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- Νεφέλη Πανδώρα Κουταλιανός
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6 Supplementary Information Supplementary Materials and Methods Multiple myeloma specimens Primary bone marrow samples from 116 MM patients and 12 patients without tumors were obtained from the 4th Department of Internal Medicine, Hiroshima Atomic-Bomb Survivors Hospital; the 1st Department of Internal Medicine, Sapporo Medical University Hospital; and the Department of Hematology, Teine Keijinkai Hospital after acquisition of informed consent from each patient. Mononuclear cells were separated from 99 samples using Ficoll-Paque (StemCell Technologies), after which we separated the CD138-positive cells using CD138 MicroBeads (Miltenyi Biotec). There were five patients whose bone marrow specimens were collected before and after treatment. All five of those patients received treatment with Dex. Of those five patients, three received bortezomib and Dex; one received vincristine, adriamysin and Dex; and one received melphalan and Dex followed by peripheral blood stem cell transplantation. Methylation analysis Combined bisulfite restriction analysis (COBRA) were performed as described previously (1). The PCR products were digested with the restriction endonuclease Hinf (TaKaRa) and precipitated with ethanol, after which the resultant DNA fragments were subjected to 2.5% agarose gel electrophoresis and stained with ethidium bromide. Bisulfite-pyrosequencing was carried out as described previously (2). The primers used for COBRA were also used for pyrosequencing. After PCR, the biotinylated PCR product was purified, made single-stranded, and used as a template in the
7 pyrosequencing reaction. Briefly, the PCR products were bound to streptavidin sepharose beads HP (Amersham Biosciences), after which beads containing the immobilized PCR product were purified, washed, and denatured using a 0.2 mol/l NaOH solution. After addition of 0.3 μmol/l sequencing primer to the purified PCR product, pyrosequencing was carried out using a PSQ96MA system (Biotage) and Pyro Q-CpG software (Biotage). MM-related genes were deemed methylated if the level of methylation was above the 75th percentile level found in control bone marrow samples. Statistics Paired t tests were used to compare differences in methylation levels before and after treatments. Pearson s and Spearman s correlation coefficients (r and rs, respectively) were calculated to evaluate the correlation between gene expression levels (log-transformed), fold expression levels, and methylation levels. Cell viability was assessed using one way ANOVA with post hoc Dunnett s test. All statistical calculations were performed using SPSSJ 15.0 (SPSS Japan Inc.). References. 1. Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 1997; 25: Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 2004; 32: e38.
8 Legends for Supplementary Figures. Supplementary Figure 1. Representative results of real-time PCR analysis of genes upregulated by DAC in MM cell lines. Expression levels of CST1 and IER3 relative to that in normal bone marrow are shown on the Y-axis. Supplementary Figure 2. Expression profile of candidate genes silenced by DNA methylation in MM. The heat map shows the expression profiles in normal colon, normal stomach and three cancer cell lines. Levels of expression are normalized to that in normal bone marrow. Supplementary Figure 3. Representative results of real-time PCR of candidate methylation target genes in MM cell lines treated with DAC. Supplementary Figure 4. Summary of gene expression in MM cells treated with DAC. Levels of expression are normalized to those in mock treated cells. Supplementary Figure 5. Bisulfite-sequencing analysis of BNIP3 and SOCS3 in MM cell lines. Open circles indicate the unmethylated CpG sites; filled circles indicate the methylated CpG sites.
9 Supplementary Table 1. Primer sequences and restriction enzymes used in this study. Primer Gene name Forward Reverse Restriction enzymes COBRA GPR30 GGTYGAGTGTTTTTGGAGGT CCTTTATCCRCTAAATATAAATAATAC TaqI FMR1NB GTTAYGGATTGTTTTGGAAG TTACCAACTCTAAATAAACCACAC MboI CST6 TTYGAGTTTYGTTTTAGTTTTAGGT TAACAACRCCAAAAAACAAAATAC HhaI TNFRSF4 TAYGGTGTTTAGTTTTAGGTTTAGGAATCCCCRACCTACCACCTTC TaqI SECTM1 GGAGTTTTAGGAAAATGAAAG CCCRTAAAAATAATAAATTCAC HhaI PTRF AGYGGGAGATTYGGAGAGAAGT CCTCCCTTTCTAAAATAAAACCAACC TaqI C16orf30 TTTTTGTTGAGAGTTAGGGAAG ACACCRACCTACTATAAATACCA MboI RASD1 GTTTTTAGTTTTAGTTYGATTTTGGA CCRCCCTTATATACAACCTACTC AfaI DAZL TGAAGAGAAAAGGAAAATTAAGAG AAACRCTACCACCCATTAAC TaqI DDX43 TAATGGTTGYGTGAAATTAGTG CCTCCRTAATAAAACATTATTCC MluI PDIA2 GGTTTTGTTTTTGTTGGGATTG AAATTTCCTCCTCTAAAAACTCCTC MluI FES TTYGGGGTTTGGGTTAATTG ACRCCTACTCTACCCCTACCTAC TaqI COX7A1 GTTTTAGTTTYGTYGYGTTTTTAG AACCCAACCCATAAAAACCTCA TaiI KRTCAP3 GGGGTYGGGTTTAGGTATAG CACRAAAAAAAACAAAATTAAATC HhaI RRAD TTGAGAAGGYGGTGGTTGTAGTAGTACCRAAACCCCTCCRAACCTAA TaqI FGFR3 GGGGTYGGGAYGTAGGAGT RCAAACAAAAACRCCCATAAC TaqI IER3 GYGTTTAGGATTGTGTATGTTAAGT TAAACCRAACRAAATATAAAACCA TaqI TKTL1 ATATTTTAGATTTTTTGATTTGTTTG TTAAAATCTAAAAACCCACTCCT TaqI CMTM3 GGTGGTTAAGAAAGTYGTAAGAAA CCACCCRTCTACCCCTATC MluI SYT11 GYGGAGTTTATTTTTTATTAGGA CTCTAAAAACCCTCRATCAATAC MboI P2RY6 GGATTTTAGYGGTGYGGAG CAACTAATCCRACCCAAATATCACT TaiI GDF15 TTTAGAGTYGTAATTTGTATAGTTAT TCRAAATACAACTCTAAAAATCC TaqI RASD1 pyro-seq1gygtttagagagtaggagtt Bisulfite-sequencing BNIP3 GATATGGYGTTAGAGGGTAATTG SOCS3 GTTTTTTTTTGTTGYGAGTAGTGAT ABL1 TTGGTTYGGGTTGGTTTTGGAAAG Y=C or T, R=G or A CCCRCCCTACCCTATAAATTC CRTCCCTCRAACTTCCCCT AAACCRAAACCCCTCTCCCAACTC 12/26/20083:09 AM
10 Supplementary Table 2. Genes up-rehulated by DAC in KMS12PE. Gene Name Systematic Name DAC VIM NM_ TNFRSF4 NM_ GPR30 NM_ VASN NM_ CST6 NM_ FMR1NB NM_ CKM NM_ AIF1 NM_ COX7A2 NM_ CMTM3 NM_ GPR68 NM_ VIM NM_ LOC XM_ RAB13 NM_ TAS1R1 NM_ EDNRB NM_ B3GALT4 NM_ CXCL9 NM_ PLAT NM_ CCRL2 NM_ TNFAIP2 NM_ ACRC NM_ LAG3 NM_ A_24_P A_24_P C16orf30 NM_ THC THC MYO1G NM_ ID2 NM_ M32220 M P2RY5 NM_ SECTM1 NM_ GPX1 NM_ IFI27 NM_ CCR9 NM_ PEPP-2 NM_ ART3 NM_
11 IFI27 NM_ ENST ENST APOE NM_ PTRF NM_ CHRDL2 NM_ CLDN19 NM_ HAS3 NM_ EPHA2 NM_ THC THC LGALS8 NM_ SPINT1 NM_ IFI27 NM_ ZP1 NM_ LOC BC THC THC AGMAT NM_ CDKN1C NM_ TNF NM_ CYB5R2 NM_ IFI27 NM_ IFI27 NM_ IFI27 NM_ IFI27 NM_ BTBD14A NM_ CCR7 NM_ TMEM49 BC AGXT2L1 NM_ ANGPT2 NM_ IFI27 NM_ NGB NM_ CNDP1 NM_ IFI27 NM_ AHNAK NM_ RAMP1 NM_ BM BM ICOS NM_ RUNX3 NM_ LXN NM_
12 SYT11 NM_ RPGRIP1 NM_ IFI27 NM_ TNNC2 NM_ FLJ25410 NM_ GLULD1 NM_ MET NM_ PROC NM_ A_24_P A_24_P ENST ENST GPR56 NM_ SPINT2 NM_ GNG11 NM_ PLXND1 NM_ BC BC RASD1 NM_ FARP1 NM_ P2RY6 NM_ LEFTY1 NM_ CD52 NM_ GDF15 NM_ A_24_P A_24_P A_23_P15226 A_23_P ENST ENST VCX2 NM_ WBP5 NM_ KITLG NM_ RHBDL3 NM_ THC THC
13 Supplementary Table 3. Genes up-regulated by DAC in RPMI8226. Gene Name Systematic Name DAC GPR30 NM_ FMR1NB NM_ DAZL NM_ DDX43 NM_ BC BC GSTP1 NM_ CST6 NM_ THC THC VCX3A NM_ VCX NM_ VCX2 NM_ THC THC SYCP3 NM_ NEFH NM_ PAGE2 NM_ VASN NM_ CXCL9 NM_ TNFRSF4 NM_ PRAME NM_ PDIA2 NM_ CKM NM_ PAGE5 NM_ DAZ2 NM_ A_24_P7750 A_24_P XIST NR_ UCHL1 NM_ BM BM ENST ENST CNDP1 NM_ ECAT8 AK LEFTY1 NM_ ENST ENST MXRA8 NM_ TAS1R1 NM_ RTP3 NM_ BEX1 NM_
14 CARD9 NM_ KRT19 NM_ LGALS2 NM_ IL18 NM_ FES NM_ RAB7B NM_ CD52 NM_ CPLX3 NM_ AIF1 NM_ P2RY5 NM_ A_23_P15226 A_23_P VCY NM_ SECTM1 NM_ ART3 NM_ FAM26A NM_ CDH1 NM_ THC THC LAG3 NM_ CYB5R2 NM_ COX7A1 NM_ CNN3 NM_ GPR68 NM_ VCX2 NM_ THC THC THC THC LY6K NM_ C8G NM_ LOC XM_ DMRTB1 NM_ A_32_P A_32_P CGREF1 NM_ CCRL2 NM_ PPAPDC3 NM_ A_24_P A_24_P ACRC NM_ MYO1G NM_ RRP22 NM_ RP13-36C9.6 NM_
15 DNALI1 NM_ HORMAD1 NM_ HSD17B1 NM_ CATSPER1 NM_ FLJ10324 NM_ CRYAA NM_ TEKT2 NM_ APOC1 NM_ PKP3 NM_ A_24_P A_24_P PTRF NM_ KRTCAP3 AY MAEL NM_ CRYBA4 NM_ RAMP1 NM_ NDRG4 NM_ ENST ENST SMARCA1 NM_ A_24_P A_24_P RP4-742C19.3 NM_ RGS1 NM_ FAM9C NM_ BCNP1 NM_ SERPINF1 NM_ RAB13 NM_ MMP13 NM_ PTGER1 NM_ C6orf154 NM_ S100A16 NM_ RRAD NM_ KRTCAP3 NM_ C16orf30 NM_ CR CR DNMT3L NM_ A_24_P93111 A_24_P RAB34 NM_ LOC AK LTK NM_
16 FGFR3 NM_ TMEM49 BC RARRES2 NM_ RRAD NM_ IER3 NM_ BCL2A1 NM_ ROBO3 NM_ BEX2 NM_ RBMY1B NM_ EDG6 NM_ A_24_P A_24_P TKTL1 NM_ CLIC3 NM_ FAM9C NM_ KRT8 NM_ PIWIL2 NM_ THC THC ZNF502 NM_ TNF NM_ C21orf105 BC ALDOB NM_ A_24_P A_24_P A_24_P A_24_P A_24_P A_24_P HSPA1A NM_ ATP4B NM_ ADAM8 NM_ COL8A2 NM_ MTMR11 NM_ CXCL1 NM_ ENST ENST CLDN4 NM_ LOC BC HSD11B1 NM_ SPOCK2 NM_ CRYAA NM_ RAB25 NM_ FRG2 NM_
17 EMILIN2 NM_ THC THC FBLIM1 NM_ KRT8 NM_
18 Supplementary Table 4. Genes up-regulated by DAC and/or Dex in OPM1. Gene Name Systematic Name Dex DAC Dex+DAC SSTR2 NM_ THC THC MAP9 NM_ ADAMTS12 NM_ NEFL NM_ BNIP3 NM_ PPP1R2P9 NR_ CXCL13 NM_ CX3CL1 NM_ KIAA2022 NM_ PARP12 NM_ CHST2 NM_ MLLT4 BC THC THC RNF43 NM_ AQP9 NM_ CYR61 NM_ VCAM1 NM_ THC THC BAIAP2L2 AK RAB31 NM_ PCSK9 NM_ CD109 NM_ TRAM1L1 NM_ AY AY THC THC LOC ENST LOC AF CMTM3 NM_ NLRP7 NM_ LILRB2 NM_ TMEM16A NM_ GNA12 NM_ PRRX2 NM_ CAPSL NM_ FILIP1L NM_
19 PSG3 NM_ BEX1 NM_ KIF26B NM_ TDRD6 NM_ MYH6 NM_ DB DB REN NM_ AK AK INSAF S CLDN19 NM_ IGFL2 NM_ ARHGAP24 AK AK AK AIF1 NM_ SLC35D2 NM_ SPIN3 NM_ TMEM45A NM_ ZNF643 NM_ DLK1 NM_ HNRNPG-T NM_ MAP9 NM_ C1orf115 NM_ BMPER NM_ MYF6 NM_ AF AF DLEC1 NM_ ENO2 NM_ RAB6IP1 NM_ KCNS1 NM_ BCL7A NM_ BU BU FN1 NM_ DMKN NM_ FLRT3 NM_ MYO7A NM_ ATP1A4 AK FGFBP2 NM_ LOC BC
20 ENST ENST RETN NM_ S100A14 NM_ C20orf186 NM_ EFEMP1 NM_ DENND2C CR ALOX5 NM_ LGALS8 NM_ THC THC AI AI CYP4B1 NM_ RAB31 NM_ SEPT10 NM_ MPN2 NM_ BC BC KIF7 NM_ CTSL2 NM_ RTP3 NM_ ELL3 NM_ A_24_P A_24_P APOC4 NM_ ZNF480 NM_ PPM1J NM_ CRISP2 NM_ HERC5 NM_ NUDT10 NM_ A_23_P11119 A_23_P BCL2L10 NM_ NAP1L5 NM_ PDLIM3 NM_ STAR NM_ A_32_P A_32_P KLHL5 NM_ TTC28 AK ATP9A NM_ ROS1 NM_ KRT18P40 XR_ THC THC
21 FLJ40473 AK ADAMTS7 AF LOC XM_ FHOD3 NM_ PHLDA2 NM_ PCDH7 NM_ BC BC AF AF NEU4 NM_ SERPINB3 NM_ GPX2 NM_ C6orf10 NM_ PCDH12 NM_ THC THC COL20A1 NM_ UNC45B NM_ DMD NM_ AF AF NDN NM_ CPVL NM_ DLX4 NM_ THC THC TSPAN6 NM_ ALX4 NM_ ZFP2 NM_ G0S2 NM_ THC THC EID3 NM_ A_24_P A_24_P ID4 NM_ FMN2 NM_ SLC16A5 NM_ THC THC PTPN20B NM_ DNAJB8 NM_ PAPSS2 NM_ PDGFA NM_ LAD1 NM_
22 FLJ39609 AK PTN NM_ KCNIP3 NM_ A_24_P A_24_P HSD3B1 NM_ KIAA1466 AB LOC AK THC THC DKFZP686A01247 NM_ PDCL2 NM_ SLC2A2 NM_ CYP2C9 NM_ ELF5 NM_ PHC2 NM_ NXPH3 AB DNAJA4 NM_ LEPREL1 NM_ USHBP1 NM_ IRX2 ENST FAM9C BX A_32_P A_32_P SORBS2 NM_ A_24_P A_24_P RPESP NM_ MAK NM_ THC THC CD19 NM_ NPY NM_ PAQR5 NM_ AW AW CDH19 NM_ ENST ENST A_24_P A_24_P THC THC THC THC COLQ NM_ DNAJA4 AF CMBL NM_
23 C10orf47 NM_ SEPSECS NM_ BC BC THC THC FAM92A3 BC ENST ENST THC THC ZNF750 NM_ THC THC AA AA DQ DQ KIAA1279 NM_ FLJ13231 NM_ PENK NM_ EIF5A2 NM_ OR7E91P NR_ FBP2 NM_ NPTX1 NM_ TCL6 NM_ C9orf70 BC PROS1 NM_ A_24_P A_24_P BC BC IL7R NM_ CRB1 NM_ RAPGEF2 AL CCL25 NM_ C18orf2 AF DB DB FLJ30679 AK THC THC ARNTL2 AF ENST ENST TEX14 NM_ BC BC THC THC BC BC FLJ22536 AK
24 C1QTNF3 AK MARK1 NM_ TMEM185B NR_ APOBEC2 NM_ RP5-821D11.2 NM_ TRPM4 NM_ HGFAC NM_ HOP NM_ A_24_P A_24_P SPAG6 NM_ INSM1 NM_ THC THC THC THC C19orf31 NM_ PPP4R1L NM_ AK AK KCNB2 NM_ AUTS2 BC BC BC TCF15 NM_ PTGIR NM_ ZSCAN18 NM_ A_24_P A_24_P ANKS6 NM_ FOXF1 NM_ AF AF MOSC1 NM_ ACTN2 NM_ PHLDA1 NM_ OR2B6 NM_ ESRRG NM_ AMIGO1 NM_ CACNB2 AK ENST ENST THC THC PHACTR1 AB NFE2 NM_ FGFBP1 NM_
25 MOCS1 NM_ CHRNG NM_ DTX4 ENST COL6A1 NM_ ENST ENST THC THC LOC XR_ MAOA NM_ LOXL4 NM_ TAC1 NM_ PARP3 NM_ SYT11 NM_ HSPA12B NM_ SLC4A4 NM_ BC BC DSP NM_ TDRD10 NM_ hcg_ AK TNFAIP8L3 NM_ ZFP14 NM_ HIST1H2AK BC A_32_P A_32_P FCER1A NM_ ENST ENST SYNGAP1 NM_ AW AW CRIP3 NM_ PHLDA1 NM_ NPNT NM_ LTB4DH NM_ ADAMTS3 NM_ THC THC KAZALD1 ENST RASD1 NM_ DKFZP434P211 AL C10orf25 NM_ AK AK A_32_P A_32_P
26 THC THC A_24_P A_24_P GLYAT NM_ PSG6 NM_ LRRC49 NM_ MGC13053 BC KRT83 NM_ A_32_P55438 A_32_P ARHGAP28 NM_ GFRA1 NM_ ENST ENST GRAMD1B AY FGFR2 NM_ LOC NR_ GDF9 NM_ MOBKL2B NM_ SPG3A NM_ KIAA2022 NM_ BC BC A2M NM_ C3orf41 AL PRRG3 NM_ FMN2 NM_ THC THC AK AK C1orf51 NM_ ENST ENST REG1A NM_ NEURL NM_ A_32_P99744 A_32_P LOC AK LOC54103 AK IL18R1 NM_ THC THC ANKRD6 NM_ A_32_P A_32_P AW AW THC THC
27 THC THC PBLD NM_ PRKCZ NM_ PDE4DIP NM_ MGC16275 BC BX BX THC THC EBF3 NM_ LOC BC THC THC LOC AL DAG1 NM_ THC THC AW AW LOC ENST NEFL NM_ FLJ33790 NM_ CTTN NM_ MMP13 NM_ PCDHGB4 NM_ HS3ST3A1 NM_ AK AK THC THC TPCN2 AL THC THC THC THC TRIM50 NM_ DHX34 D AL AL DMD NM_ LOC XR_ ELSPBP1 NM_ ALDOB NM_ GATA4 AF ANGPT2 ENST KIAA1841 BC FLJ13224 AK AK AK
28 GPR109B NM_ TCF7 NM_ RAPH1 NM_ ENST ENST A_24_P A_24_P CEACAM19 AK KLRF1 NM_ CTDSPL NM_ NO145 NM_ THC THC C22orf31 NM_ ENST ENST LOC NR_ CBR4 AK BC BC FABP3 NM_ A_23_P A_23_P A_24_P A_24_P TKTL2 NM_ FAM110B NM_ THC THC FCAMR AY A_32_P72682 A_32_P DEFB104A NM_ KIAA1598 NM_ A_32_P13202 A_32_P ABL1 NM_ A_24_P A_24_P LOC NR_ TMEM132A NM_ AK AK ENST ENST ADAMTS18 NM_ KCNH5 NM_ AK AK THC THC DA DA PDCD4 NM_
29 NRSN1 NM_ NR1H4 NM_ AK AK WFDC5 NM_ THC THC C3orf64 NM_ TMEM35 NM_ ANGPT1 NM_ CCDC65 NM_ SYCP2 NM_ THC THC GPR126 NM_ TMPRSS12 NM_ AK AK TITF1 NM_ AK AK C1orf210 NM_ FOXD1 NM_ ENST ENST BC BC GREM2 NM_ LMCD1 NM_ SLFN11 NM_ AK AK AW AW AK3L2 NM_ A_24_P A_24_P RHOV ENST MOV10L1 NM_ KRT18P41 XR_ MGC45438 NM_ PURG NM_ BC BC NOPE NM_ THC THC ARHGAP6 NM_ LIPG NM_ AW AW
30 LILRA5 NM_ ABCC9 NM_ ENST ENST ARHGEF4 NM_ OR4S1 NM_ A_23_P A_23_P GZMK NM_ C3orf50 BC MERTK U AK AK CDS1 NM_ ENST ENST ANGPT2 NM_ HOXB5 NM_ THC THC KCNJ9 ENST BG BG CB CB LOC NM_ PLA2G3 NM_ AK AK THC THC MIER2 AB AK AK TGFBI NM_ ABCC11 NM_ HMGCS2 NM_ THC THC KIF19 NM_ BAPX1 NM_ BG BG KLRG2 ENST BC BC BU BU TCEAL7 NM_ RPRM NM_ SNED1 NM_ LOC XR_
31 LOC NM_ AK AK PRDM9 NM_ BI BI THC THC PKD1L2 NM_ SLC40A1 NM_ ENST ENST ZCCHC2 ENST BCAR4 CR LEMD1 NM_ THC THC A_24_P A_24_P BC BC GLUL NM_ LOC NM_ THC THC RAB3C NM_ ANKRD22 NM_ BU BU PAPSS2 NM_ SAA3P AY THC THC DNM3 NM_ LOC AK KCNE1 NM_ AA AA CHRNB4 NM_ CFTR NM_ C22orf24 AL SRD5A2 NM_ LOC NM_ ENST ENST FLJ13236 NM_ BC BC GPR161 NM_ PRL NM_ AF AF
32 THC THC LOC89944 NM_ HOXA4 NM_ A_23_P A_23_P A_24_P A_24_P MAGED4 NM_ ARHGAP20 NM_ A_32_P A_32_P AK AK CXCL12 NM_ CR CR C1orf115 NM_ GJA4 NM_ GCH1 NM_ DUSP13 NM_ CR CR COL13A1 NM_ ROR2 NM_
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