Supplementary Table 1. mirna modulated in D HF and ND HF patients. Table shows in a linear scale the same values of Fig. 2.
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1 Supplementary Table 1. mirna modulated in D HF and ND HF patients. Table shows in a linear scale the same values of Fig. 2. mir D HF VS CTR ND HF VS CTR D HF VS ND HF FOLD CHANGE p FOLD CHANGE p FOLD CHANGE mir *** 10.9 *** 2.1 ns mir *** 2.7 * 2.3 ns mir-34c 5.5 *** 1.3 ns 4.2 *** mir ** 2.8 ** 1.4 ns mir-34b 3.4 ** 1.3 ns 2.7 ** mir-199b 2.8 ** 1.4 ns 2.0 * mir-199a 2.5 ** 1.3 ns 1.5 ns mir-485-5p 2.4 ** 1.6 * 1.4 ns mir * -1.3 ns 2.4 ** mir ns -1.8 ** 2.1 * mir ns -2.7 * 1.2 ns mir ns -2.3 ** 1.2 ns mir-133a -1.9 ** -1.7 ** -1.1 ns mir-10a -2.3 ns -2.5 ** 1.1 ns mir ns -6.9 ** 2.4 ** mir ** -5.4 *** 1.1 ns mir *** -4.4 *** -1.4 ns Statistically significant differences are in bold (*p=0.05; **p=0.01; ***p=0.001) p
2 Supplementary Table 2. Comparison of D HF deregulated mirnas expression in the remote and border zones of HF patients mir D HF vs CTR ND HF vs CTR D HF vs ND HF REM BZ REM BZ REM BZ mir-34c mir-34b mir mir-199b mir mir Legend: D HF=diabetic heart failure; CTR=control; ND HF=non-diabetic heart failure; REM=remote zone; BZ=border zone. Statistically significant differences are in bold (D HF, n=10; ND HF, n=23)
3 Supplementary Table 3. mirna-mrna interactions Gene expression profiles were measured and Class Comparison analysis identified differentially expressed genes. Then, we performed a bioinformatic analysis adopting the most widely used mirna target prediction algorithms and identified a list of putative direct target genes for each of the significantly modulated mirnas. Next, we intersected predicted mirna target genes and differently expressed genes, to discriminate those significantly deregulated genes that were putative targets of the identified mirnas. Given the inhibitory nature of mirna/mrna interaction, we applied a further selection criterion and considered only those predicted targets displaying opposite modulation with their corresponding mirna. Finally, we carried out a statistical correlation analysis between the expression levels of the HF deregulated mirnas in each patient and the HF-deregulated putative targets. We identified 140, 329 and 11 mirna/mrna interactions in the D-HF vs CTR, ND-HF vs CTR and D-HF vs ND-HF groups, respectively mirna-mrna interactions mir-10a mir-133a mir-182 mir-199a mir-199b mir-204 mir-210 mir-216 mir-221 mir-222 mir-223 mir-338 mir-372 mir-485-5p mir-650 ABCB4 ARHGAP6 HIST1H2AC JAGN1 ACPL2 AASS PARK7 AASS ANO1 HIST2H2BA AKAP7 ACADL ABRA AASS CCDC122 ALG10B ATXN7L1 RPS4Y1 REPS1 C16orf68 ABRA ACPL2 APBB2 IAH1 AMY1A AGPHD1 ACPL2 ANKRD2 DPM1 AMY1A CABLES1 YIPF7 C20orf24 ACPL2 ANKRD2 ART4 RAD54B AMY2A ALG10B CXorf40B ARAF EYA3 C20orf26 CTSK COPS6 ARAF ARAF BICD1 SPAM1 ANKRD31 AMY1A DHX16 FBXO10 IQUB CEP192 ELMO1 DHCR24 C19orf47 CAV2 BMP6 SPEF2 ASZ1 ANKRA2 DOCK3 FCN3 KLRD1 GABARAPL2 ENTPD1 DNAJC8 COBL DHX16 C7orf41 C1orf129 ANKRD20A2 ELL2 GLUL RGPD4 IAH1 ITGA8 FAM96B COPS6 DNAI2 CENPF C21orf99 ANKRD20B FADS1 NDUFS5 SLC6A15 IGBP1 MLL FBXO10 CSDA FBXO10 CPE C3orf57 ARHGAP19 MYOT PDE4D NBAS PIK3CG HMOX2 DHX16 FGF7 CRISPLD1 CALCR ATP6AP1L PALLD PHTF2 NUDT6 POGZ JAGN1 DOCK3 LPL HTR4 CASC2 ATXN7L1 RCAN1 RGAG1 PPIL6 PRELP MTCH1 FBXO10 PALLD HYDIN CLEC9A AVIL SLC7A2 SGPP2 PRTG RGL1 PRDX1 FCN3 PDE4D KCTD7 CMYA5 C12orf47 SRPX SLCO4A1 RGPD4 SESN3 PRDX6 FGF7 REPS1 KIAA1377 CPB2 C12orf63 THOC4 SSSCA1 SLCO4C1 SLC16A4 REPS1 HSPB7 RGAG1 MCOLN2 DCAF6 C1orf129 ABRA YIPF7 SUMO1 ZFP37 ASXL3 LPL SLC7A2 MGC21881 DDX4 C20orf19 ACPL2 AASS ZNF626 ALMS1 LRRFIP1 STRAP MYH10 DENND1B C21orf34 DHX16 ANKRD2 ARRDC3 MURC STXBP6 NEB DPH5 C21orf49 ELL2 ARAF ATXN7L1 PALLD TTC38 NOV DPP10 C21orf99 FADS1 CSRP3 C7orf58 PHB TUBA3D NRG1 EIF2B3 C3orf57 MYOT FCN3 CD302 PHTF2 UBE2D3 NRK EZH1 C5 RCAN1 GLUL CTSK REPS1 YBX1 POLQ GDPD1 C5orf4 SAP30BP LCLAT1 HAPLN1 SLC7A2 AASS RIN2 GTF2H5 C5orf56 SLC7A2 NRAP HLA-DPA1 SSR3 ACPL2 SLC16A9 HSFY2 C9orf93 SRPX PDE4D HLA-DRA STRAP ANKRD2 SMOC2 MAEL CASC1 THOC4 PHTF2 ITGA8 STXBP6 ARAF SPEF2 MGC21881 CCDC122 PSMD2 MAB21L1 TNIK CAV2 SSX2 MPPED2 CD96 RGAG1 MLL TTC38 DHX16 TMEM140 NAALAD2 CDH9 SGPP2 OLFML1 UGP2 FGF7 TTLL7 ODZ3 CDKL3 SLCO4A1 OMD AASS KBTBD10 WASF3 OR14A16 CEP120 WDR74 PDK3 ABRA NRAP ZNF479 PCDH15 CMYA5 PHC3 ACPL2 NUDT4P1 ZNF625 PLCZ1 CRYGS POGZ ARAF PDE4D APBB2 PMCHL2 DNAH14 PRELP C19orf47 PPA1 ART4 PRKAR2B DPH5 SEMA3D COBL PVR C7orf41 PXMP4 DPM1 SESN3 CSDA REPS1 CNTN4 RAD54B DPP10 SLC16A4 DHX16 RGAG1 CPE RGPD4 EFCAB6 ZFP37 EIF5AL1 SLC7A2 CRISPLD1 RNPC3 EFCAB7 ZFP90 FAM78B STRAP DSG1 RPS6KA6 EFHA2 ZNF280D FCN3 STXBP6 HDAC9 SCN2A EIF1AY FGF7 TUBA3D HMGB2 SCN3A EPHA7 HMGA1 HTR4 SLC26A7 FAM13A HSPB7 HYDIN SPRYD5 FAM72D KBTBD10 IAH1 SUMO1 FAM82A1 LRRFIP1 KCNMA1 USH2A FBXO32 D HF VS CTR MEIS1 KCTD7 WDR49 FDFT1 ND HF VS CTR PHTF2 KIAA1377 AMY2A FLJ37201 D HF VS ND HF PPA1 KLRK1 C3orf57 GABARAPL2 PPM1G LRP1B CPB2 GARNL3 PVR MCOLN2 DENND1B GBP5 REPS1 MGC21881 GTF2H5 GDPD1 SLC22A5 MYH10 MAEL GPAM SLC7A2 NEB ODZ3 GPLD1 SNRPB NETO1 SPRYD5 HEXA SSR3 NOV SUMO1 HRH4 STRAP NRK HSFY2 STXBP6 OR4K1 HTR4 TNIK OR5H14 IAH1 UGP2 POLQ IAPP PRKG2 IFT80 RAD54B IGBP1 RANBP17 IQUB RGPD4 IRAK1BP1 RHOBTB1 KIAA1009 RIN2 KIAA1109 SLC16A9 KIAA1377 SMOC2 KLRF1 SPEF2 LRP1B STON1 LRRC37A3 TMEM140 LY75 TRPC1 MGC21881 TTLL7 MKL2 WDR17 NAALAD2 ZNF479 NETO1 NME5 OR10A7 OR14A16 OR4K1 OR8K5 PEX7 PHF3 PLCZ1 PMS2L5 POLI POLQ POTED POTEF PRTG RAD54B RERGL RGPD4 SCN2A SLC16A9 SLCO4C1 SLFN13 SOX5 SPAM1 SPEF2 SPRYD5 SUMO1 SYT14L TAAR8 TDRD3 TFDP2 TRPC1 ZBBX ZBTB20 ZFP37 ZNF253 ZNF442 ZNF564 ZNF610 ZNF626 ZNF627 ZNF66 ZNF705D ZNF766 ZNF844 ZNF98
4 Supplementary Table 4. Regulation of mir-199a/b, and mir-210 by hypoxia and high glucose in vitro mir HF ncms HCAEC ND D HG HYP HG+ HYP HG HYP HG+ HYP mir-199a mir-199b mir Legend: HF=heart failure; ND=non-diabetic; D=diabetic; ncms=neonatal mouse cardiomyocytes; HG=high glucose; HYP=hypoxia; HCAEC= human coronary artery endothelial cells. Statistically significant differences are in bold (ncms, n=8; HCAEC, n=8).
5 Supplementary Table 5. Comparison with previous studies mir DISEASE MODULATION REF 10a Aortic Stenosis/Hypertrophic Cardiomyopathy DOWN Ikeda et al, a End Stage Dilated/Ischemic-Cardiomyopathy DOWN Sucharov et al, 2008 End Stage Dilated/Ischemic-Cardiomyopathy DOWN Schipper et al, 2008 Aortic Stenosis/Hypertrophic-Cardiomyopathy DOWN Carè et al, 2007 Aortic Stenosis/Hypertrophic-Cardiomyopathy DOWN Duisters et al, 2010 End Stage Dilated/Ischemic-Cardiomyopathy UP Matkovich et al, End Stage Dilated-Cardiomyopathy DOWN Thum et al, a End Stage Dilated-Cardiomyopathy UP van Rooij et al, 2006 End Stage Dilated/Ischemic Cardiomyopathy UP Matkovich et al, b End Stage Ischemic-Cardiomyopathy UP Da Costa Martins et al, End Stage Dilated-Cardiomyopathy UP Thum et al, End Stage Dilated/Ischemic-Cardiomyopathy DOWN Ikeda et al, End Stage Ischemic-Cardiomyopathy UP van Rooij et al, b End Stage Dilated-Cardiomyopathy UP Thum et al, End Stage Dilated-Cardiomyopathy UP Thum et al, 2007
6 Supplementary Figure 1. mrnas unsupervised hierarchical clustering analysis. Unsupervised hierarchical clustering analysis is displayed as 3-dimensional representation with a cloud of spheres in which each sphere represents a single sample.
7 Supplementary Figure 2. mrna Class Comparison Validation. After Class Comparison analysis the expression of some of the mrnas significantly deregulated was measured by qpcr in the relevant patient groups: D HF vs CTR (A); ND HF vs CTR (B) and ND HF vs D HF (C), (*p=0.05, **p=0.01, ***p=0.001; ND HF, n=16; D HF, n=6 and CTR, n=15).
8
9 Supplementary Figure 3. In vitro validation of mir-204 and -216a predicted targets HEK293 cells were transfected with plasmidic expression vectors encoding the precursor of mir-216a and mir-204, two of the most strongly modulated mirnas in our study. Expression vectors also carried puromycin as selection marker. The two pre-mirna encoding vectors or the null-vector as control, were transfected by Fugene 6 and fast selected by 2? g/ml puromycin for 24 hours, to decrease the influence of nontransfected cells. mrna expressions of nine selected targets were measured by qpcr. We found that seven transcripts were significantly down-regulated after mir-204 or mir-216 transfection (n=4).
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