DKK1
TGFBR2
PPARA
LEFTY1
BTG1
BMPR2
TIMP3
RBL1
TP63
RUNX1
PKD1
RUNX3
PDCD4
RB1
CD44 / Dickkopf-1
Transforming growth factor beta receptors Ⅱ
Peroxisome proliferator-activated receptor alpha
Left-right determination factor 1
B-cell translocation gene 1
Bone morphogenetic protein receptor, type II
T-cell lymphoma invasion and metastasis 1
Retinoblastoma-like 1 (p107)
Tumor protein p63
Runt-related transcription factor 1
Polycystic Kidney Disease 1
Runt-related transcription factor 3
Programmed cell death 4
Retinoblastoma 1
CD44 adhesion molecule / Antagonist of the Wnt signaling; tumor suppressor
Tumor suppressor; antagonist of the Wnt signaling
Can act as a tumor suppressor
Activated by Oct4 and Sox2 in ESCs; antagonizing the Wnt and Nodal signaling; important in stem cell control and development
Antiproliferation; tumor suppressor; regulated by the Wnt signaling
Act as a classic tumor suppressor gene
Tumor suppressor in colon cancer; Wnt signaling inhibitor
Tumor suppressor; interact with the Wnt network
Tumor suppressor
Interacts with the canonical Wnt signaling; classical tumor suppressor
Inhibits tumor cell migration and invasion; inhibits the Wnt signaling.
Tumor suppressor; negatively regulates the Wnt signaling
Tumor suppressor; negatively regulates Wnt-dependent transcription.
Tumor suppressor; interacts with the Wnt signaling pathway
Known target of miR-373.
Supplementary Table 1Candidate targets of miR-372/373 selected for validating by dual
luciferase assay.
Supplementary Table 2 Oligonucleotidesused for promoter reporter constructs, EMSA, 5’ and 3’RACE, chromatinimmunoprecipitation (ChIP) and lentiviral constructs.
Experiment / Sequence (5’-3’)Promoter constructs
(The restriction sites of Kpn I and HindⅢ are underlined) / F1: GGGGTACCAGAGATCATCCAGGTGGTTC (KpnI)
F2: GGGGTACCTTTGAGTTCCTCTCTATGCC
F3: GGGGTACCAGTGCAGTGGTGTGATCTCG
F4: GGGGTACCTGTGTCTGACTAAGGCAAGC
F5: GGGGTACCGAATGAGAGGGTGGGAAGGG
F6: GGGGTACCAGTTGAGTGGGCTCTCACCC
F0: CCCAAGCTTGCAATACTCACAGAGGCCAA (HindⅢ)
5’RACEand3’RACE / 5-Race-outer: CAAGCGGTAACACTCAAAAGATG
5-Race-inner: AAAAATCACAGAAATAGAACAAAA
3-Race-outer: CTGGGCGACAGAGCAAGACT
3-Race-inner: TTGAGTAGGGCATCACGAACCAT
EMSA andsite-directedmutagenesis
(TCF/LEF1 binding elements are underlined. The mutant nucleotides in the TCF/LEF1 binding elementsare in shadow) / TBE1-wt-F: AAGATCAAAGCAAGAATTTT
TBE1-wt-R: AAAATTCTTGCTTTGATCTT
TBE1-mt-F: AAGACTAAGCCAAGAATTTT
TBE1-mt-R: AAAATTCTTGGCTTAGTCTT
TBE3-wt-F: AGGGTTCAAAGGATTCTCCC
TBE3-wt-R: GGGAGAATCCTTTGAACCCT
TBE3-mt-F: AGGGTCTAAGCGATTCTCCC
TBE3-mt-R: GGGAGAATCGCTTAGACCCT
TBE4-wt-F: AAGCTAGGATCAAAGGGAGC
TBE4-wt-R: TGCTCCCTTTGATCCTAGCT
TBE4-mt-F: AAGCTAGGATTCAATGGAGC
TBE4-mt-R: TGCTCCATTGAATCCTAGCT
Chromatin immunoprecipitation / CHIP-TBE1-F: AGGTGGTTCACAAATAGCAG
CHIP-TBE1-R: ACTCCAGCCTGGGTGATAGA
CHIP-TBE3-F: AATGTTCACTGCCGCACTGT
CHIP-TBE3-R: ACACCTATAATCCCTGCTAC
CHIP-TBE4-F: GAGTGGATGACTGGTGGAAT
CHIP-TBE4-R: ATAGGATTAAAGCCCACCCC
CHIP-Myc-F: CCC AAA AAAAGGCACGGA A
CHIP-Myc-R: TATTGGAAATGCGGTCATGC
CHIP-neg-F: ACGGCCAACATGCTCAGG
CHIP-neg-R: TGTTTGCAACTGCTGCGTTAG
Lentiviral constructs
(The restriction sites ofEcoR IandBamH Iare underlined) / Pre-miR-372-F: CGGAATTCAACCTGCGGAGAAGATACCA (EcoR I)
Pre-miR-372-R: CGGGATCCTCCTTTCACCATCTAACCCA (BamH I)
Pre-miR-373-F: CGGAATTCCGACAGAGCAAGACTCATTC (EcoR I)
Pre-miR-373-R: CGGGATCCCTAAAATGGAGCATACAGAC (BamH I)
shDKK1-F1:
GATCCGGAATAAGTACCAGACCATTCTTCCTGTCAGAAATGGTCTGGTACTTATTCCTTTTTG
shDKK1-R1:
AATTCAAAAAGGAATAAGTACCAGACCATTTCTGACAGGAAGAATGGTCTGGTACTTATTCCG
shDKK1-F2: GATCCAGGTGCTGCACTGCCTATTCTTCCTGTCAGAAATAGGCAGTGCAGCACCTTTTTTG
shDKK1-R2:AATTCAAAAAAGGTGCTGCACTGCCTATTTCTGACAGGAAGAATAGGCAGTGCAGCACCTG
Supplementary Table 3 Primers for reverse transcriptasePCR and quantitativereal-time PCR.
Primer / Sequence (5’-3’)miR-371-3p / RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTAT TCGCACTGGATACGACACACTC
Forward: ACCGCTAAGTGCCGCCATCTTTTG
miR-371-5p / RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTGCC
Forward: GCCGCCACTCAAACTGTGGGG
miR-372 / RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGCTC
Forward: GGTCATAAAGTGCTGCGACATTTG
miR-373 / RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACACCC
Forward: TTCATGAAGTGCTTCGATTTTGG
Universe miRNA Reverse primer / miR-REV: GTGCAGGGTCCGAGGT
U6 / Forward: CGCTTCGGCAGCACATATAC
Reverse: ACGGCCAACATGCTCAGG
cyclin D1 / Forward: AACTACCTGGACCGCTTCCT
Reverse: CCACTTGAGCTTGTTCACCA
c-Myc / Forward: TGGTCTTCCCCTACCCTCTCAAC
Reverse: GATCCAGACTCTGACCTTTTGCC
c-Jun / Forward: ACGGCCAACATGCTCAGG
Reverse: TGTTTGCAACTGCTGCGTTAG
CTNNB1 / Forward: GAGCCTGCCATCTGTGCTCT
Reverse: ACGCAAAGGTGCATGATTTG
DKK1 / Forward: CGGGAATTACTGCAAAAATGGA
Reverse: GCACAGTCTGATGACCGGAGA
TGFBR2 / Forward: ATTTTCCACCTGTGACAACC
Reverse: ATTGCACTCATCAGAGCTAC
BTG1 / Forward: AGGAGCTGCTGGCAGAACATT
Reverse: TGCTCAGTCCAATCCGCTGTG
Beta-actin / Forward: GAAACTACCTTCAACTCCATC
Reverse: CGAGGCCAGGATGGAGCCGCC
Supplementary Table 4 Oligonucleotidesusedfor the construction of 3’ UTR reporter plasmids.Overhangs after digestionby XhoI (CTCGAG) orNot I(CGCCGGCG)arein bold. Nucleotides complementary to the seed sequence of miR-372373 are underlined. The mutant nucleotides in the mutant-type oligonucleotidesare in shadow.
Target / Oligo sequence (5’-3’)DKK1 / WT / F: TCGAGTAAATTACTGTATTGTAAATTCTCAGTGTGGCACTTACCTGTAAATGCAATGC
R: GGCCGCATTGCATTTACAGGTAAGTGCCACACTGAGAATTTACAATACAGTAATTTAC
MT / F: TCGAGTAAATTACTGTATTGTAAATTCTCAGTGTCTTAAACCCCTGTAAATGCAATGC
R: GGCCGCATTGCATTTACAGGGGTTTAAGACACTGAGAATTTACAATACAGTAATTTAC
TGFBR2 / WT / F: TCGAGTATATACTCTTTTTATATCAAAAGTCTCAAGCACTTATTTTTATTCTATGCGC
R: GGCCGCGCATAGAATAAAAATAAGTGCTTGAGACTTTTGATATAAAAAGAGTATATAC
MT / F: TCGAGTATATACTCTTTTTATATCAAAAGTCTCACTGAAACCTTTTTATTCTATGCGC
R: GGCCGCGCATAGAATAAAAAGGTTTCAGTGAGACTTTTGATATAAAAAGAGTATATAC
LEFTY1 / WT / F: TCGAGATTCTTATTATTCACTGCACTATATTCTAAGCACTTACATGTGGAGATACTGC
R: GGCCGCAGTATCTCCACATGTAAGTGCTTAGAATATAGTGCAGTGAATAATAAGAATC
MT / F: TCGAGATTCTTATTATTCACTGCACTATATTCTACTTAAACCCATGTGGAGATACTGC
R: GGCCGCAGTATCTCCACATGGGTTTAAGTAGAATATAGTGCAGTGAATAATAAGAATC
BTG1 / WT / F: TCGAGGTACTGGAAATTTTACAAGACTTTTACCTAGCACTTAAATATGTATAAATGGC
R: GGCCGCCATTTATACATATTTAAGTGCTAGGTAAAAGTCTTGTAAAATTTCCAGTACC
MT / F: TCGAGGTACTGGAAATTTTACAAGACTTTTACCTCTTAAACCAATATGTATAAATGGC
R: GGCCGCCATTTATACATATTGGTTTAAGAGGTAAAAGTCTTGTAAAATTTCCAGTACC
CD44 / WT / F:TCGAGAAGCATGATATGTATATTGCTGAGTTGAAAGCACTTATTGGAAAATATTAAGC
R:GGCCGCTTAATATTTTCCAATAAGTGCTTTCAACTCAGCAATATACATATCATGCTTC
MT / F: TCGAGAAGCATGATATGTATATTGCTGAGTTGAACCTAAACCTTGGAAAATATTAAGC
R: GGCCGCTTAATATTTTCCAAGGTTTAGGTTCAACTCAGCAATATACATATCATGCTTC