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
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
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
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
Supplementary Table 3. Affymetric Genechip Data Gene Symbol RefSeq Transcript ID Ratio Fold-Change Tmed6 NM_025458 0.24-4.22 Lgals2 NM_025622 0.24-4.09 Serpina1a NM_009243 0.25-4.00 Ugt2b34 NM_153598 0.28-3.57 Sgk2 NM_013731 0.29-3.51 Sult1d1 NM_016771 0.29-3.43 Serpina1c NM_009245 0.30-3.30 Serpina1b NM_009244 0.36-2.75 Slc25a44 NM_001145876 0.38-2.63 Psap NM_001146120 0.38-2.62 Asb5 NM_029569 0.40-2.48 Pik3c2g NM_011084 0.42-2.38 Igfbp4 NM_010517 0.43-2.33 Fam134c NM_026501 0.43-2.31 Gc NM_008096 0.44-2.28 Il13ra1 NM_133990 0.45-2.24 Os9 NM_001171026 0.45-2.22 Cited4 NM_019563 0.45-2.21 Kif5b NM_008448 0.45-2.21 Hmox1 NM_010442 0.46-2.19 Hfe NM_010424 0.46-2.18 Mycbp NM_019660 0.46-2.16 Cspp1 NM_026493 0.46-2.16 Dhx36 NM_028136 0.46-2.16 Laptm4b NM_033521 0.47-2.15 Ccdc86 NM_023731 0.47-2.15 Pkdcc NM_134117 0.47-2.14 Hpgds NM_019455 0.47-2.11 Cdk16 NM_011049 0.48-2.08 Cmtm6 NM_026036 0.48-2.07 Crp NM_007768 0.48-2.06 Avl9 NM_030235 0.49-2.05 Gm11276 NM_001177544 0.49-2.05 Ppargc1a NM_008904 0.49-2.03 Tsc22d3 NM_001077364 0.49-2.03 Mpdu1 NM_011900 0.50-2.00 Scarb1 NM_016741 0.50-2.00 Ang NM_001161731 0.50-1.99 Rnf186 NM_025786 0.51-1.97 Twf1 NM_008971 0.51-1.97 Mcrs1 NM_001164156 0.51-1.97 Nup210 NM_018815 0.51-1.96
Cdx2 NM_007673 0.51-1.96 Ptgs2 NM_011198 0.51-1.95 Luc7l2 NM_001170848 0.51-1.95 Ttc28 NM_024477 0.51-1.95 2810432L12Rik NM_025944 0.51-1.95 Clcn3 NM_007711 0.51-1.95 Tor2a NM_152800 0.52-1.94 2900062L11Rik NM_029823 0.52-1.94 Cpeb3 NM_198300 0.52-1.94 Kcnip1 NM_027398 0.52-1.91 Pea15a NM_011063 0.52-1.91 Ap1m2 NM_001110300 0.53-1.90 Ptdss2 NM_013782 0.53-1.90 Slc29a4 NM_146257 0.53-1.90 Fam117a NM_172543 0.54-1.87 Ugt1a1 NM_013701 0.54-1.87 Slc2a2 NM_031197 0.54-1.85 Purb NM_011221 0.54-1.85 Ret NM_001080780 0.54-1.85 Galns NM_016722 0.54-1.84 Erp29 NM_026129 0.55-1.83 Dpp4 NM_001159543 0.55-1.83 3110049J23Rik NM_026085 0.55-1.83 Glg1 NM_009149 0.55-1.83 Rnf138 NM_019706 0.55-1.83 Pppde2 NM_134095 0.55-1.83 Tmem192 NM_001163747 0.55-1.83 Plagl1 NM_009538 0.55-1.82 Rnase4 NM_021472 0.55-1.82 Gpd2 NM_001145820 0.55-1.82 Ptprf NM_011213 0.55-1.81 5930434B04Rik NM_029862 0.55-1.81 Snw1 NM_025507 0.55-1.80 Pcdha1 NM_001003671 0.56-1.80 Rtn2 NM_001025364 0.56-1.80 Nek4 NM_011849 0.56-1.79 2510039O18Rik NM_029841 0.56-1.79 Bcat1 NM_001024468 0.56-1.78 Tacr3 NM_021382 0.56-1.78 Stk11ip NM_027886 0.56-1.78 Arhgef16 NM_001112744 0.56-1.78 Hip1r NM_145070 0.56-1.77 Reep6 NM_139292 0.56-1.77 Chd4 NM_145979 0.56-1.77 Igf1r NM_010513 0.56-1.77
Ufm1 NM_026435 0.56-1.77 Crtac1 NM_145123 0.57-1.76 Stx18 NM_026959 0.57-1.76 Peg3 NM_008817 0.57-1.76 St6galnac5 NM_012028 0.57-1.76 Lifr NM_001113386 0.57-1.76 Adcy6 NM_007405 0.57-1.75 Atp7a NM_001109757 0.57-1.75 Grb7 NM_010346 0.57-1.75 Xpo7 NM_023045 0.57-1.75 Neu1 NM_010893 0.57-1.75 Wrnip1 NM_030215 0.57-1.74 Zswim7 NM_027198 0.57-1.74 Slc4a10 NM_033552 0.57-1.74 Rbm39 NM_133242 0.57-1.74 Ddr1 NM_007584 0.58-1.74 Flt3l NM_013520 0.58-1.73 Rab4b NM_029391 0.58-1.73 Rab20 NM_011227 0.58-1.72 Bbx NM_027444 0.58-1.72 Lpcat3 NM_145130 0.58-1.72 Tpr NM_133780 0.58-1.72 Serf2 NM_011354 0.58-1.72 Fam120a NM_001033268 0.58-1.71 Abhd8 NM_022419 0.58-1.71 Ubfd1 NM_138589 0.58-1.71 Rassf7 NM_025886 0.58-1.71 Gla NM_013463 0.58-1.71 Sephs2 NM_009266 0.59-1.71 Dpp7 NM_031843 0.59-1.71 D11Wsu99e NM_138598 0.59-1.71 Gpatch4 NM_001110809 0.59-1.71 Eif4ebp1 NM_007918 0.59-1.70 2810405K02Rik NM_025582 0.59-1.70 Plk3 NM_013807 0.59-1.70 Chpf NM_001001565 0.59-1.69 Hhip NM_020259 0.59-1.69 Wipi1 NM_145940 0.59-1.69 Sez6l NM_019982 0.59-1.69 Rhou NM_133955 0.59-1.69 Tsta3 NM_031201 0.59-1.69 Git1 NM_001004144 0.59-1.68 Vat1 NM_012037 0.59-1.68 Frk NM_001159544 0.60-1.68 Arhgef12 NM_027144 0.60-1.68
Nr1h4 NM_001163504 0.60-1.68 Egfl6 NM_019397 0.60-1.68 Smc6 NM_025695 0.60-1.68 Cnot6 NM_212484 0.60-1.68 Vps53 NM_026664 0.60-1.67 Neat1 NR_003513 0.60-1.67 Prkar1b NM_008923 0.60-1.67 Gfer NM_023040 0.60-1.67 Ccnd3 NM_001081635 0.60-1.67 Tmem93 NM_001168470 0.60-1.67 G3bp2 NM_001080794 0.60-1.67 Derl1 NM_024207 0.60-1.67 Lpl NM_008509 0.60-1.66 Extl2 NM_001163514 0.60-1.66 Ssbp2 NM_024186 0.60-1.66 Hsbp1 NM_024219 0.60-1.66 B930041F14Rik NM_178699 0.60-1.66 Ogfr NM_031373 0.60-1.66 Gdap10 XM_895068 0.60-1.66 Hs1bp3 NM_021429 0.60-1.66 Cxcl12 NM_001012477 0.60-1.66 Aars NM_146217 0.60-1.65 Seh1l NM_001039088 0.61-1.65 Dio1 NM_007860 0.61-1.65 Dmpk NM_032418 0.61-1.65 H1f0 NM_008197 0.61-1.65 Dnajc19 NM_001026211 0.61-1.65 Cdkn1b NM_009875 0.61-1.65 BC005537 NM_024473 0.61-1.64 Fxyd3 NM_008557 0.61-1.64 Ung NM_001040691 0.61-1.64 Sult1c2 NM_026935 0.61-1.64 Sirpa NM_001177646 0.61-1.64 Sub1 NM_011294 0.61-1.64 Zxdc NM_030260 0.61-1.64 Fam3c NM_138587 0.61-1.64 Amz2 NM_025275 0.61-1.64 Id2 NM_010496 0.61-1.64 Hadh NM_008212 0.61-1.64 Furin NM_001081454 0.61-1.63 Slc25a23 NM_025877 0.61-1.63 Mtdh NM_026002 0.61-1.63 Myo1d NM_177390 0.61-1.63 Tubb5 NM_011655 0.61-1.63 Nrk NM_013724 0.61-1.63
Bcl9 NM_029933 0.61-1.63 Tnfsf12 NM_001034097 0.61-1.63 Cpt1a NM_013495 0.61-1.63 Herc1 NM_145617 0.61-1.63 Zfp568 NM_001033355 0.62-1.62 2510009E07Rik NM_001001881 0.62-1.62 Syt2 NM_009307 0.62-1.62 Myo10 NM_019472 0.62-1.62 2610002M06Rik NM_025921 0.62-1.62 3200002M19Rik NM_027532 0.62-1.62 Depdc6 NM_001037937 0.62-1.62 Idh2 NM_173011 0.62-1.62 Alg2 NM_019998 0.62-1.62 Tbl1x NM_020601 0.62-1.62 Hist1h4a NM_175655 0.62-1.61 Xbp1 NM_013842 0.62-1.61 Nphp1 NM_016902 0.62-1.61 Ccpg1 NM_001114328 0.62-1.61 Cend1 NM_021316 0.62-1.61 Srpk2 NM_009274 0.62-1.61 Sema4a NM_001163489 0.62-1.61 Usp48 NM_130879 0.62-1.61 Egfr NM_007912 0.62-1.60 Adcyap1 NM_009625 0.62-1.60 Ccdc56 NM_026618 0.62-1.60 1110059E24Rik NM_025423 0.62-1.60 Parp3 NM_145619 0.62-1.60 Pofut2 NM_030262 0.62-1.60 Mat1a NM_133653 0.62-1.60 Ptprd NM_001014288 0.63-1.60 Wars NM_001164314 0.63-1.60 4921506J03Rik NM_001033474 0.63-1.60 Grina NM_023168 0.63-1.59 Wnt4 NM_009523 0.63-1.59 Arf3 NM_007478 0.63-1.59 Zdhhc6 NM_001033573 0.63-1.59 Gpx2 NM_030677 0.63-1.59 Dleu2 NR_028264 0.63-1.59 Mapk8ip1 NM_011162 0.63-1.59 Ppp1r1a NM_021391 0.63-1.59 Slc22a23 NM_001033167 0.63-1.59 Thrap3 NM_146153 0.63-1.58 Dner NM_152915 0.63-1.58 Sbno1 NM_001081203 0.63-1.58 Tmem206 NM_025864 0.63-1.58
Spt1 NM_009267 0.63-1.58 Tacc3 NM_001040435 0.63-1.58 Htatsf1 NM_028242 0.63-1.58 Abcf1 NM_013854 0.63-1.58 Mll5 NM_026984 0.63-1.58 Tbc1d8 NM_018775 0.63-1.58 Wnk4 NM_175638 0.63-1.58 Rnf11 NM_013876 0.63-1.58 Gpr133 NM_001081342 0.64-1.57 Agbl3 NM_178630 0.64-1.57 Hus1 NM_008316 0.64-1.57 Vamp7 NM_011515 0.64-1.57 Pvrl3 NM_021495 0.64-1.57 Stk40 NM_001145827 0.64-1.57 Scamp5 NM_020270 0.64-1.57 Smarcc1 NM_009211 0.64-1.57 Herpud1 NM_022331 0.64-1.57 Wdr33 NM_001170966 0.64-1.57 Cntfr NM_001136056 0.64-1.56 Naa15 NM_053089 0.64-1.56 Fam102a NM_153560 0.64-1.56 Gbf1 NM_178930 0.64-1.56 Ndst1 NM_008306 0.64-1.56 Nfix NM_001081981 0.64-1.56 Thoc2 NM_001033422 0.64-1.56 Cnpy2 NM_019953 0.64-1.56 0610031J06Rik NM_020003 0.64-1.56 Edil3 NM_001037987 0.64-1.56 Rufy3 NM_027530 0.64-1.56 Mia3 NM_177389 0.64-1.56 Sphk1 NM_001172472 0.64-1.56 Spock1 NM_001166463 0.64-1.56 Gm5595 NM_001008427 0.64-1.55 Sct NM_011328 0.64-1.55 2510039O18Rik NM_029841 0.64-1.55 Arhgap5 NM_009706 0.64-1.55 Hsd17b4 NM_008292 0.65-1.55 Pdap1 NM_001033313 0.65-1.55 Nras NM_010937 0.65-1.55 Flcn NM_146018 0.65-1.55 Gmfb NM_022023 0.65-1.55 H2-Ke2 NM_010385 0.65-1.55 Mef2a NM_001033713 0.65-1.55 Stk24 NM_145465 0.65-1.54 Dusp18 NM_173745 0.65-1.54
1810019J16Rik NM_001083916 0.65-1.54 Elf5 NM_001145813 0.65-1.54 Cpn1 NM_030703 0.65-1.54 Cyp51 NM_020010 0.65-1.54 Isca1 NM_026921 0.65-1.54 Bmp2 NM_007553 0.65-1.54 Wdr12 NM_021312 0.65-1.54 Epha1 NM_023580 0.65-1.54 Gad1 NM_008077 0.65-1.54 Rad23a NM_009010 0.65-1.54 Phip NM_001081216 0.65-1.54 Nono NM_023144 0.65-1.53 Smagp NM_001033872 0.65-1.53 Pvrl2 NM_001159724 0.65-1.53 Gm8615 /// NM_011937 /// NR_028061 /// Gnpda1 XM_990406 0.65-1.53 Cdh1 NM_009864 0.65-1.53 B3gnt3 NM_028189 0.65-1.53 Kif12 NM_010616 0.65-1.53 Kl NM_013823 0.65-1.53 Mtmr9 NM_177594 0.65-1.53 Trib3 NM_175093 0.65-1.53 Ifnz NM_197889 0.65-1.53 Zfp81 NM_207541 0.65-1.53 Nmnat3 NM_144533 0.65-1.53 Hk1 NM_001146100 0.65-1.53 Selm NM_053267 0.65-1.53 Tmem135 NM_028343 0.65-1.53 Cbs NM_144855 0.66-1.53 Maff NM_010755 0.66-1.53 E2f1 NM_007891 0.66-1.53 Brd4 NM_020508 0.66-1.53 Slc40a1 NM_016917 0.66-1.53 Rnf114 NM_030743 0.66-1.53 Ccdc12 NM_028312 0.66-1.52 Tmprss3 NM_001163776 0.66-1.52 Rab27a NM_023635 0.66-1.52 Lypla2 NM_011942 0.66-1.52 Gatsl2 NM_030719 0.66-1.52 Socs4 NM_080843 0.66-1.52 Soat2 NM_146064 0.66-1.52 Vldlr NM_001161420 0.66-1.52 Slc7a8 NM_016972 0.66-1.52 Taok3 NM_001081308 0.66-1.52 Reep2 NM_144865 0.66-1.52
Cdo1 NM_033037 0.66-1.52 Psen1 NM_008943 0.66-1.52 Ccnd2 NM_009829 0.66-1.52 Smad1 NM_008539 0.66-1.52 Rala NM_019491 0.66-1.52 Mrpl36 NM_053163 0.66-1.52 Tm2d1 NM_053157 0.66-1.52 G6pdx NM_008062 0.66-1.52 Spast NM_001162870 0.66-1.51 3110070M22Rik NM_026084 0.66-1.51 Pcgf2 NM_001163307 0.66-1.51 Helz NM_198298 0.66-1.51 H6pd NM_173371 0.66-1.51 Pdxk NM_172134 0.66-1.51 Smc4 NM_133786 0.66-1.51 Mrc2 NM_008626 0.66-1.51 Rnf2 NM_011277 0.66-1.51 Rap2c NM_172413 0.66-1.51 Snx5 NM_024225 0.66-1.51 Nol3 NM_030152 0.66-1.51 Syt13 NM_030725 0.66-1.51 Sh3glb1 NM_019464 0.66-1.51 F2rl1 NM_007974 0.66-1.51 Gnai2 NM_008138 0.66-1.51 H2afx NM_010436 0.66-1.51 1110067D22Rik NM_173752 0.66-1.51 Fads1 NM_146094 0.66-1.51 Hnf4a NM_008261 0.66-1.51 Tob2 NM_020507 0.66-1.51 Agfg1 NM_010472 0.66-1.51 Atm NM_007499 0.66-1.51 Sptlc2 NM_011479 0.66-1.51 Nudt19 NM_033080 0.66-1.51 P4hb NM_011032 0.66-1.51 Tor3a NM_023141 0.66-1.51 Cyb5r3 NM_029787 0.66-1.51 Mrpl11 NM_025553 0.66-1.50 Stau2 NM_001111272 0.67-1.50 Fem1a NM_010192 0.67-1.50 Mllt1 NM_022328 0.67-1.50
Supplementary Table 4. Genes down regulated by mir-24 over expression in MIN6 cells
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).
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.
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.
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).
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.
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.