Kozar et al. 1
Cross-linking ligation and sequencing of
hy-brids (qCLASH) reveals an unpredicted miRNA
targetome in melanoma cells
Ines Kozar
1, Demetra Philippidou
1, Christiane Margue
1, Lauren A. Gay
2,
Rolf Renne
2, Stephanie Kreis
11 Department of Life Sciences and Medicine, University of Luxembourg, 6, avenue du Swing,
L-4367 Belvaux, Luxembourg
2 Department of Molecular Genetics and Microbiology, University of Florida, 1200 Newell
Drive, Gainesville, FL 32610, USA
A
D
A375_XP A375_miR509 Mel501
AX1 AX2 AX3 A509_1 A509_2 A509_3 M1 M2 M3
0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 Biological replicates Propor tion of miRNA − mRNA h ybr ids Binding Region 3'UTR 5'UTR 5'UTR−CDS CDS CDS−3'UTR unknown
C
B
miR−17 miR−24 miR−320a miR−22 miR−23a miR−31 miR−125b miR−16 miR−92a let−7a 0 25 50 75 Number of hybrids Binding Region 3'UTR 5'UTR 5'UTR−CDS CDS CDS−3'UTR Top 10 miRNAs in A375_miR509E
F
G
miR−100 miR−31 miR−17 miR−22 miR−24 miR−92a miR−125b miR−23a miR−16 let−7a 0 50 100 Number of hybrids Binding Region 3'UTR 5'UTR 5'UTR−CDS CDS CDS−3'UTR Top 10 miRNAs in 501MelmiR−320a miR−17 miR−24 miR−31 miR−125b miR−22 miR−23a miR−92a miR−16 let−7a 0 25 50 75 100 125 Number of hybrids Binding Region 3'UTR 5'UTR 5'UTR−CDS CDS CDS−3'UTR Top 10 miRNAs in A375_XP
A375_miR509 A509_1 A375_miR509 A509_2 A375_miR509 A509_3 A375_XP AX1 A375_XP AX2 A375_XP AX3 Mel501 M1 Mel501 M2 Mel501 M3 All 5pr ime 3pr ime All 5pr ime 3pr ime All 5pr ime 3pr ime All 5pr ime 3pr ime All 5pr ime 3pr ime All 5pr ime 3pr ime All 5pr ime 3pr ime All 5pr ime 3pr ime All 5pr ime 3pr ime 0.00 0.25 0.50 0.75 1.00 Biological replicates Propor tion of miRNA − mRNA h ybr ids Seed match 2−7 0mm 2−8 0mm 2−8 1mm 2−8 2mm Other A375_XP AX1 A375_XP AX2 A375_XP AX3 A375_miR509 A509_1 A375_miR509 A509_2 A375_miR509 A509_3 Mel501 M1 Mel501 M2 Mel501 M3 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 2 − 7 0mm 2 − 8 0mm 2 − 8 1mm 2 − 8 2mm Other 0.00 0.25 0.50 0.75 1.00 Biological replicates Propor tion of miRNA − mRNA h ybr ids Strength Strong Moderate Weak Absent 374 378 183 227 332 143 424 342 214
A375_XP A375_miR509 Mel501
AX1 AX2 AX3
A509_1 A509_2 A509_3
M1 M2 M3 0 100 200 300 400 0 100 200 300 0 100 200 300 Biological replicates Number of miRNA − mRNA h ybr ids
Figure S1: (Caption on next page.)
Kozar et al. 3
Figure S1: (Previous page.) miRNA-mRNA hybrid data of additional melanoma cells. (A) Number of miRNA-mRNA hybrids identified in the three biological replicates of additional BRAF-mutant melanoma cell lines. A375 XP (Vemurafenib-resistant) and A375 miR509 (over-expressing miR-509) are cells derived from the parental cell line A375. 501Mel is an independent highly pigmented BRAF-mutant melanoma cell line. (B) Propor-tion of miRNA-mRNA hybrids across three biological replicates of the different melanoma cells with miRNA binding sites mapped to the mRNA sequence and the number of identified hybrid sequences in each replicate. (C) The type of seed pairing (5’ miRNA sequence) was divided into classes consisting of nt 2-7 at 5’ end of miRNA with zero mismatches (2-7 0mm), nt 2-8 with zero mismatches (2-8 0mm), one mismatch (2-8 1mm), or two mismatches (2-8 2mm), and the remaining binding modes were classified as other. The proportion of miRNA-lncRNA hybrids across the three biological replicates and for all hybrids, 5’ and 3’ hybrids was plotted for the different seed categories. (D) miRNA base-pairing via the 3’ portion of the miRNA (supplementary 3’ or non-seed pairing). The strength of 3’ sequence binding was classified as strong (>8 bound nt), moderate (5-8 bound nt), weak (1-4 bound nt), and absent (zero bound nt) and was plotted for each seed sequence type across the biological replicates. Top 10 miRNAs that occur in miRNA-mRNA hybrids showing the number of hybrids detected and the corresponding binding region on the mRNA in (E) A375 XP cells, (F) A375 miR509 cells (stably transfected with miR509), and (G) 501Mel cells.
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
let−7a miR−100 miR−320a miR−34a
AXL CD68 CDK6 CIZ1 DICER1 ERBB3 FOSL1 INTS3 JUNB LRP8 MDM2 MDM4 MYC NF1 PKM2 RHOBTB3 RPS4X RPS6 RPS6KA3 SERPINE2 SPR Y2 SPR Y4 TUSC2 XYL T2 strong moder ate w eak absent strong moder ate w eak absent strong moder ate w eak absent strong moder ate w eak absent 2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
Binding Region ● ● ● ● 5'UTR 5'UTR−CDS CDS 3'UTR Average target mRNA expression ● ● ● ● ● 2500000 5000000 7500000 10000000 12500000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
let−7a miR−100 miR−320a miR−34a
AXL CD68 CDK6 CIZ1 DICER1 ERBB3 FOSL1 INTS3 JUNB LRP8 MDM2 MDM4 MYC NF1 PKM2 RHOBTB3 RPS4X RPS6 RPS6KA3 SERPINE2 SPR Y2 SPR Y4 TUSC2 XYL T2 strong moder ate w eak absent strong moder ate w eak absent strong moder ate w eak absent strong moder ate w eak absent 2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
2−7 no mismatches 2−8 no mismatches2−8 one mismatch 2−8 two mismatchesother
Binding Region ● ● ● ● 5'UTR 5'UTR−CDS CDS 3'UTR Average target mRNA expression ● ● ● ● ● 2500000 5000000 7500000 10000000 12500000
Figure S2: (Caption on next page.)
Kozar et al. 5
Figure S2: (Previous page.) Overview of binding characteristics of miRNA-mRNA hybrids. Selected miRNA-mRNAs with the corresponding mRNA expression levels in melanoma patient data (TCGA2015). The binding characteristics (seed match, supplemental 3’ pairing or strength, and binding region on mRNA) are illustrated as well as the average target mRNA expression levels.
A
B
Figure S3: (Caption on next page.)
Kozar et al. 7
Figure S3: (Previous page.) Expression of selected genes in melanoma patient TCGA data. Expression of selected genes found in hybrids in TCGA melanoma patient data according to (A) tissue origin (normal solid tissue, primary solid tumor, metastatic tumor) and (B) mutational subtype (BRAF-mutant, NF1-mutatnt, RAS-mutant, or triple wild type melanoma tumours).
A
B
let−7a−5p miR−320a−3p miR−100−5p miR−34a−5p
NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p 0 2000 4000 0 50 100 0 5000 10000 15000 20000 0 1000 2000 3000 F old Change Condition 48h_25nM 48h_50nM 72h_25nM 72h_50nM
SPRY2 SPRY4 TUSC2
RPS4X RPS6 RPS6KA3 SERPINE2
MDM4 MYC PKM2 RHOBTB3
FOSL1 INTS3 LRP8 MDM2
AXL CD68 CIZ1 ERBB3
NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p 0.0 0.4 0.8 1.2 0.0 0.5 1.0 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 1.5 0.0 0.3 0.6 0.9 1.2 0.0 0.5 1.0 1.5 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 F old Change Condition 48h_25nM 48h_50nM 72h_25nM 72h_50nM
SPRY2 SPRY4 TUSC2
RPS4X RPS6 RPS6KA3 SERPINE2
MDM4 MYC PKM2 RHOBTB3
FOSL1 INTS3 LRP8 MDM2
AXL CD68 CIZ1 ERBB3
NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p NCM let − 7a − 5p miR − 320a − 3p miR − 100 − 5p miR − 34a − 5p 0.0 0.4 0.8 1.2 0.0 0.5 1.0 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 1.5 0.0 0.3 0.6 0.9 1.2 0.0 0.5 1.0 1.5 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.00 0.25 0.50 0.75 1.00 1.25 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 F old Change Condition 48h_25nM 48h_50nM 72h_25nM 72h_50nM
Figure S4: (Caption on next page.)
Kozar et al. 9
Figure S4: (Previous page.) Expression of selected target mRNAs following miRNA mimic transfection. (A) The expression of four selected miRNAs in A375 cells transfected with 25nM and 50nM miRNA mimic for 48h and 72h compared to cells treated with negative control mimic (NCM). Represented are the mean and standard deviation of three technical replicates (n=1). (B) The expression fold change of selected target mRNA upon miRNA mimic transfection was normalised to the corresponding negative control mimic (NCM) in each condition (25nM and 50nM mimic transfection for 48h and 72h). Represented are the mean and standard deviation of three technical replicates.
NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 CD68 Fold change ns ns * **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change CIZ1 ** ns ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change INTS3 ns ns ns *
IGR37
NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change FOSL1 *** ns *** ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change DICER1 **** * *** *** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change JUNB ns ns ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change LRP8 ns * * ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change MDM4 ns ** ns ** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change ERBB3 ns ns ns *** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change MYC * ** ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change NF1 ns ns ns * NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change SPRY4 ns ns *** **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change MDM2 * ns ** **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change PKM2 *** ns * *** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change RPS4X ns * ** *** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change TUSC2 ns ns ns ** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 2.5 Fold change RHOBTB3 * ns ns **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change RPS6 ns ns ns * NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0 2 4 6 Fold change RPS6KA3 * ns ** **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change SERPINE2 ns ns * **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 2.5 Fold change SPRY2 ns ns *** ****A
Figure S5: (Caption on next page.)
Kozar et al. 11
501Mel
NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change CD68 **** ns **** **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change CIZ1 ns ns ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change INTS3 ns ns ns * NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0 1 2 3 Fold change FOSL1 *** ns ** ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change DICER1 ** ns * ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change JUNB ns ns ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change LRP8 ns * ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change MDM4 ns ** ns ns NCM let-7 a-5p mim ic miR -100 -5p m imic miR -320 a-3p mim ic miR -34a -5p m imic 0 1 2 3 Fold change ERBB3 ns ns **** **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change MYC ns *** ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change NF1 ns ns ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change SPRY4 ns ns ns *** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0 1 2 3 Fold change MDM2 ns * **** **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change PKM2 ns ns ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change RPS4X ns ns **** ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change TUSC2 ns ns ns ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 2.5 Fold change RHOBTB3 ns ns ns *** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change RPS6 ns ns ** ns NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 Fold change RPS6KA3 ns ns ns ** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0 1 2 3 4 Fold change SERPINE2 * ns ** **** NCM let-7 a-5p mim ic miR -100 -5p mim ic miR -320 a-3p mim ic miR -34a -5p mim ic 0.0 0.5 1.0 1.5 2.0 2.5 Fold change SPRY2 ns ns **** ***B
Figure S5: (Caption on next page.)
Figure S5: (Previous page.) Expression of selected target mRNAs following trans-fection with 4 different miRNA mimics in IGR37 (A) and 501Mel (B). The expression levels were normalized to the corresponding negative control mimic (NCM). Illustrated are the mRNA expression levels of 21 genes upon 50nM mimic treatment for 72h. Statistical sig-nificance was determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test with ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Kozar et al. 13 MIR503HG MIR34AHG MIR3142HG MIR29B2CHG MIRLET7BHG A1 A2 A3 MIR191 MIR191 MIR125B1 MIR425 MIR197 MIR15B MIR30B MIR222 MIR125B1 MIRLET7A2 MIR100 MIR191 MIR21 MIR29A MIR6087 MIR574 MIRLET7F2 MIR125A MIR140 MIR320A MIR26A1 MIR30D MIRLET7G MIR25 MIR181A1 MIRLET7D MIR27B MIR99A MIR221 MIR93 MIR30E MIR130A MIR15B MIR26B MIR182 MIR30B MIR30C1 MIR222 MIR125B1 MIRLET7A2 MIR100 MIR191 MIR21 MIRLET7I MIR29A MIR6087 MIR425 MIR574 MIRLET7F2 MIRLET7A1 MIR125A MIR197 MIR140 MIR374B MIR1307 MIR30A MIR125B2 MIR320A MIR26A1 MIR30D MIRLET7G MIR16−2 MIR193A MIR99B MIR204 MIR374A MIR342 MIR25 MIR181A1 MIRLET7F1 MIRLET7D MIR27B MIR10B MIR99A MIRLET7C MIR221 MIR103A2 MIR31 MIR32 MIR320D1 MIR151A MIR193B MIR93 MIR23B MIR30E MIR130A MIR15B MIR455 MIR935 MIRLET7E MIR3620 MIR106B MIR328 MIR199B MIR484 MIR128−1 MIR196A2 MIR331 MIR423 MIR4286 MIR6511A2 MIR181A2 MIR320C1 MIR324 MIR671 MIR615 MIR6511B1 MIR98 MIR138−1 MIR185 MIR2110 0 30 60 90 150 100 50 0 60 40 20 0
Number of occurences in miRNA−lncRNA hybrids
MIR3 14 2HG MIR3 4A HG MIR5 03 HG
Figure S6: (Caption on next page.)
Figure S6: (Previous page.) Detailed overview of pri-miRNA-miRNA interac-tions. Detailed overview of miRNAs that have been found to interact with the most fre-quent pri-miRNAs (lncRNAs: MIRLET7BHG, MIR29B2CHG, MIR3142HG, MIR503HG, MIR34AHG). The number of occurrences of the corresponding miRNAs in miRNA-lncRNA hybrids is illustrated across three biological replicates and pri-miRNA genes.
