SUPPLEMENTARY DATA SEQUENCES (5-3 ) NAME

Átírás

1 Supplementary Table 1. Primers for cloning. Thirty-six genes down-regulated by mir-24 in MIN6 cells. Two methods (mirna microarray and qrt-pcr) were used to quantify mrna levels with or without mir-24 over expression in MIN6 cells. qrt-pcr was also utilized to determine the mrna levels of these genes relative to control cells after silencing Hnf1a and Neurod1. Prediction of MREs was conducted using miranda and TargetScanMouse 4.2. After combining results of bioinformatic and experimental analyses, 36 genes confirmed to be down regulated were divided into three groups: (1) genes containing the mir-24 seed, but with no targets of Hnf1a or Neurod1; (2) genes not containing the mir-24 seed, but with targets of Hnf1a or Neurod1; (3) genes not containing the mir-24 seed or targets of Hnf1a or Neurod1. NAME Forward SEQUENCES (5-3 ) Reverse wt-hmox1 CTAGT TAAAAGCAGC TGTTCTGAGC CC A AGCTT GGGCTCAGAA CAGCTGCTTT TA A mt-hmox1 CTAGT TAAAAGGACG ACTTGACTCG GC A AGCTT GCCGAGTCAA GTCGTCCTTT TA A wt-slc25a44 CTAGT TCTTCCCTGC CTCTACTGAG CCT A AGCTT AGGCTCAGTA GAGGCAGGGA AGA A mt-slc25a44 CTAGT TCTACGGACC GACTTGACTC GGT A AGCTT ACCGAGTCAA GTCGGTCCGT AGA A wt-slc25a44-2 CTAGT TATCTCCTCA GGCCAGCTGA GCCA A AGCTT TGGCTCAGCT GGCCTGAGGA GATA A wt-igfbp4 CTAGT CCCTACAGGA TAAGCCTGAG CCT A AGCTT AGGCTCAGGC TTATCCTGTA GGG A mt-igfbp4 CTAGT CCCAAGAGCA AATTCGACTC GGT A AGCTT ACCGAGTCGA ATTTGCTCTT GGG A wt-os9 CTAGT CGGTACCTGT GCTTCTGAGC CC A AGCTT GGGCTCAGAA GCACAGGTAC CG A mt-os9 CTAGT CGCAAGGACG GCTTGACTCG GC A AGCTT GCCGAGTCAA GCCGTCCTTG CG A wt-cdk16 CTAGT TTGTTGCCTG CTTACCTGCC TGAGCCA A AGCTT TGGCTCAGGC AGGTAAGCAG GCAACAA A mt-cdk16 CTAGT TTCAAGGGAC GATTCCTGCG ACTCGGA A AGCTT TCCGAGTCGC AGGAATCGTC CCTTGAA A wtlaptm4b CTAGT CACTTCCATG ATGAACTCTC TGAGCCA A AGCTT TGGCTCAGAG AGTTCATCAT GGAAGTG A mt-laptm4b CTAGT CACAAGGAAC AACTTCTCTG ACTCGGT A AGCTT ACCGAGTCAG AGAAGTTGTT CCTTGTG A wt-mycbp CTAGT TCAAACATAT TTTTTTGAGC CT A AGCTT AGGCTCAAAA AAATATGTTT GA A wt-avl9 CTAGT CATTTCAAGT CAGGTACTGA GCCA A AGCTT TGGCTCAGTA CCTGACTTGA AATG A mt-avl9 CTAGT CATAAGAACT GACTTTGACT CGGT A AGCTT ACCGAGTCAA AGTCAGTTCT TATG A wt-avl9-2 CTAGT CTACTTTTAC TTCATTCTGA GCCT A AGCTT AGGCTCAGAA TGAAGTAAAA GTAG A wt-psap CTAGT GGCAGGAGCA TTTTCTGAGC CC A AGCTT GGGCTCAGAA AATGCTCCTG CC A mt-psap CTAGT GGCAGGAGCA TTTTGACTCG GC A AGCTT GCCGAGTCAA AATGCTCCTG CC A wt-cited4 CTAGT GCCGCCTTGC CCCAATCTGA GCCA A AGCTT TGGCTCAGAT TGGGGCAAGG CGGC A mt-cited4 CTAGT GCCGCGGACC GCCTTTGACT CGGT A AGCTT GCCGAGTCAA AATGCTCCTG CC A wt-g6pdx CTAGT CCACCACTGG CCCTATCTGA GCCA A AGCTT TGGCTCAGAT AGGGCCAGTG GTGG A mt-g6pdx CTAGT CCACCAGACG CGCTTTGACT CGGT A AGCTT ACCGAGTCAA AGCGCGTCTG GTGG A wt-tsc22d3 CTAGT AGAGATGGGA GAGATTGAGC CA A AGCTT TGGCTCAATC TCTCCCATCT CT A wt-cmtm6 CTAGT ATTCTTAATT CCTTCTGAGC CA A AGCTT TGGCTCAGAA GGAATTAAGA AT A mt-cmtm6 CTAGT ATTCTTAATT CCTTGACTCG GT A AGCTT ACCGAGTCAA GGAATTAAGA AT A wt-mpdu1 CTAGT CTCTAACTGC AGCCTCCTGA GCCA A AGCTT TGGCTCAGGA GGCTGCAGTT AGAG A mt-mpdu1 CTAGT CTCTAAGACG ACCCTCGACT CGGT A AGCTT ACCGAGTCAA AGCGCGTCTG GTGG A wt-neurod1 CTAGT CTATTCATGT AATAAATCTG AGCCT A AGCTT AGGCTCAGAT TTATTACATG AATAG A mt-neurod1 CTAGT CTAAAGAACG AAAATTTGAC TCGGT A AGCTT ACCGAGTCAA ATTTTCGTTC TTTAG A wt-hnf1a CTAGT CACTGCCTGG AGGATATCTG AGCCT A AGCTT AGGCTCAGAT ATCCTCCAGG CAGTG A mt-hnf1a CTAGT CACAGGGACG ACTTTATGAC TCGGT A AGCTT ACCGAGTCAT AAAGTCGTCC CTGTG A

2 wt-hnf1b CTAGT GTGTGTGCTA ATGAACTGAG CCA A AGCTT TGGCTCAGTT CATTAGCACA CAC A mt-hnf1b CTAGT GTCAGAGGAA AACTTGACTC AAT A AGCTT ATTGAGTCAA GTTTTCCTCT GAC A wt-pdx1 CTAGT TTGCTCCTTT CAAGGTCTGA GCCT A AGCTT AGGCTCAGAC CTTGAAAGGA GCAA A mt-pdx1 CTAGT TTCCAGGAGA CTTGGTGACT CGGT A AGCTT ACCGAGTCAC CAAGTCTCCT GGAA A mt-cdx2 CTAGT ACAGCAATCC CAGGCTGAGC CA A AGCTT TGGCTCAGCC TGGGATTGCT GT A mt-cdx2 CTAGT ACAGCAAACG CATTGACTCG GT A AGCTT ACCGAGTCAA TGCGTTTGCT GT A wt-parp1 CTAGT CCCTAACTGG GGCGACTGAG CCT A AGCTT AGGCTCAGTC GCCCCAGTTA GGG A mt-parp1 CTAGT CCCTAAGACG GCCTTGACTC GGT A AGCTT ACCGAGTCAA GGCCGTCTTA GGG A NeuroD1-3'UTR GAGCTC AAACGAATCCACTGTGCGT ACGCGT CATCACATCTCAAACAGCAC mt-neurod1-3' UTR TCATGTAATAAATGTGTGCGTAAATTTATTCAGG CCTGAATAAATTTACGCACACATTTATTACATGA kcnj8-3'utr ACTAGT GCAGGATG ACCGGAGACA AAGCTT CCGTGTGGTTCAATGGCTCA kcnj11-3'utr ACTAGT CGTGGAAAGTGAAGTATGGTAT GAGCTC GAGCCAGGATTTGAACCAAT Neurod1-cds CATATG ACCAAATCATACAGCGAGAGC GGATCC CTAATCGTGAAAGATGGCAT Hnf1a-cds GAAGATCT ATGGTTTCTAAGCTGAGC GATATC TTACTGGGAAGAGGAGG Ccnd3-cds CATATG GAGCTGCTGTGTTGCGAG GGATCC CTACAGGTGAATGGCTGT Cdk4-cds CATATG GCTGCCACTCGATATGAAC GGATCC TCACTCTGCGTCGCTTT

3 Supplementary Table 2. Primers for qrt-pcr NAME SEQUENCES (5-3 ) Forward Reverse mir-124a ACACTCCAGCTGGGTAAGGCACGCGGTG CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGCATTCA mir-127 ACACTCCAGCTGGGTCGGATCCGTCTGAG CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAAGCT mir-130a ACACTCCAGCTGGGCAGTGCAATGTTAAAA CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATGCCCTT mir-15a ACACTCCAGCTGGGTAGCAGCACATAATGG CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACAAACC mir-181a ACACTCCAGCTGGGAACATTCAACGCTGTCG CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACTCACCG mir-21 ACACTCCAGCTGGGTAGCTTATCAGACTGA CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCAACATC mir-24 ACACTCCAGCTGGGTGGCTCAGTTCAGCAG CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTGTTCCT mir-30c ACACTCCAGCTGGGTGTAAACATCCTACACT CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCTGAGAG mir-375 ACACTCCAGCTGGGTTTGTTCGTTCGGCTC CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCACGCGA mir-376a ACACTCCAGCTGGGATCGTAGAGGAAAAT CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACGTGGAT mir-7 ACACTCCAGCTGGGTGGAAGACTAGTGATT CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAACAAAA mir-146a ACACTCCAGCTGGGTGAGAACTGAATTCCA CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACCCATG mir-34a ACACTCCAGCTGGGTGGCAGTGTCTTAGCTG CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACAACCA URP TGGTGTCGTGGAGTCG U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT Slc25a44 ATGGAGGACAAACGGAACATC GTGAACGGGTACACGCTGA Psap GTGGTTGACTCTTACCTGCCT TCTTGTTGGACTCAAGCTGTTT Igfbp4 AGAAGCCCCTGCGTACATTG TGTCCCCACGATCTTCATCTT Os9 GGTGTCGGGAGCCTGAATTT CCTCTCTTTCACGTTGGAAGTG Cited4 GAGGGCTACTGCCTGCTAC GCGCCTCTTCGTCGATGAG Hmox1 AAGCCGAGAATGCTGAGTTCA GCCGTGTAGATATGGTACAAGGA Mycbp GCTGGACACGCTGACGAAA TCTAGGCGAAGCAGCTCTATTT Laptm4b GCTCCCTGGACTCGGTTCTA GCAGGACCACAGCATTGATGA Cdk16 AGATAAGACCAATGGTGTCCCT CACTCTCCCCATCAGATCCCA Cmtm6 ATGGAGAACGGAGCGGTCTA CACACTCGGACACAACCTCT Avl9 GGTCGGCTTTCACCACAAGAA CACCATCTGGTAAGGCAAGGA Tsc22d3 CGTGAGCAACTTTCGGCAG AGAGCATGGTCTGGTCTATGT Mpdu1 ACAGAGGAGAGACCGTGAAAG GAGCAGAGTGACTACAGCCA G6pdx CACAGTGGACGACATCCGAAA AGCTACATAGGAATTACGGGCAA Tmed6 TGACCAGATGGGATACCTCTACT AGAAGCCTGTTTCTTGTGTAGC Lgals2 AACATGAAACCAGGGATGTCC CGAGGGTTAAAATGCAGGTTGAG Serpina1a CTCCTCCAAACCCTCAACAG AGAAGGAAGACAGCAGTGGC Ugt2b34 ATCCTGGCAGATCCCTTCATAC ATTCTGACATAACAACAGGCACA Sgk2 CCATTGGTTACCTTCACTCTCTC GTCTCCTCAGGCTCTACACAT Sult1d1 TCTGGAACAACTTGGGTCAGT GCTGAACAGGAAGGTGTGTTT

4 Serpina1b GAGCATTGGCACAGCGTTTG AAGCGATGGTTGGATGTCAGC Pik3c2g CCATCTATCAGCTAATCGACGTG GGAACACACTGTGAAGCTCAG Gc CCTGCTGGCCTTAGCCTTT TGCTCAAATGTGCTACTGGAAA Il13ra1 ATGCTGGGAAAATTAGGCCATC ATTCTGGCATTTGTCCTCTTCAA Cspp1 AGGAAGAAGTAGGCATTTCTGGT TCGGCTCTGCAATGTTGTCTG Pkdcc CGGTCACCCTGCTTGACTTC GGCATTGTAGAGGTTCCGTTTC Crp GTGCTGAAGTACGATTCATGGT GGTTTCCCATCAATCCAGAACT Slc2a2 TCAGAAGACAAGATCACCGGA GCTGGTGTGACTGTAAGTGGG Fam134c TGTGGGATCGAGCCTATGTG CCCTGGCAACAGTAGAATCATC Kif5b GCGGAGTGCAACATCAAAGTG CATAAGGCTTGGACGCGATCA Dhx36 TAGAGATGCAGCGTTTCAGAAAA CCACAACCAGTTTCACCACTTA Ccdc86 CGGATCACCCGGTCCTAGA GGGGAGGTAATTTGGATGGCT Hpgds GGGCAGACTTCTACTGGGATA GACTTTGTTCCGTAATGACACCA Ppargc1a ATGTGTCGCCTTCTTGCTCT ATCTACTGCCTGGGGACCTT Hfe CACCGCGTTCACATTCTCTAA CTGGCTTGAGGTTTGCTCC Cdkn1b GGCCCGGTCAATCATGAA CATATCCCGGCAGTGCTTCT Ccnd3 CGAGCCTCCTACTTCCAGTG GGACAGGTAGCGATCCAGGT ccnd1 CAGAAGTGCGAAGAGGAGGTC TCATCTTAGAGGCCACGAACAT ccnd2 GAGTGGGAACTGGTAGTGTTG CGCACAGAGCGATGAAGGT cdk4 ATGGCTGCCACTCGATATGAA TCCTCCATTAGGAACTCTCACAC p15 CCCTGCCACCCTTACCAGA CAGATACCTCGCAATGTCACG p21 CCTGGTGATGTCCGACCTG CCATGAGCGCATCGCAATC kcnj8 AAGAGCATCATCCCGGAGGA ATGTTCTTGTGTGCCAGGTTG β-actin AGGCCAACCGTGAAAAGATG AGAGCATAGCCCTCGTAGATGG

5 Supplementary Table 3. Affymetric Genechip Data Gene Symbol RefSeq Transcript ID Ratio Fold-Change Tmed6 NM_ Lgals2 NM_ Serpina1a NM_ Ugt2b34 NM_ Sgk2 NM_ Sult1d1 NM_ Serpina1c NM_ Serpina1b NM_ Slc25a44 NM_ Psap NM_ Asb5 NM_ Pik3c2g NM_ Igfbp4 NM_ Fam134c NM_ Gc NM_ Il13ra1 NM_ Os9 NM_ Cited4 NM_ Kif5b NM_ Hmox1 NM_ Hfe NM_ Mycbp NM_ Cspp1 NM_ Dhx36 NM_ Laptm4b NM_ Ccdc86 NM_ Pkdcc NM_ Hpgds NM_ Cdk16 NM_ Cmtm6 NM_ Crp NM_ Avl9 NM_ Gm11276 NM_ Ppargc1a NM_ Tsc22d3 NM_ Mpdu1 NM_ Scarb1 NM_ Ang NM_ Rnf186 NM_ Twf1 NM_ Mcrs1 NM_ Nup210 NM_

6 Cdx2 NM_ Ptgs2 NM_ Luc7l2 NM_ Ttc28 NM_ L12Rik NM_ Clcn3 NM_ Tor2a NM_ L11Rik NM_ Cpeb3 NM_ Kcnip1 NM_ Pea15a NM_ Ap1m2 NM_ Ptdss2 NM_ Slc29a4 NM_ Fam117a NM_ Ugt1a1 NM_ Slc2a2 NM_ Purb NM_ Ret NM_ Galns NM_ Erp29 NM_ Dpp4 NM_ J23Rik NM_ Glg1 NM_ Rnf138 NM_ Pppde2 NM_ Tmem192 NM_ Plagl1 NM_ Rnase4 NM_ Gpd2 NM_ Ptprf NM_ B04Rik NM_ Snw1 NM_ Pcdha1 NM_ Rtn2 NM_ Nek4 NM_ O18Rik NM_ Bcat1 NM_ Tacr3 NM_ Stk11ip NM_ Arhgef16 NM_ Hip1r NM_ Reep6 NM_ Chd4 NM_ Igf1r NM_

7 Ufm1 NM_ Crtac1 NM_ Stx18 NM_ Peg3 NM_ St6galnac5 NM_ Lifr NM_ Adcy6 NM_ Atp7a NM_ Grb7 NM_ Xpo7 NM_ Neu1 NM_ Wrnip1 NM_ Zswim7 NM_ Slc4a10 NM_ Rbm39 NM_ Ddr1 NM_ Flt3l NM_ Rab4b NM_ Rab20 NM_ Bbx NM_ Lpcat3 NM_ Tpr NM_ Serf2 NM_ Fam120a NM_ Abhd8 NM_ Ubfd1 NM_ Rassf7 NM_ Gla NM_ Sephs2 NM_ Dpp7 NM_ D11Wsu99e NM_ Gpatch4 NM_ Eif4ebp1 NM_ K02Rik NM_ Plk3 NM_ Chpf NM_ Hhip NM_ Wipi1 NM_ Sez6l NM_ Rhou NM_ Tsta3 NM_ Git1 NM_ Vat1 NM_ Frk NM_ Arhgef12 NM_

8 Nr1h4 NM_ Egfl6 NM_ Smc6 NM_ Cnot6 NM_ Vps53 NM_ Neat1 NR_ Prkar1b NM_ Gfer NM_ Ccnd3 NM_ Tmem93 NM_ G3bp2 NM_ Derl1 NM_ Lpl NM_ Extl2 NM_ Ssbp2 NM_ Hsbp1 NM_ B930041F14Rik NM_ Ogfr NM_ Gdap10 XM_ Hs1bp3 NM_ Cxcl12 NM_ Aars NM_ Seh1l NM_ Dio1 NM_ Dmpk NM_ H1f0 NM_ Dnajc19 NM_ Cdkn1b NM_ BC NM_ Fxyd3 NM_ Ung NM_ Sult1c2 NM_ Sirpa NM_ Sub1 NM_ Zxdc NM_ Fam3c NM_ Amz2 NM_ Id2 NM_ Hadh NM_ Furin NM_ Slc25a23 NM_ Mtdh NM_ Myo1d NM_ Tubb5 NM_ Nrk NM_

9 Bcl9 NM_ Tnfsf12 NM_ Cpt1a NM_ Herc1 NM_ Zfp568 NM_ E07Rik NM_ Syt2 NM_ Myo10 NM_ M06Rik NM_ M19Rik NM_ Depdc6 NM_ Idh2 NM_ Alg2 NM_ Tbl1x NM_ Hist1h4a NM_ Xbp1 NM_ Nphp1 NM_ Ccpg1 NM_ Cend1 NM_ Srpk2 NM_ Sema4a NM_ Usp48 NM_ Egfr NM_ Adcyap1 NM_ Ccdc56 NM_ E24Rik NM_ Parp3 NM_ Pofut2 NM_ Mat1a NM_ Ptprd NM_ Wars NM_ J03Rik NM_ Grina NM_ Wnt4 NM_ Arf3 NM_ Zdhhc6 NM_ Gpx2 NM_ Dleu2 NR_ Mapk8ip1 NM_ Ppp1r1a NM_ Slc22a23 NM_ Thrap3 NM_ Dner NM_ Sbno1 NM_ Tmem206 NM_

10 Spt1 NM_ Tacc3 NM_ Htatsf1 NM_ Abcf1 NM_ Mll5 NM_ Tbc1d8 NM_ Wnk4 NM_ Rnf11 NM_ Gpr133 NM_ Agbl3 NM_ Hus1 NM_ Vamp7 NM_ Pvrl3 NM_ Stk40 NM_ Scamp5 NM_ Smarcc1 NM_ Herpud1 NM_ Wdr33 NM_ Cntfr NM_ Naa15 NM_ Fam102a NM_ Gbf1 NM_ Ndst1 NM_ Nfix NM_ Thoc2 NM_ Cnpy2 NM_ J06Rik NM_ Edil3 NM_ Rufy3 NM_ Mia3 NM_ Sphk1 NM_ Spock1 NM_ Gm5595 NM_ Sct NM_ O18Rik NM_ Arhgap5 NM_ Hsd17b4 NM_ Pdap1 NM_ Nras NM_ Flcn NM_ Gmfb NM_ H2-Ke2 NM_ Mef2a NM_ Stk24 NM_ Dusp18 NM_

11 J16Rik NM_ Elf5 NM_ Cpn1 NM_ Cyp51 NM_ Isca1 NM_ Bmp2 NM_ Wdr12 NM_ Epha1 NM_ Gad1 NM_ Rad23a NM_ Phip NM_ Nono NM_ Smagp NM_ Pvrl2 NM_ Gm8615 /// NM_ /// NR_ /// Gnpda1 XM_ Cdh1 NM_ B3gnt3 NM_ Kif12 NM_ Kl NM_ Mtmr9 NM_ Trib3 NM_ Ifnz NM_ Zfp81 NM_ Nmnat3 NM_ Hk1 NM_ Selm NM_ Tmem135 NM_ Cbs NM_ Maff NM_ E2f1 NM_ Brd4 NM_ Slc40a1 NM_ Rnf114 NM_ Ccdc12 NM_ Tmprss3 NM_ Rab27a NM_ Lypla2 NM_ Gatsl2 NM_ Socs4 NM_ Soat2 NM_ Vldlr NM_ Slc7a8 NM_ Taok3 NM_ Reep2 NM_

12 Cdo1 NM_ Psen1 NM_ Ccnd2 NM_ Smad1 NM_ Rala NM_ Mrpl36 NM_ Tm2d1 NM_ G6pdx NM_ Spast NM_ M22Rik NM_ Pcgf2 NM_ Helz NM_ H6pd NM_ Pdxk NM_ Smc4 NM_ Mrc2 NM_ Rnf2 NM_ Rap2c NM_ Snx5 NM_ Nol3 NM_ Syt13 NM_ Sh3glb1 NM_ F2rl1 NM_ Gnai2 NM_ H2afx NM_ D22Rik NM_ Fads1 NM_ Hnf4a NM_ Tob2 NM_ Agfg1 NM_ Atm NM_ Sptlc2 NM_ Nudt19 NM_ P4hb NM_ Tor3a NM_ Cyb5r3 NM_ Mrpl11 NM_ Stau2 NM_ Fem1a NM_ Mllt1 NM_

13 Supplementary Table 4. Genes down regulated by mir-24 over expression in MIN6 cells

14 Supplementary Figure 1. Characteristics of diabetic db/db mice and their littermate controls. A-D: Comparison of body weight (A), blood glucose (B), GSIS (C), and insulin content (D) in islets from 8- weeks-old db/db mice and littermates control. Eight-week-old db/db mice exhibited high body weight, hyperglycemia, defective GSIS and reduced insulin content compared with their littermates. Secreted insulin was normalized to total protein concentration. E: Expression of the insulin gene was decreased in islets of db/db mice with age (8- to 12-weeks-old).

15 Supplementary Figure 2. Palmitate induces MIN6 cell dysfunction. A-D: MIN6 cells were incubated with or without palmitate for 24 h when GSIS (A) and KSIS (C) assays were performed. After preincubation for 1 h, supernatants containing released insulin from low glucose (2 mmol/l) (white), high glucose (20 mmol/l) (black) and KCl (50 mmol/l) (black) groups were collected sequentially for further determination using RIA. No obvious changes were detected in the low glucose challenged group, while palmitate treated cells displayed significantly reduced insulin release in response to high glucose or KCl. Secreted insulin was normalized to total protein concentration. The GSIS index (B) and KSIS index (D) were ratios of stimulus-induced insulin secretion relative to the basal low glucose level. E: MIN6 cells were treated with or without palmitate for 36 h when cell viability was measured by the WST-1 assay. Palmitate inhibited cell proliferation.

16 Supplementary Figure 3. mir-24 impairs MIN6 cell function. A: No obvious changes were observed at the G1 phase when mir-24 was over expressed in MIN6 cells at indicated concentrations. B-E: GSIS (B) and KSIS (C) assays were performed in MIN6 cells over expressing mir-24 (50 nmol/l) for 48 h. Results were similar to that in palmitate damaged cells. D and E: Rat insulin promoter luciferase activity (D) and insulin mrna (E) were significantly repressed by mir-24.

17 Supplementary Figure 4. Bioinformatic analysis of genes downregulated by mir-24. A-C: DAVID Bioinformatics Resources 6.7 was used to perform statistical analysis of over represented GO terms for 351 down regulated genes caused by elevated mir-24 in MIN6 cells. Genes with at least 1.5-fold change were clustered. Cellular processes were sorted by score (-log[p value]), and clustered genes were divided into three subsets accordingly. The highly positive score set included genes involved in the phosphate metabolic process, vesicle-mediated transport and response to stress, hormone stimulus and nutrient levels (A). Cell cycle genes were clustered in the middle subset with a score of 1.13 (B). The lowest scored gene subset also represented important biological functions (C).

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19 Supplementary Figure 5. mir-24 directly down regulates several functional genes. A: The mir-24 MREs in 3'UTR sequences of 36 down regulated gene were predicted by miranda and TargetScanMouse 4.2. Fourteen genes containing 16 mir-24 MREs were aligned with mir-24. The paired seeds are indicated as vertical lines. Twelve mutant (mt) sequences are also listed beneath each mir-24 sequence. B and C: Except for Mycbp and Tsc22d3, activities of all of the other 14 MREs were detected by dual luciferase reporter assay. Avl9 and Slc25a44 both contained two active MREs. Reporter activities of genes containing mutations in seed sequences showed little or no differences from that of the vector control. D and E: mrnas levels of the 16 genes were down regulated to varying degrees.

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21 Supplementary Figure 6. Cyclind3 and Cdk4 are major regulatory molecules of the cell cycle. A: Expression levels of Cyclind3 and Cdk4 were analyzed by Western blotting. B: Cyclind3 and Cdk4 expressed from pcmv5-myc vectors were detected by using a myc-tag antibody and shown to be highly elevated. Moreover, these protein levels were obviously decreased in cells with elevated mir-24 relative to the negative control. β-tubulin was used as an internal control. C: BrdU labeling showed a significant decrease in the level of DNA in cells co-transfected with vector control and mir-24 to that co-tranfected with vector control and Neg sirna. Over expression of Cyclind3 and Cdk4 fully restored cell proliferation in the presence of over expressed mir-24.

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