Supplementary Table 1 Clinicopathological Characteristics

Supplementary Tables

Supplementary Table S1. Clinicopathological Characteristics. Characteristic Number of patients (%) Age, median(range) 61(38-84)years Gender Male 143(77.3) Female 42(22.7) Smoking status Never 88(47.6) Former/current 97(52.4) Surgical approach Sweet approach 130(70.3) Ivor Lewis approach 34(18.4) Ivor Lewis approach+cervical incision 16(8.6) Sweet approach+cervical incision 2(1.1) Minimally invasive esophagectomy 3(1.6) Tumor stage Ⅰ A 3(1.6) Ⅰ B 10(5.4) Ⅱ A 50(27.0) Ⅱ B 45(24.3) Ⅲ A 44(23.8) Ⅲ B 23(12.4) Ⅲ C 10(5.4) Tumor differentiation Well/moderate 105(56.8) Poor 80(43.2) Charlson comorbidity index (CCI) <3 138 (74.6) ≥3 47 (25.4) Note: Tumor stage was determined according to 7th edition of UICC-AJCC TNM staging system

Supplementary Table S2. Differently expressed miRNAs between ECs and NMs. miRNA Sequence ECs vs.NMs

1 Log2FC Style P-Value FDR hsa-miR-126-3p tcgtaccgtgagtaataatgcg -1.647483494 down 0 0 hsa-miR-451a aaaccgttaccattactgagtt -1.65895591 down 0 0 hsa-miR-99b-5p cacccgtagaaccgaccttgcg -1.66539343 down 0 0 hsa-miR-26a-5p ttcaagtaatccaggataggct -1.683017076 down 0 0 hsa-miR-26b-5p ttcaagtaattcaggataggt -1.709578252 down 0 0 hsa-miR-126-5p cattattacttttggtacgcg -1.816966633 down 0 0 hsa-miR-100-5p aacccgtagatccgaacttgtg -1.824441371 down 0 0 hsa-miR-10a-5p taccctgtagatccgaatttgtg -1.867781043 down 0 0 hsa-miR-143-5p ggtgcagtgctgcatctctggt -1.892258222 down 0 0 hsa-miR-143-3p tgagatgaagcactgtagctc -2.197916019 down 0 0 hsa-miR-195-5p tagcagcacagaaatattggc -2.217941529 down 0 0 hsa-miR-1 tggaatgtaaagaagtatgtat -2.351785345 down 0 0 hsa-miR-133a tttggtccccttcaaccagctg -2.74123279 down 0 0 hsa-miR-145-5p gtccagttttcccaggaatccct -2.968242676 down 0 0 hsa-miR-145-3p ggattcctggaaatactgttct -3.024082135 down 0 0 hsa-miR-139-5p tctacagtgcacgtgtctccagt -2.226581077 down 1.07E-261 8.90E-260 hsa-miR-4454 ggatccgagtcacggcacca -1.740270003 down 4.18E-217 3.08E-215 hsa-miR-125a-5p tccctgagaccctttaacctgtga -1.827815583 down 2.49E-205 1.74E-203 hsa-miR-375 tttgttcgttcggctcgcgtga -1.915837949 down 1.06E-169 6.36E-168 hsa-miR-548a-5p aaaagtaattgcgagttttacc -2.523150543 down 4.77E-148 2.66E-146 hsa-miR-4328 ccagttttcccaggatt -2.697970737 down 1.07E-141 5.83E-140 hsa-miR-28-5p aaggagctcacagtctattgag -1.601477789 down 1.62E-129 8.41E-128 hsa-miR-218-5p ttgtgcttgatctaaccatgt -1.936428345 down 2.01E-114 9.65E-113 hsa-miR-136-3p catcatcgtctcaaatgagtct -1.759250561 down 1.70E-66 6.11E-65 hsa-miR-3195 cgcgccgggcccgggtt -1.673316592 down 1.81E-60 6.01E-59 hsa-miR-4741 cgggctgtccggaggggtcggct -1.858183604 down 4.58E-52 1.35E-50 hsa-miR-335-5p tcaagagcaataacgaaaaatgt -1.59031393 down 8.31E-49 2.39E-47 hsa-miR-3681-3p acacagtgcttcatccactact -2.078739976 down 4.14E-45 1.13E-43 hsa-miR-133b tttggtccccttcaaccagcta -2.172907377 down 2.76E-42 7.34E-41 hsa-miR-4532 ccccggggagcccggcg -1.760169355 down 2.14E-41 5.60E-40 hsa-miR-378i actggactaggagtcagaagg -2.058668597 down 4.76E-36 1.15E-34 hsa-miR-10b-3p acagattcgattctaggggaat -2.24764374 down 2.27E-33 5.14E-32 hsa-miR-337-5p gaacggcttcatacaggagtt -1.974437863 down 8.17E-31 1.71E-29 hsa-miR-4455 agggtgtgtgtgttttt -2.024008714 down 2.50E-21 3.86E-20 hsa-miR-10a-3p caaattcgtatctaggggaata -1.966466764 down 8.89E-21 1.34E-19 hsa-miR-4770 tgagatgacactgtagct -2.038610548 down 1.84E-20 2.71E-19 hsa-miR-4509 actaaaggatatagaaggtttt -2.397678635 down 6.57E-17 8.55E-16 hsa-miR-4745-3p tggcccggcgacgtctcacggtc -1.736815844 down 4.60E-16 5.76E-15 hsa-miR-4638-3p cctggacaccgctcagccggccg -1.692441591 down 2.00E-12 2.08E-11 hsa-miR-378h actggacttggtgtcagatgg -1.603674809 down 2.13E-07 1.47E-06 hsa-miR-4691-3p ccagccacggactgagagtgcat -1.743000579 down 5.33E-06 3.07E-05 hsa-miR-376b-5p cgtggatattccttctatgttt -1.648405924 down 0.0011277 0.0042316

2 hsa-miR-302a-3p taagtgcttccatgttttggtga -1.951900185 down 0.005660 0.017287 8 hsa-miR-302b-3p taagtgcttccatgttttagtag -1.702520189 down 0.016806 0.0421777 1 hsa-miR-7-5p tggaagactagtgattttgttgt 2.022229924 up 0 0 hsa-miR-21-3p caacaccagtcgatgggctgt 1.843075582 up 0 0 hsa-miR-5588-5p actggcattagtgggactttt 1.829174581 up 0 0 hsa-miR-141-3p taacactgtctggtaaagatgg 1.806832619 up 0 0 hsa-miR-96-5p tttggcactagcacatttttgct 1.787574712 up 0 0 hsa-miR-3529-3p aacaacaaaatcactagtcttcca 2.099750195 up 4.53E-262 3.79E-260 hsa-miR-224-5p caagtcactagtggttccgtt 2.061727489 up 1.91E-242 1.52E-240 hsa-miR-18a-5p taaggtgcatctagtgcagatag 1.9798533 up 5.75E-190 3.86E-188 hsa-miR-135b-5p tatggcttttcattcctatgtga 2.328348343 up 8.72E-185 5.74E-183 hsa-miR-34c-5p aggcagtgtagttagctgattgc 2.381382549 up 9.93E-170 5.98E-168 hsa-miR-1260b atcccaccactgccaccat 1.645866709 up 3.66E-122 1.86E-120 hsa-miR-182-5p tttggcaatggtagaactcacact 1.662900442 up 5.55E-96 2.44E-94 hsa-miR-200a-5p catcttaccggacagtgctgga 1.701196601 up 6.22E-76 2.36E-74 hsa-miR-196a-5p taggtagtttcatgttgttggg 1.889754261 up 1.02E-72 3.83E-71 hsa-miR-944 aaattattgtacatcggatgag 1.747372199 up 6.25E-59 2.04E-57 hsa-miR-4533 tggaaggaggttgccggacgct 2.524792369 up 1.85E-52 5.49E-51 hsa-miR-1305 ttttcaactctaatgggagaga 2.040124142 up 4.42E-30 9.14E-29 hsa-miR-3136-5p ctgactgaataggtagggtcatt 1.786858781 up 6.27E-28 1.21E-26 hsa-miR-18b-5p taaggtgcatctagtgcagttag 1.955033645 up 9.23E-26 1.66E-24 hsa-miR-3978 gtggaaagcatgcatccagggtg 1.611424083 up 6.54E-25 1.14E-23 t hsa-miR-1269a ctggactgagccgtgctactgg 1.821194054 up 1.95E-22 3.08E-21 hsa-miR-562 aaagtagctgtaccatttgc 1.824669665 up 5.84E-22 9.12E-21 hsa-miR-5687 ttagaacgttttagggtcaaat 2.12039056 up 1.31E-19 1.86E-18 hsa-miR-18a-3p actgccctaagtgctccttctgg 1.83199315 up 5.09E-13 5.44E-12 hsa-miR-135b-3p atgtagggctaaaagccatggg 1.757722262 up 3.44E-12 3.51E-11 hsa-miR-4643 gacacatgaccataaatgctaa 1.730131518 up 8.76E-12 8.69E-11 hsa-miR-3975 tgaggctaatgcactacttcac 2.098725147 up 9.20E-12 9.11E-11 hsa-miR-6512-5p taccattagaagagctggaaga 1.938090549 up 2.13E-10 1.92E-09 hsa-miR-539-5p ggagaaattatccttggtgtgt 1.643218724 up 6.95E-09 5.55E-08 hsa-miR-518d-3p caaagcgcttccctttggagc 1.766620338 up 8.25E-08 5.97E-07 hsa-miR-888-5p tactcaaaaagctgtcagtca 1.631917707 up 8.85E-08 6.37E-07 hsa-miR-5197-5p caatggcacaaactcattcttga 2.044739998 up 1.23E-06 7.67E-06 hsa-miR-4759 taggactagatgttggaatta 1.811917848 up 6.08E-05 3.00E-04 hsa-miR-5692b aataatatcacagtaggtgt 1.773095835 up 0.003528 0.0115246 7

3 Supplementary Table S3. Univariate and Multivariate analysis of factors associated with disease- specific survival.

Variables Univariate Analysis Multivariate Analysis HR 95% CI P HR 95% CI P Gender (female vs. 0.791 0.494-1.267 0.329

male) Age(≥60 vs. <60) 1.044 0.709-1.538 0.826 Smoking status 0.734 0.498-1.082 0.118

(non-smokers vs.

smokers) Tumor 1.245 0.846-1.833 0.266

differentiation(Poor

vs. Well/Moderate) Surgical approach 0.127 Ivor Lewis 1.179 0.720-1.932 0.512

approach vs. Sweet

approach Other approach * 0.510 0.246-1.057 0.070

vs. Sweet approach CCI(≥3 vs. <3) 1.522 1.003-2.310 0.049 1.749 1.146-2.668 0.009 Tumor stage (Ⅲ-IV 2.567 1.736-3.795 <0.001 2.573 1.732-3.824 <0.001

vs. Ⅰ-Ⅱ) MiRNA-126 1.607 1.090-2.370 0.017 1.554 1.050-2.301 0.028

(negative vs.

positive) Abbreviations and Note: 95%CI, 95% confidence interval; CCI, Charlson comorbidity index; Other approach include Ivor Lewis approach+cervical incision, Sweet approach+cervical incision and Minimally invasive esophagectomy.

Supplementary Table S4. A short list of functionally relevant genes significantly downredulated in 126- EC cells vs. NC-EC cells.

G e n e S y m b o l Gene Description miR-126/control TargetScan Pictar miRanda miRDB

style p-

4 value(<0.05)

ADAM9 ADAM metallopeptidase domain 9 down 6.68E-58 + + + +

PIK3R2 phosphoinositide-3-kinase, regulatory subunit 2 (beta) down 1.67E-37 - - + -

SYDE1 synapse defective 1, Rho GTPase, homolog 1 (C. elegans) down 7.12E-17 - - + -

GPR64 G protein-coupled receptor 64 down 8.87E-07 - - - +

CHST11 carbohydrate (chondroitin 4) sulfotransferase 11 down 1.24E-05 - - + -

TMEM128 transmembrane protein 128 down 1.63252E-05 - - - +

IGSF9B immunoglobulin superfamily, member 9B down 5.09E-05 - - + -

OPLAH 5-oxoprolinase (ATP-hydrolysing) down 5.83E-05 - - + -

PAG1 phosphoprotein associated with glycosphingolipid microdomains down 7.27E-05 - - + -

LARP6 La ribonucleoprotein domain family, member 6 down 0.000283627 - - + -

KSR1 kinase suppressor of ras 1 down 0.001202221 - - + +

PIK3CD phosphoinositide-3-kinase, catalytic, delta polypeptide down 0.001629352 - - + -

CALHM3 calcium homeostasis modulator 3 down 0.001769405 - - - +

IGF2BP2 insulin-like growth factor 2 mRNA binding protein 2 down 0.00193895 - - + -

ZXDA zinc finger, X-linked, duplicated A down 0.002413057 - - - +

MYH11 myosin, heavy chain 11, smooth muscle down 0.005472495 - - + -

ZBTB7C zinc finger and BTB domain containing 7C down 0.005873784 - - - +

ZC3H12B zinc finger CCCH-type containing 12B down 0.006536688 - - + +

LRP1B low density lipoprotein receptor-related protein 1B down 0.011267139 - - - +

SLC12A3 solute carrier family 12 (sodium/chloride transporters), member 3 down 0.013367434 - - + -

TMEM40 transmembrane protein 40 down 0.014538934 - - + -

HIST1H2BN histone cluster 1, H2bn down 0.018131111 - - - +

APC2 adenomatosis polyposis coli 2 down 0.020848075 - - + -

ZNF333 zinc finger protein 333 down 0.020848075 - - - +

RAPGEF3 Rap guanine nucleotide exchange factor (GEF) 3 down 0.02449727 - - - +

TCL6 T-cell leukemia/lymphoma 6 (non-protein coding) down 0.024841435 - - + -

UCN2 urocortin 2 down 0.027078987 - - + -

PIGR polymeric immunoglobulin receptor down 0.027964082 - - + -

SOBP sine oculis binding protein homolog (Drosophila) down 0.043127863 - - + -

GJB2 gap junction protein, beta 2, 26kDa down 0.043433224 - - - +

ENO4 enolase family member 4 down 0.047004076 - - - +

5 +: genes presented as potential targets of miR-126 in TargetScan, PicTar, miRanda or miRDB. Down: genes downregulated in miR-126 overexpressed EC-109 cells vs. control EC-109 cells.

Supplementay Table S5. Primer sequences (5’-3’).

6 PCR primers for Methylation-specific PCR (MSP) methylated forward GGGTTTGAAAGTTAGTAAAGGGTT Egfl7 reverse GCGACCCGCCAACACC Unmethylated forward TGGGGGGTTTGAAAGTTAGTAA Egfl7 reverse ACCTCTAAACCACAACCCAC Primers for ADAM9-UTR and DNMT1-UTR clones ADAM9 forward GCTCTAGAGTATTCACGCAGTTACTCGCT

reverse TATAGGCCGGCCCTTCCTTCTTTCTCCAGCCT

DNMT1 forward GCTCTAGA CTCCCGTCACCCCTGT

reverse TATAGGCCGGCCAAGTCTTAATTTCCACTCATACAG Primers for real time PCR

ADAM9 forward GCTAGTTGGACTGGAGATTTGG

reverse GTGGCTCCTTGAACACACTG

DNMT1 forward TCTACCAGGGAGAAGGACAGG

reverse CTCACAGACGCCACATCGC

PIK3R2 forward AATGCAGCAAGGAATACCTGG

reverse GCTCTCATGGATCTCGGCAA

VEGFA forward CAGAAGGAGGAGGGCAGAAT

reverse CTCGATTGGATGGCAGTAGC

IRS1 forward CTGCACAACCGTGCTAAGG

reverse CGTCACCGTAGCTCAAGTCC

Crk forward GCGGAGTAGCTGGTACTGG

reverse GCGCGAGTTCTCTGAGACG

GAPDH forward TGCACCACCAACTGCTTAGC

reverse GGCATGGACTGTGGTCATGAG siRNA sequence si-DNMT1 sense CGAGUUGCUAGACCGCUUCTT

antisense GAAGCGGUCUAGCAACUCGTT si-ADAM9-1 sense CCAAUGGAAACCUGAUCAA dTdT (S1) antisense dTdT GGUUACCUUUGGACUAGUU si-ADAM9-2 sense CCAGAAUAACAAAGCCUAU dTdT (S2) antisense dTdT GGUCUUAUUGUUUCGGAUA si-ADAM9-3 sense GCGAAGGAAGUACCUGUAA dTdT (S3) antisense dTdT CGCUUCCUUCAUGGACAUU

7 Supplementary Table S6. The primary antibodies for western blot and IHC.

Antibody Concentration Concentration Specificity Company Catalog #

for WB for IHC

DNMT1 1:1000 / Rabbit monoclonal CST 5032S

PIK3R2 1:1000 / Rabbit polyclonal Abcam ab71052

ADAM9 1:1000 / Rabbit polyclonal CST 2099S

/ 1:100 Rabbit polyclonal Abcam ab186833

PEGFR 1:1000 / Rabbit monoclonal Epitomics 1727-1

(Abcam) (ab40815)

EGFR 1:1000 / Rabbit monoclonal Epitomics 2116-1

(Abcam) (ab76153) PAKT 1:1000 / Rabbit monoclonal Epiomics 2118-1

(Abcam) (ab81283) AKT 1:1000 / Rabbit monoclonal Epitomics 1085-1

(Abcam) (ab32038)

HRP-GAPDH 1:10000 / Mouse monoclonal KangChen KC-5G5

Abbreviations: WB, western blot; IHC, immunohistochemistry; CST, Cell Signaling Technology