Supplemental Information Loss of Estrogen Regulated MIR135A1 at 3p21.1 Promotes Tamoxifen Resistance in Breast Cancer Weijie Zhang, Mingming Wu, Qing-Yun Chong, Min Zhang, Xiao Zhang, Lan Hu, Yanghao Zhong, Pengxu Qian, Xiangjun Kong, Sheng Tan, Gaopeng Li, Keshuo Ding, Peter E. Lobie, and Tao Zhu. Supplemental data include 5 supplemental tables, and 7 supplemental figures and legends. Supplemental Tables Supplementary Table S1. Sequences of the miRNA mimics/siRNAs, shRNAs, and primers for cloning, ChIP assays and qRT-PCR analysis. miRNAs Sense Strand (5'-3') Antisense Strand (5'-3') hsa-miR-135a UAUGGCUUUUUAUUCCUAUGU ACAUAGGAAUAAAAAGCCAUA mimics GA UU siERK1 GAAACUACCUACAGUCUCUTT AGAGACUGUAGGUAGUUUCTT siERK2 GUGCUCUGCUUAUGAUAAUTT AUUAUCAUAAGCAGAGCACTT siAKT GCACCUUCAUUGGCUACAATT UUGUAGCCAAUGAAGGUGCTT Negative Control (NC) UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT shRNAs Sense Strand (5'-3') CCGGCTACAGGCCAAATTCAGATAACTCGAGTTATCTGAATTTGGC shESR1 CTGTAGTTTTT CCGGGTGAATGCACTGGTGTCTCATCTCGAGATGAGACACCAGTGC shESRRA ATTCACTTTTT CCGGCGGAAGTCAGATTGTAGCCAACTCGAGTTGGCTACAATCTGA shNCOA1 CTTCCGTTTTT CCGGGCTTGACTGGTTTGAGACACACTCGAGTGTGTCTCAAACCAG shPIM2 TCAAGCTTTTT CCGGCCACCTCTCAATGTCGACAAACTCGAGTTTGTCGACATTGAG shMRAS AGGTGGTTTTTG CCGGGTCATCAAGATTGGGTTGTTTCTCGAGAAACAACCCAATCTTG shLCP1 ATGACTTTTTG CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTA shScr GG Primers for plasmid construction Sense Strand (5'-3') Antisense Strand (5'-3') pri-miR-135a-1 CGAATTCAGGAACTCCATGCCA AATGTCGACCCTGAAGTCAGCC cloning primer GCACAG CTTCAGTG TCACATAGGAATTTAAGCCATACGATTCACATAGGAATTTAAGCCA TAACCGGTTCACATAGGAATTTAAGCCATATCACTCACATAGGAAT TTAAGCCATACCCGGTGCATGACTAAGCTAGATAATCACATAGGAA TTTAAGCCATACGATTCACATAGGAATTTAAGCCATAACCGGTTCA CATAGGAATTTAAGCCATATCACTCACATAGGAATTTAAGCCATAA miR-135a Sponge CGCGTCGACCTAAGTATCCTCCTCTTGGA ESR1 3’UTR TACTCGAGAGCCCTGGGGCATG GTGCGGCCGCGCTATGGCTTGG cloning primer GAGCTGAAC TTAAACATATC ESR1 3’UTR Mut GCAGATATGTTTAACCGCCCAT CTCAAAAGGCATGGGCGGTTAA primer GCCTTTTGAG ACATATCTGC ESRRA 3’UTR ATCTCGAGAGGGGTGGGACTG CCGCGGCCGCTTTCTCTTTGCAG cloning primer GTGGGGG CAAATATAC ESRRA 3’UTR CAGTGCTGCCCCTTGCACGTGC CACTCTGGGGGCACGTGCAAGG MutA primer CCCCAGAGTG GGCAGCACTG GTAGGGGGCCTTGCGGGGGGG CATCCCGTGCAGCCCCCCCGCA ESRRA 3’UTR CTGCACGGGATG AGGCCCCCTAC MutB primer NCOA1 3’UTR CGCGCTCGAGCCACTTTTAAAG ATGCGGCCGCCTTCAGGGATGC cloning primer GAATGTGAAA TTTATTATCC NCOA1 3’UTR CAGACTTCAAGGAAGTCCCCA AGGAAGGACCTGGGGACTTCCT MutA primer GGTCCTTCCTTCTGCATCTC TGAAGTCTGAGTCCATGC NCOA1 3’UTR CATTTCCAGCTTGAAAGCCTAC GGTAGGGACTAGAGTAGGCTTT MutB primer TCTAGTCCCTACC CAAGCTGGAAATG PIM2 3’UTR ATCTCGAGAATGGTCAGAAGA GCGCGGCCGCCTATTACTTTTAT cloning primer GCCATCCCA TAAATAGTGGG PIM2 3’UTR Mut GCCTGGATTATTTAAATGTGGA ATAGTGGGTTTCCACATTTAAA primer AACCCACTAT TAATCCAGGC MRAS 3’UTR ATCTCGAGCAGGCCTGAGGCCC GCGCGGCCGCCACTCGTTTCAC cloning primer TGGGCACA GACAATGGCC MRAS 3’UTR CATACAAAGTACAAAACAACA GGAAAGAAGCTTGTTGTTTTGT MutA primer AGCTTCTTTCC ACTTTGTATG MRAS 3’UTR CAGCACCAAGACCTGTTCCCTT CAGTTGACTCCAAGGGAACAGG MutB primer GGAGTCAACTG TCTTGGTGCTG LCP1 3’UTR ATCTCGAGGGCCCAATGGGGCT GCGCGGCCGCGGCTACAGTAAG cloning primer GGGTGGGA TTCAATCTGAAG LCP1 3’UTR Mut GCTCCAGGGATGATTCCCAAAG CCAAGTTGAACTTTGGGAATCA primer TTCAACTTGG TCCCTGGAGC Promoter L GCGCTCGAGAGCAGGGAAGTG TATAAGCTTTCAGGGAGCTCTT cloning primer GCTCATTATTTC GGCTCAGGGAG Promoter S ATACTCGAGTGAGAGAGACCC TATAAGCTTCCCTGCTGTTTGCA cloning primer TCAGGGGGACCT ACTCTGGGCA Promoter L Mut CGGCACCGTCCAGGGGCACAA CACAGGCACTTGTGCCCCTGGA primer GTGCCTGTG CGGTGCCG Primers for ChIP assay Sense Strand (5'-3') Antisense Strand (5'-3') Site 1 Primer ACTTCGCGGCCACTCATC CCTCACTGCCCATAGTCCTG Site 2 Primer GGAACAGTGTATGCCCAGTCCT GGAGTAGCCCCAGCACATTCCA Site 3 Primer AGGCTCACAGGTTTTACTCCA ACTGCAGGAATATCTAGGCTCT Site 4 Primer TCGCACATGACCCAGACCC TGGCACTAGGCAGGCACCA Site 5 Primer ACCCAGTGCCAATACTCATGCT CTGTGCTGGCATGGAGTTCCT Site 6 Primer CAGAGCCCGCTTACCACGTT GGCTGGCAGGACTTTCCCC ACGGCTCCAATCCCTATATGAG Site 7 Primer GGCCCCTCCACACCCTCAG T TFF1 site Primer CCGGCCATCTCTCACTATGAA CCTCTTTCCCATGGGAGTCTC NC site Primer ATGGTTGCCACTGGGGATCT TGCCAAAGCCTAGGGGAAGA Primers for RNA Pull down assay Sense Strand (5'-3') Antisense Strand (5'-3') ESR1 site GCCGTAATGATTCTATAATGCC AAATCAAAGACTGCACTTCC ESRRA site CCATAACGTGCCCCCAGAGTGT TAGGTTTCTGCCTCCCACGCATC NCOA1 site TGAAAGCCAGCCATATTACTCT TCTGCGCGTCTAAATATCACA PIM2 site GACCCTACTCTGTTATCCCAA CTTATTTCCCCTAGCCCAT MRAS site 1 CAAAGTACAAAACAAGCCAT CTATAAAAGACATCCAAGCTCA MRAS site 2 TTCATGCTGGATCTTAACCAA TCTGCTCATTGACTAGGCTT LCP1 site GGATGATTCAAGCCATTCCAA CTCCTGCAAAGAATGAAGCAC GAPDH site TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG Primers for qRT-PCR Sense Strand (5'-3') Antisense Strand (5'-3') GTCGTATCCAGTGCAGGGTCCG miR-135a RT AGGTATTCGCACTGGATACGAC Primer TCACAT miR-135a qRT-PCR Primer CGGCTATGGCTTTTTATTCC GTGCAGGGTCCGAGGT TGGAACGATACAGAGAAGATT snRNA U6 AGCA AACGCTTCACGAATTTGCGT pri-miR-135a-1 TATAGGGATTGGAGCCG GCAGCCTGGGTTGAGA pri-miR-135a-2 TAAAGTCTCATGTAGGGATG GCTCCACAGGCTAACG TFF1 ATACCATCGACGTCCCTCCA AAGCGTGTCTGAGGTGTCCG NR3C2 GGCCAGCTTAATATTGTCCAG GTGCCTGAACATGAATGCTT APC GAGGTCAGCCTAAACCCAT ACCGGAGTATTTTCAATAGCAA MTSS1 AGGCAGATCAGCAGGAGT ACAGAAGGCTTGGTGGAA JAK2 GAATAGCCAAAGAAAACGAT ATCTGTACCTTATTCGCTTC SIAH1 CAGCAGTTCTTCGCAATCGT TCGCCTATGACCATTTAGCTC HOXA10 CTTCCGAGAGCAGCAAAGCCT CTTCCGACCACTCTTTGCCGTGA ESR1 GCATTCTACAGGCCAAATT CACAGGACCAGACTCCATAA ESRRA CGTGGCTACCCTCTGTGACCT GCACCTCCATCCACACGCTCT NCOA1 GAGCCAGGGACCGAGAA GCCGTGCAATACAAATCAG PIM2 CACTGCCATTCCCGTGGAGT AAGCAGGGCACCAGAACCAA MRAS AAAGCCTTCCATGACCTCGTT GTCTCCCCGCCATTTGGTT TCCCAATGAACCCAAACACGA LCP1 A TCATCAATTGTGTCTGGCACT GAPDH TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG Supplementary Table S2. List of antibodies used in western blot and chromatin immunoprecipitation assay. Incubation Protein Assay Antibody Origin Dilution period ERα WB mmab #sc-8005, Santa Cruz 1:500 Over night ERα IHC mmab #sc-8005, Santa Cruz 1:50 Over night pERα (S118) WB mmab #2511, Cell Signaling 1:1000 Over night pERα #64508, Cell (S167) WB rmab Signaling 1:1000 Over night ERα ChIP mmab #sc-8005, Santa Cruz 5 ug Over night ESRRA WB mmab #sc-65718, Santa Cruz 1:500 Over night ESRRA IHC mmab #sc-65718, Santa Cruz 1:50 Over night NCOA1 WB rpab #sc-8995, Santa Cruz 1:500 Over night NCOA1 IHC rpab #sc-8995, Santa Cruz 1:50 Over night PIM2 WB mmab #sc-13514, Santa Cruz 1:500 Over night PIM2 IHC mmab #sc-13514, Santa Cruz 1:50 Over night #sc-292844, Santa MRAS WB rpab Cruz 1:500 Over night #sc-292844, Santa MRAS IHC rpab Cruz 1:50 Over night #sc-133218, Santa LCP1 WB mmab Cruz 1:500 Over night #sc-133218, Santa LCP1 IHC mmab Cruz 1:50 Over night pAKT (S473) WB rpab #9271, Cell Signaling 1:1000 Over night pAKT #sc-7985-R, Santa (S473) WB rpab Cruz 1:1000 Over night pAKT (S473) IHC rpab #9271, Cell Signaling 1:100 Over night AKT1 WB mmab #sc-5298, Santa Cruz 1:1000 Over night pERK1/2 (T202/Y204) WB mmab #9106, Cell Signaling 1:1000 Over night pERK1/2 (T202/Y204) IHC mmab #9106, Cell Signaling 1:100 Over night tERK1/2 WB mmab #13-6200, Zymed 1:1000 Over night TWIST1 WB rpab #sc-15393, Santa Cruz 1:1000 Over night EZH2 WB rpab #5246, Cell Signaling 1:1000 Over night FN1 WB mmab # 610077, BD 1:5000 Over night #13409-1-AP, Occludin WB rpab Proteintech 1:1000 Over night γ-catenin WB mmab #sc-8415, Santa Cruz 1:1000 Over night #60008-1-Ig, β-actin WB mmab Proteintech 1:5000 Over night Abbreviations: WB, Western blot; ChIP, chromatin immunoprecipitation; mmab, mouse monoclonal antibody; rmab, rabbit monoclonal antibody; rpab, rabbit polyclonal antibody. Supplementary Table S3. The correlation of MIR135A1 deletion with the clinicopathologic features of breast cancer in the TCGA breast tumor dataset Parameter n MIR135A1 loss, n (%) p value Menopause pre 213 78 (37) 0.072 peri 35 14 (40) post 649 190 (29) Age <=35 29 15 (52) 0.006 35-55 401 139 (35) >55 573 162 (28) Vital status alive 906 277 (31) 0.052 dead 97 39 (40) Recurrence free 746 225 (30) 0.294 recurred 63 23 (37) Histological type IDC 730 265 (36) 0.000 ILC 177 33 (19) Other types 95 18 (19) Pathologic T T1 262 57 (22) 0.000 T2 582 213 (37) T3* 156 45 (29) Pathologic stage I 169 40 (24) 0.052 II 570 191 (34) III* 246 79 (32) Estrogen receptor － 215 112 (52) 0.000 ＋ 744 192(26) Progesterone receptor － 308 151 (49) 0.000 ＋ 648 153 (24) HER2 low 306 104 (34) 0.447 high 525 165 (31) * includes higher categories. Supplementary Table S4. The correlation of pri-miR-135a-1 expression with the clinicopathologic features of breast cancer in the TCGA breast tumor dataset Parameter n Lower pri-miR-135a-1, n (%) p value Menopause pre 213 78 (37) 0.000 period 35 22 (63) post 649 358 (55) Age <=35 29 13 (45) 0.041 35-55 401 182 (45) >55 573 306 (53) Vital status alive 906 453 (50) 0.989 dead 97 48 (50) Recurrence free 746 369 (50) 0.353 recurred 63 35 (56) Histological type IDC 730 366 (50) 0.989 ILC 177 88 (50) Other types 95 47 (50) Pathologic T T1 262 132 (50) 0.746 T2 582 294 (51) T3* 156 75 (48) Pathologic stage I 169 79 (47) 0.148 II 570 285 (50) III* 230 129 (56) Estrogen receptor － 215 132 (61) 0.000 ＋ 744 339 (46) Progesterone receptor － 308 193(63) 0.000 ＋ 648 277 (43) HER2 low 306 174 (57) 0.004 high 525 244 (47) * includes higher categories. Supplementary Table S5. Gene List EMT markers examined in the Oxford breast tumor set CDH1, CDH2, VIM, FN1, SNAI1, SNAI2, TCF3, TWIST1, ZEB1, JUP, CTNNB1, ZEB2, TJP1, OCLN, GJA1, GJB1, CTNNA1, FOXC2, MMP2, MMP9, EZH2 ERα cofactors and interacting proteins AHR, BCAR1, BRCA1, CAV1, CCND1, EFNA5, ESR1, ESR2, ESRRA, ESRRB, ESRRG, IGF1, MTA1, NCOA1, NCOA2, NCOA3, NCOR1, NCOR2, NR0B1, NR0B2, NRIP1, PELP1, PHB2, SAFB,