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Supporting Information
Supplementary Figures S1-S11
Figure S1. Overexpression of miR-183-96-182 cluster miRNAs in U2OS cells.
Figure S2. Effects of miR-183-96-182 cluster miRNAs on cisplatin-induced foci formation of RAD51, RPA2, FANCD2 and BRCA1.
Figure S3. Overexpression of miR-96 sensitizes cancer cells to cisplatin and AZD2281.
Figure S4. Overexpression of miR-96 sensitizes HR-deficient cancer cells to cisplatin.
Figure S5. MiR-96 is highly conserved across species.
Figure S6. Conservation of miR-96 binding sites and surrounding regions in the transcripts of REV1 and RAD51.
Figure S7. Combinational treatment with miR-96 and cisplatin delayed tumor growth.
Figure S8. Effects of miR-183-96-182 cluster miRNA mimics on cellular sensitivity to cisplatin and AZD2281.
Figure S9. Effects of IR on the expression of miR-183-96-182 cluster miRNAs.
Figure S10. Effect of miR-183-96-182 cluster miRNAs on BRCA1 and REV1 expression.
Figure S11. Effect of miR-183-96-182 cluster miRNAs on cell growth.
Supplementary Tables S1-S2
Table S1. Top 100 predicted targets of miR-96 by each miRNA prediction algorithm.
Table S2. Predicted miR-96 targets by at least two prediction algorithms
Figure S1. Overexpression of miR-183-96-182 cluster miRNAs in U2OS cells. (A) Alignment of miR-183-96-182 cluster miRNAs. (B) U2OS cells were transfected with miRNA mimics and harvested for RNA extraction and real-time RT-PCR two days later.
Figure S2. Effects of miR-183-96-182 cluster miRNAs on cisplatin-induced foci formation of RAD51, RPA2, FANCD2 and BRCA1. U2OS cells were transfected with individual miRNA mimics. Two days later, cells were exposed to 2 µM cisplatin for 24 hours and then fixed for immunofluorescent staining. Quantitation of relative percentage of cells with at least 10 foci (normalized to miR-neg-transfected cells) is shown.
Figure S3. Overexpression of miR-96 sensitizes cancer cells to cisplatin and AZD2281. (A-C) HeLa and PEO1 C4-2 (C4-2) cells were transfected with miRNA mimics. Two days later, cells were re-seeded for survival assays (A-B) or processed for western blot analysis (C). (D) HeLa cells transfected with miRNA mimics or BRCA2 siRNA were plated for clonogenic cisplatin sensitivity assay (n=4, mean+/-SEM). Representative images of crystal violet-stained colonies are also shown (Note: 525 and 1050 cells/well were seeded for miR-neg and miR-96, respectively.) (E-G) U2OS cells were infected with lentivirus expressing non-targeting control (NSC) or miR-96. Cells were prepared for real-time RT-PCR, western blot (E) or survival assays (F-G). (H) U2OS cells transfected with miRNA mimics were plated for paclitaxel sensitivity assay.
Figure S4. Overexpression of miR-96 sensitizes HR-deficient cancer cells to cisplatin. (A-C) BRCA1-deficient HCC1937 breast cancer cells were infected with lentivirus producing miR-96 or shREV1. Two days later, cells were processed for either survival assays or western blot analysis. (D-F) BRCA2-deficient PEO1 ovarian cancer cells were transfected with miRNA mimics or siRNAs as indicated. Two days later, cells were harvested for survival assays or western blot analysis.
Figure S5. MiR-96 is highly conserved across species. Sequences in box are mature miR-96 sequence.
Figure S6. Conservation of miR-96 binding sites and surrounding regions in the transcripts of REV1 and RAD51. (A) The miR-96 binding site (8-mer) within REV1 3’UTR is highly conserved across species. Numbers (127-177) indicates the sequences included from human REV1 3’-UTR. (B) The miR-96 binding site (7-mer) inside RAD51 coding region is conserved in vertebrates. Numbers (986-1039) indicates the sequences included from human RAD51 coding sequence.
Figure S7. Combinational treatment with miR-96 and cisplatin delayed tumor growth. MDA-MB-231 cells stably expressing either control (NSC) or miR-96 (pLe-miR-96) were injected subcutaneously into the flank of mice. When tumor grew up to around 40-50 mm3, mice were treated with either PBS or cisplatin. Tumor volume was monitored and the time of tumor volume reaching 200 mm3 was calculated as described in materials and methods. (The graph is a part of the graph shown in Figure 6D.)
Figure S8. Effects of miR-183-96-182 cluster miRNA mimics on cellular sensitivity to cisplatin and AZD2281. BRCA2-proficient PEO1 C4-2 ovarian cancer cells were transfected with miRNA mimics. Two days later, cells were re-seeded for survival assays.
Figure S9. Effects of IR on the expression of miR-183-96-182 cluster miRNAs. U2OS cells were irradiated with 5 Gy IR and harvested at indicated time for quantitative RT-PCR analysis.
Figure S10. Effect of miR-183-96-182 cluster miRNAs on BRCA1 and REV1 expression. U2OS cells were transfected with miRNA mimics. Two days later, cells were harvested for western blot analysis.
Figure S11 Effect of miR-183-96-182 cluster miRNAs on cell growth. U2OS cells were transfected with miRNA mimics and then re-seeded at 1x104 cells/well in 12-well for 5 days. Cells were fixed and stained with crystal violet. Relative cell growth was determined by the amount of absorbed dye that was resolubilized and measured spectrophotometrically. * p < 0.05
Supporting Tables
Table S1. Top 100 predicted targets of miR-96 by each miRNA prediction algorithm.
Gene name MiRanda TargetScan PicTar
LRCH2 + + +
RGPD2 +
RGPD1 + +
CMAS +
SLC1A1 + + +
ADCY6 + + +
DPY19L3 + +
ADK + +
JMJD1C + + +
DGKH +
LRRC3 +
NOVA1 + +
FLJ43390 +
HLA-F +
CYYR1 +
AK3 + +
LPPR4 + + +
SPRY3 + +
RDH11 + +
C17orf104 +
GAD2 + +
SOX5 + + +
ARHGAP6 + +
PTPMT1 +
MTRR + +
LOC646241 +
MSN + +
PCDH11X +
PRRX1 + +
OXGR1 + +
COL25A1 +
LOC339862 +
PCDH11Y +
CAMK2N1 + +
LIPG +
FOXF2 + + +
HERC2P2 +
LPHN2 +
ZFP36L1 + + +
MDM2 +
MTSS1 + + +
SPIN1 + +
ANKRD32 +
ITPR1 + + +
NTN4 + +
GPC6 +
RAPGEF4 + +
DNAJC30 +
SDC2 + +
ZNF175 + +
LRRC7 + +
MYRIP + +
UNC13C + +
NEUROD4 + +
PROK2 + +
KDM5D +
WISP3 +
CNOT6L +
DYNLT3 +
SLC39A1 + +
TBX5 +
STK17A + +
C14orf169 +
ST7OT3 +
RAB35 +
REV1 + + +
CLSPN +
FN1 +
ANKRD27 + +
PHF20L1 +
GPC3 + +
ZCCHC11 + +
GPR135 + +
FAM49B +
GAP43 +
CCDC67 + +
NIPBL +
SPAG17 + +
CHST1 + +
SPEN +
SH3BP5 + + +
CELF2 +
ZCCHC3 + +
FRAP1 +
TAC1 +
MORF4L2 +
KIAA0907 +
FHL1 +
BOD1L +
ABCC6P1 +
RNLS +
FOXO1 + +
BRPF3 + +
PSME4 +
KLHL7 +
RAD51 + (+)a
PRKCE + + +
LILRA1 +
PSMD10 +
HAS2 + + +
KIAA2022 +
SIN3B +
FRS2 +
DLAT +
SLC38A4 +
DEPDC1 +
SOX6 +
NLGN2 + +
GRHL2 +
SPAG8 +
TMEM170B +
MITF + +
RGS17 +
ZBTB41 +
HBEGF +
BRWD1 +
PRKAR1A + +
MTR +
SNX30 +
SPAST +
GLT8D3 +
CD164 +
HOOK3 +
CHIC1 +
MYO5B +
GNA13 +
ELTD1 +
EOMES +
CALCRL +
MED14 +
ULBP1 +
ATXN1 + +
DIXDC1 +
PLCB4 +
EPM2AIP1 +
KIAA2018 +
TMEM169 +
RAB8B +
INSIG2 + +
GLE1 +
DENND2C +
USP45 +
SH3KBP1 +
CRYGS +
ALKBH1 +
DMN +
C21orf91 +
PGAP1 +
PROSC +
LDB3 +
GJC1 +
ZKSCAN2 +
CTDSP1 +
KIAA1576 +
CGI-72 +
PRRG3 +
STK19 +
HTR4 +
DTR +
PBX2 +
DKFZP434J154 +
RARG +
RASA1 +
TTYH3 +
USP6 +
TOPORS +
FOXO3A +
DSIPI +
SLC1A2 +
FLJ22169 +
MGC23401 +
CBFA2T3 +
VAT1 +
GNAQ +
MTM1 +
TSK +
OSR1 +
FLJ90013 +
MYOHD1 +
GRK6 +
TFCP2L3 +
BRUNOL6 +
PPP3R1 +
ABCD1 +
CACNB1 +
PB1 +
C8orf13 +
ADD3 +
SLC6A6 +
LASS2 +
KIAA0992 +
HBP1 +
TENS1 +
HDAC9 +
DGCR2 +
AKAP1 +
CUL5 +
CACNA2D2 +
DKFZp761B1514
+
EDNRB +
LMNA +
APG16L +
MAP3K3 +
EIF5 +
OVOL1 +
SNX16 +
LUZP1 +
FAD104 +
TRIM46 +
DKFZp761H1710
+
DKFZp667B0210
+
RAB10 +
BCL2L12 +
INPP5A +
C6orf134 +
SPG4 +
TAF15 +
VAMP3 +
DLGAP2 +
TM9SF4 +
CAV1 +
PPM1F +
PRR3 +
PLOD3 +
RAFTLIN +
BNC2 +
RNF130 +
PFN1 +
GPRASP2 +
FLOT2 +
Note: a RAD51 is also a predicted target of miR-96 with lower score.
Table S2 Predicted miR-96 targets by at least two prediction algorithms.
Gene name MiRanda TargetScan PicTar LRCH2 + + + RGPD1 + + SLC1A1 + + + ADCY6 + + + DPY19L3 + + ADK + + JMJD1C + + + AK3 + + LPPR4 + + + SPRY3 + + RDH11 + + GAD2 + + SOX5 + + + ARHGAP6 + +
MTRR + + MSN + + OXGR1 + + CAMK2N1 + + FOXF2 + + + ZFP36L1 + + + MTSS1 + + + SPIN1 + + ITPR1 + + + NTN4 + + RAPGEF4 + + SDC2 + + ZNF175 + + LRRC7 + + MYRIP + + UNC13C + + NEUROD4 + + PROK2 + + SLC39A1 + + STK17A + + REV1 + + + ANKRD27 + + GPC3 + + ZCCHC11 + + GPR135 + +
CCDC67 + + SPAG17 + + CHST1 + + SH3BP5 + + + ZCCHC3 + + FOXO1 + + BRPF3 + + PRKCE + + + HAS2 + + + NOVA1 + + PRRX1 + +
NLGN2 + +
MITF + +
PRKAR1A + +
ATXN1 + +
INSIG2 + +
Note: Only top 100 predicted targets were included.
