-tgtcccgggataggttcagggag-3' 1,911/1,933 ICAM1 S/Kosac-NotI 5'-atgcggccgcgccaccatggctcccagcagcccccggccc-3' 320/343 NM_ ICAM1 AS-XhoI 5'-atctcgagtcagggaggcgtggcttgtgtg-3' 1,897/1,918 Maspin Maspin S 5'-aatggatgccctgcaactagcaaa-3' 67/90 NM_ Maspin AS 5'-tttctgccaactaccccggacaat-3' 1,471/1,494 Maspin S/Kozac-NotI 5'-atgcggccgcgccaccatggatgccctgcaactagcaa-3' 68/90 NM_ Maspin AS-XbaI 5'-attctagattaaggagaacagaatttgccaa-3' 1,173/1,195 Cloning of RUN6 RUN6 RUN6/S 5'-gtgctcgcttcggcagcacatata-3' NR_ RUN6/AS 5'-aaaatatggaacgcttcacgaatttg-3' 81/106 2

3 Suppl. Table 2 3

4 Suppl. Table 2 contd. 4

5 Suppl. Table 2 contd. 5

6 Suppl. Table 2 contd. 6

7 Suppl. Table 3 Transcript Predicted seed sequence (3'-UTR) position (nucleotide) RefSeq CCR1 UAAGCU NM_ PLAT AUAGGCU NM_ PTX3 AUAAGUU NM_ TNFAIP3 GAUAAGCU; AUGAGCU ; NM_ ICAM1 AUAGGCU NM_ IL1B AUAAGCU NM_