Supplementary data to: The mir-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2
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- Ὑπατος Αγγελίδης
- 5 χρόνια πριν
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1 Supplementary data to: The mir-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB and ZEB2 Sun-Mi Park, Arti B. Gaur 3, Ernst Lengyel 2 and Marcus E. Peter,4 The Ben May Department for Cancer Research, 2 Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, The University of Chicago and 3 Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire.
2 Supplementary figure legends: 2 Figure S. Expression of 207 different mirnas in the NCI60 cells ranked according to expression in either epithelial or mesenchymal cell lines. P-values are the result of a two-sample t-test analysis. High expression is indicated in red and low expression in blue. Fold change (log2) is indicated. Figure S2. Comparison of the ratio of E-cadherin/Vimentin protein expression with expression of either mir-4, mir-200a, mir-200b or mir-200c in the NCI60 cells. Pearson correlation coefficients (r) and p-values (p) are reported. Figure S3. (A) Normalized ΔCTs of the expression of the functional cluster mir-200a/mir-4 mirnas were added and plotted against the sum of the ΔCTs of mir-200b and mir-200c. (B) Normalized ΔCTs of the expression of the genetic cluster mir-200a and mir-200b mirnas were added and plotted against the sum of the ΔCTs of mir-200c and mir-4. Residual variances (R 2 ) are reported. Figure S4. Effects of only 5 nm of mir-200 on MDA-MB-23 cells. Cells were transfected with either 5 nm scrambled oligo (S) or a mixture of 2.5 nm mir-200a and 2.5 nm mir-200c and after 3 (D3) or 6 days (D6) expression of E-cadherin, ZEB and ZEB2 mrna was determined using real-time PCR. Figure S5. (A) Analysis of the expression of let-7d and mir-200c in 59 of the NCI60 cells. In the left half, the expression of let-7d in the Type I and Type II cells previously identified among the NCI60 cells (Algeciras-Schimnich et al. 2003) and the SC and SC2 cell lines among the 59 NCI60 cells are shown. In the right half the expression of mir-200c in the same cells is shown. (B) Comparison of the ratio of E-cadherin/Vimentin protein expression with expression of let-7d in the NCI60 cells. Cells with either high E-cadherin and no Vimentin (I), E- cadherin/vimentin ratio of around one (II) and with high Vimentin and low E-cadherin expression (III) are labeled with a stippled oval. (C) Comparison of the ratio of E- cadherin/vimentin protein expression with expression of mir-200c in the NCI60 cells. Group I, group II and group III cells are labeled. SC and SC2 cells within the epithelial group I are labeled in blue and red, respectively. Residual variances (R 2 ) are given. References Algeciras-Schimnich, A., E.M. Pietras, B.C. Barnhart, P. Legembre, S. Vijayan, S.L. Holbeck, and M.E. Peter Two CD95 tumor classes with different sensitivities to antitumor drugs. Proc Natl Acad Sci U S A 00:
3 Figure S mirna ID K562 HL60TB MOLT4 CCRFCEM SW620 IGROV NCI-H522 SR OVCAR5 NCI-H23 CAKI ADR-RES PC3 BT549 ACHN SN2 OVCAR8 HOP62 LOXIMVI SNB9 SF295 MDAMB435 TK0 MDAMB23 U25 SKMEL M4 A498 SF268 SNB75 UACC62 RXF393 SKMEL28 SF539 HS578 HOP92 OVCAR4 NCI-H322M HCC2998 HT29 T47D COLO205 HCT5 MCF7 KM2 OVCAR3 HCT6 epithelial mesenchymal 0-0 p-value hsa-mir-200c hsa-mir-4 hsa-mir-200b hsa-mir-200a hsa-mir-203 hsa-mir-55 hsa-mir-40 hsa-mir-46 hsa-mir-204 hsa-mir-05 hsa-mir-375 hsa-mir-299 hsa-mir-37 hsa-mir-32 hsa-mir-2 hsa-mir-34 hsa-mir-27 hsa-mir-223 hsa-mir-22 hsa-mir-34a hsa-mir-97 hsa-mir-25b hsa-mir-23 hsa-mir-8b hsa-mir-30a-3p hsa-mir-95 hsa-mir-7 hsa-mir-00 hsa-mir-29a hsa-mir-99b hsa-mir-96b hsa-mir-205 hsa-mir-28 hsa-mir-94 hsa-let-7a hsa-mir-32 hsa-mir-30e-3p hsa-mir-96 hsa-mir-99a hsa-mir-92 hsa-mir-27a hsa-mir-424 hsa-mir-92 hsa-mir-2 hsa-mir-8a hsa-mir-30a-5p hsa-mir-32 hsa-mir-35b hsa-mir-25 hsa-mir-30c hsa-mir-48a hsa-mir-07 hsa-mir-24 hsa-mir-30an hsa-mir-39 hsa-mir-20 hsa-mir-324-5p hsa-mir-335 hsa-mir-28b hsa-mir-30a hsa-mir-53 hsa-mir-35a hsa-mir-9 hsa-mir-302b hsa-mir-92 hsa-mir-222 hsa-let-7d hsa-mir-22 hsa-mir-24a hsa-mir-99a hsa-mir-372 hsa-mir-5 hsa-mir-98 hsa-mir-302d hsa-mir-342 hsa-mir-52 hsa-mir-345 hsa-mir-37 hsa-mir-302a hsa-mir-86 hsa-mir-26 hsa-mir-24b hsa-mir-30b hsa-mir-302c hsa-mir-373 hsa-mir-338 hsa-mir-48b hsa-mir-28a hsa-mir-325 hsa-mir-33b hsa-mir-339 hsa-mir-42-5p hsa-mir-29c hsa-mir-9 hsa-mir-425 hsa-mir-82 hsa-mir-28 hsa-let-7f hsa-mir-330 hsa-mir-30b hsa-mir-24 hsa-mir-23a hsa-mir-36 hsa-mir-29 hsa-mir-326 hsa-mir-47 hsa-mir-30e hsa-mir-99b hsa-mir-54 hsa-mir- hsa-mir-99-s hsa-mir-38 hsa-mir-0b hsa-mir-368 hsa-mir-380-3p hsa-mir-384 hsa-mir-224 hsa-mir-36 hsa-mir-380-5p hsa-mir-377 hsa-mir-8c hsa-mir-383 hsa-mir-38 hsa-mir-422a hsa-mir-33 hsa-mir-337 hsa-mir-26 hsa-mir-04 hsa-mir-93 hsa-mir-29 hsa-mir-22a hsa-mir-44 hsa-mir-98 hsa-mir-208 hsa-mir-27 hsa-mir-220 hsa-mir-369 hsa-mir-89 hsa-let-7i hsa-mir-23b hsa-mir-43 hsa-mir-03 hsa-mir-27b hsa-mir-88 hsa-mir-26a hsa-mir-33 hsa-mir-9a hsa-mir-45 hsa-let-7g hsa-mir-9b hsa-mir-84 hsa-mir-29b hsa-mir-367 hsa-mir-296 hsa-mir-346 hsa-mir-340 hsa-mir-20n hsa-mir-30 hsa-mir-6 hsa-mir-7-5p hsa-mir-5a hsa-mir-422b hsa-mir-324-3p hsa-mir-374 hsa-mir-95 hsa-mir-328 hsa-mir-423 hsa-mir-87 hsa-mir-0a hsa-mir-42-3p hsa-mir-85 hsa-mir-33a hsa-mir-90 hsa-mir-20 hsa-mir-49 hsa-mir-83 hsa-mir-382 hsa-mir-5b hsa-mir-25 hsa-mir-93 hsa-mir-26b hsa-mir-320 hsa-mir-34b hsa-mir-376a hsa-mir-7-3p hsa-mir-0 hsa-mir-25a hsa-mir-06a hsa-mir-30d hsa-mir-34c hsa-mir-370 hsa-mir-50 hsa-mir-206 hsa-mir-8 hsa-mir-379 hsa-mir-3 hsa-let-7e hsa-mir-378 hsa-mir-22 hsa-mir-06b hsa-mir-96a hsa-mir-323 hsa-let-7c hsa-let-7b hsa-mir
4 Figure S E-cadherin/Vimentin R= p< mir R= p= mir-200a R= p= R=0.563 p< mir-200b mir-200c
5 Figure S3 A mir-200a + mir-4 (ΔCTs) 00 0 B R 2 = mir-200b + mr-200c (ΔCTs) mir-200a + mir-200b (ΔCTs) 00 0 R 2 = mir-200c + mir-4 (ΔCTs)
6 Figure S4 25 D3 D6.2 D3 D6 D3 D Fold change S 200a/c S 200a/c 0 S 200a/c S 200a/c S 200a/c S 200a/c E-cadherin ZEB ZEB2
7 A let-7d mir-200c p= p= p=0.06 p= Figure S5 00 % of highest expression B E-cadherin/Vimentin I II 2 I II 2 Type supercluster Type supercluster II 0-3 III let-7d I R 2 =0.08 C 0 6 OVCAR T47D OVCAR3 III COLO205 HT29 II DU45 H322M HCC2998 HCT5 MCF7 HCT mir-200c I KM2 R 2 =0.656
8 Table S: Genes downregulated >0.5 in MBA-MD-23 cells transfected with either mir-200a or mir-200c mir-200a vs scr mir-200c vs scr TargetScan TargetRank Gene log2 change log2 change mir-4/200a mir-200b,c/429 mir-200a mir-200c Descriptions ICEBERG gb:nm_0257. /DB_XREF=gi: /GEN=ICEBERG / QKI X X X X gb:al0378 /DB_XREF=gi: /FEA=FLmRNA /CNT= LOX X X gb:l6895 /DB_XREF=gi: /FEA=DNA /CNT=3 /TID=H WIPF X X X X gb:aw /DB_XREF=gi: /DB_XREF=wy59c0.x LHFP X X X gb:nm_ /DB_XREF=gi: /GEN=LHFP /FEA= Unknown gb:af /DB_XREF=gi: /FEA=FLmRNA /CNT ZEB X X X X gb:ai37366 /DB_XREF=gi: /DB_XREF=qz3b0.x Unknown Cluster Incl. U79256:Human clone 2379 mrna sequence /c Unknown gb:be /DB_XREF=gi: /DB_XREF=7e57e09.x Unknown gb:al33706 /DB_XREF=gi: /DB_XREF=DKFZp76 APS X X gb:af /DB_XREF=gi: /FEA=FLmRNA /CN ZEB () X X X X gb:ai80674 /DB_XREF=gi: /DB_XREF=wf06h03.x RPS gb:aa42942 /DB_XREF=gi:72320 /DB_XREF=zl43c04.s TAXBP gb:al /DB_XREF=gi: /DB_XREF=DKFZp586 Unknown gb:av /DB_XREF=gi: /DB_XREF=AV70339 Unknown gb:aw93 /DB_XREF=gi: /DB_XREF=xd89b09.x ZEB X X X X gb:nm_ /DB_XREF=gi: /GEN=ZFHXB /FE PPAP2B X X gb:aa /DB_XREF=gi: /DB_XREF=af39f2.s Unknown gb:nm_ /DB_XREF=gi: /GEN=FLJ2432 / Unknown gb:bf /DB_XREF=gi: /DB_XREF= Unknown gb:al04976 /DB_XREF=gi: /FEA=FLmRNA /CNT=8 ATP2C gb:af /DB_XREF=gi: /GEN=ATP2C /FEA= Unknown gb:aa97899 /DB_XREF=gi: /DB_XREF=ol76e05.s Unknown gb:h95263 /DB_XREF=gi:02896 /DB_XREF=yu20e04.s /C Unknown gb:al /DB_XREF=gi: /FEA=mRNA /CNT=2 Unknown gb:nm_ /DB_XREF=gi: /GEN=FLJ2903 / Unknown gb:nm_0283. /DB_XREF=gi: /GEN=LOC57826 PCDH X X gb:ai52425 /DB_XREF=gi: /DB_XREF=th09d.x ALS2CR gb:ab /DB_XREF=gi: /GEN=ALS2CR2 /FE TRIP gb:bf /DB_XREF=gi: /DB_XREF=7h02f04.x Unknown gb:bf59387 /DB_XREF=gi:6864 /DB_XREF=nac05a0 Unknown gb:ai /DB_XREF=gi: /DB_XREF=tc57a04.x Unknown gb:aa /DB_XREF=gi: /DB_XREF=nn22f08.s MTND gb:nm_7374. /DB_XREF=gi: /GEN=MTND6 /TID Unknown gb:ak /DB_XREF=gi: /FEA=FLmRNA /CNT PAPPA X X X gb:aa48534 /DB_XREF=gi:72559 /DB_XREF=zl06d06.s COP gb:bc /DB_XREF=gi:3846 /FEA=FLmRNA /CN LSM gb:au53477 /DB_XREF=gi:04998 /DB_XREF=AU5347 Unknown gb:al /DB_XREF=gi: /GEN=DKFZp586C6
9 THBD gb:nm_ /DB_XREF=gi: /GEN=THBD /FEA= IGFBP gb:nm_ /DB_XREF=gi: /GEN=IGFBP /FEA IDH gb:d3889. /DB_XREF=gi:4648 /GEN=Id-H /FEA=FLmR ZEB () X X X X gb:u270. /DB_XREF=gi:52972 /FEA=FLmRNA /CNT=2 / Unknown gb:ab /DB_XREF=gi: /GEN=KIAA0874 /FE DNAJC X gb:aa /DB_XREF=gi: /DB_XREF=ak09g0.s ATM X gb:u82828 /DB_XREF=gi: /FEA=mRNA /CNT=3 /T Unknown gb:ak /DB_XREF=gi: /GEN=FLJ00002 /FE RPS gb:aw /DB_XREF=gi: /DB_XREF=wv03e06.x MTAP gb:nm_ /DB_XREF=gi: /GEN=MTAP44 /FE Unknown gb:ai /DB_XREF=gi: /DB_XREF=cong.P. LOC gb:nm_ /DB_XREF=gi: /GEN=LOC55828 /F Unknown gb:al /DB_XREF=gi: /GEN=DKFZp434E3 Unknown gb:nm_ /DB_XREF=gi: /GEN=KIAA0798 /F Unknown gb:ai74220 /DB_XREF=gi:50498 /DB_XREF=wg39c02.x Unknown gb:aa77568 /DB_XREF=gi: /DB_XREF=zf3a02.s ADAMTS gb:ak /DB_XREF=gi: /FEA=mRNA /CNT= MAIL gb:be /DB_XREF=gi: /DB_XREF=7e89c0.x TRIM gb:af /DB_XREF=gi: /GEN=TRIM6 /FEA= CS gb:af00540 /DB_XREF=gi: /DB_XREF=AF00540 Unknown gb:aw52589 /DB_XREF=gi: /DB_XREF=xf76g02.x Unknown gb:bg /DB_XREF=gi:34683 /DB_XREF= Unknown gb:aa /DB_XREF=gi: /DB_XREF=af43e03.s CALM gb:al04607 /DB_XREF=gi:5434 /DB_XREF=DKFZp434 PTMA gb:ai0886 /DB_XREF=gi: /DB_XREF=HA0306 /FE Unknown gb:aw34977 /DB_XREF=gi: /DB_XREF=UI-H-BI-a Unknown gb:ai /DB_XREF=gi: /DB_XREF=wd73d06.x Unknown gb:ai /DB_XREF=gi: /DB_XREF=tc32d2.x Unknown gb:ai76003 /DB_XREF=gi: /DB_XREF=wh83a07.x Unknown gb:ai /DB_XREF=gi: /DB_XREF=wp72g2.x Unknown gb:bg93589 /DB_XREF=gi: /DB_XREF=60287 Unknown gb:al /DB_XREF=gi: /DB_XREF=AL SFRS gb:be /DB_XREF=gi: /DB_XREF=MR3-CT0 H05E gb:u8267 /DB_XREF=gi: /FEA=DNA_9 /CNT=6 /TID Unknown gb:n7074 /DB_XREF=gi: /DB_XREF=za86d.s /C THBD gb:nm_ /DB_XREF=gi: /GEN=THBD /FEA= TM7SF gb:nm_ /DB_XREF=gi: /GEN=TM7SF /FE APAF X X gb:nm_ /DB_XREF=gi: /GEN=APAF /FEA HAS gb:nm_ /DB_XREF=gi: /GEN=HAS2 /FEA= H4FE gb:nm_ /DB_XREF=gi:45029 /GEN=H4FE /FEA Unknown gb:ab /DB_XREF=gi: /GEN=KIAA0877 /FE MKPX gb:nm_ /DB_XREF=gi:99043 /GEN=MKPX /FEA= Unknown Cluster Incl. AA5065:zl06h03.r Homo sapiens cdna, 5 en
10 PRO gb:ai /DB_XREF=gi: /DB_XREF=tj5d04.x / TASR gb:ai /DB_XREF=gi: /DB_XREF=wq32b06.x PRO gb:ai /DB_XREF=gi: /DB_XREF=tc92a05.y Unknown gb:n76327 /DB_XREF=gi: /DB_XREF=yv39c02.r /C Unknown gb:n32832 /DB_XREF=gi:5323 /DB_XREF=yw85g06.s / NNMT gb:nm_ /DB_XREF=gi: /GEN=NNMT /FEA= Unknown gb:ab /DB_XREF=gi: /GEN=KIAA098 /FE PTTG gb:nm_ /DB_XREF=gi: /GEN=PTTG3 /FE GJA gb:nm_ /DB_XREF=gi: /GEN=GJA /FEA= Unknown gb:nm_ /DB_XREF=gi: /GEN=KIAA076 /F Unknown gb:au5338 /DB_XREF=gi:04659 /DB_XREF=AU533 Unknown gb:ak /DB_XREF=gi: /FEA=mRNA /CNT= Unknown gb:nm_ /DB_XREF=gi: /GEN=FLJ20730 /F Unknown gb:ai97246 /DB_XREF=gi: /DB_XREF=wr39d0.x KBRAS gb:ai97020 /DB_XREF=gi: /DB_XREF=wq89d09.x () The online versions of both TargetScan and TargetRank are based on an incomplete 3'UTR sequence for ZEB of 542 bp. The complete ZEB 3'UTR contains 954 bp (gb:u270) and is predicted to be a target of both mir-200a and mir-200c by TargetScan and TargetRank.
11 Table S2: The top 20 genes most downregulated in MDA-MB-23 cells transfected with only mir-200a mir-200a mir-200c TargetScan TargetRank GenBank Gene Fold down Fold down mir-4/200a mir-200b,c/429 mir-200a mir-200c Accession # Unknown 2.3 NC NM_ DLG NC NM_ Unknown 9.85 NC BC SDR-O 8.57 NC NM_ Unknown 7.46 NC AF MKP 6.06 NC AB Unknown 6.06 NC AL KCNJ NC X AB PRO NC NM_0420. Unknown 2.00 NC AA66276 Unknown 2.00 NC AL NAPL3.87 NC X NM_ H4F2.87 NC NM_ TIMELESS.74 NC AK Unknown.74 NC AA Unknown.74 NC AI42320 Unknown.74 NC BC Unknown.74 NC AK IL.62 NC NM_ Unknown.62 NC AI38223
12 Table S3: The top 20 genes most downregulated in MDA-MB-23 cells transfected with only mir-200c mir-200a mir-200c TargetScan TargetRank GenBank Gene Fold down Fold down mir-4/200a mir-200b,c/429 mir-200a mir-200c Accession # Unknown NC 3.93 AI26576 Unknown NC 3.00 AW H3FA NC 2.3 NM_ Unknown NC 2.3 AF ADRB NC 9.85 X AI Unknown NC 9.9 BF Unknown NC 8.57 AI Unknown NC 7.46 BF47693 Unknown NC 7.46 AI23346 RASGRP2 NC 6.50 AI68882 CQR NC 6.06 W72082 Unknown NC 4.92 AL Unknown NC 4.92 AA56795 Unknown NC 4.59 NM_ TCRA NC 4.29 M5565. Unknown NC 4.29 BC COMP NC 3.03 NM_ Unknown NC 2.64 AI96235 Unknown NC 2.30 NM_ Unknown NC 2.30 BC00275.
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