1 2 3 4 5 6 7 8 Supplemental Materials and Methods Cell proliferation assay Cell proliferation was measured with Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). GCs were plated at 96-well plates at approximately 1 1.5 x 10 4 cells per well and cultured in the growth medium. After 48h transfection, each well were added with 10 μl of the Cell Counting Kit-8 solution and then incubated at 37 ºC for 2 h. Cell number assays were performed in a 96-well format plate reader (ELX 800 universal microplate Reader; BioTek, Inc., Highland Park, IL) by measuring the absorbance at a wavelength of 450 nm (OD450). 9 10 11 12 13 14 15 16 17 18 19 20 Supplemental Figures 1
21 2
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Supplemental Fig. S1. Expression and function of mir-383 in GCs. A, mir-383 expression levels in mouse ovarian preantral follicles. Total RNA was extracted from the follicles with different diameters and then subjected to real-time PCR. Data obtained from the follicles with 60 80 μm in diameter were arbitrarily set at 1.0. B, Hematoxylin and eosin (HE) staining of mouse ovarian tissues collected at 0, 4, 12, 24 and 48h post PMSG injection. C, Serum E 2 levels in PMSG-treated mice at 0, 4, 12, 24 and 48h. D, mir-383 expression levels in the ovaries from PMSG-treated mice at 0, 4, 12, 24 and 48h. E and F, The efficacy of mir-383 mimics or inhibitors was evaluated by real-time PCR. mgcs were transfected with the mir-383 mimics (120nM) (E) or mir-383 inhibitors (150nM) (F) for 48h. The intracellular expression levels of mir-383 were examined by real-time PCR. G, Both mir-383 mimics and inhibitors did not alter GC proliferation. mgcs were transfected with 120 nm each of mir-383 mimics and mimics NC, or 150nM each of mir-383 inhibitors and inhibitors NC for 24, 48 and 72h. Cell viability was measured by the cell counting kit-8 assay after transfection. H, mir-383 did not alter apoptosis of GCs. The apoptosis rate was measured using flow cytometry with Annexin V and PI staining after transfection either with 120 nm each of mir-383 mimics and mimics NC, or 150nM each of mir-383 inhibitors and inhibitors NC for 48h. I and J, mir-383 mimics enhanced Cyp19a1 protein expression, while knockdown of endogenous mir-383 suppressed Cyp19a1 protein expression in GCs. GCs were transfected with oligonucleotides for 48 h, and Cyp19a1 protein levels were determined by Western blotting. Representative Western blot for Cyp19a1 (upper bands) and GAPDH (lower bands) (I) and the corresponding densitometric analysis (J) are shown. The ratio of the Cyp19a1 band intensity over the GAPDH band intensity in controls was arbitrarily set at 1.0. K, Both mir-383 mimics and inhibitors did 42 not alter Cyp11a1 mrna expression levels. mgcs were transfected with oligonucleotides for 48 h. Total 3
43 44 45 46 47 RNA was extracted from GCs, and the Cyp11a1 mrna levels were determined by real-time PCR analysis. L, mir-383 expression levels in GCs, MEF and RAW164.7 cells. Total RNA was extracted from GCs, MEF and RAW164.7 cells and then subjected to real-time PCR. Data obtained from the MEF was arbitrarily set at 1.0. In A, C-H and J-L, data shown represent the mean ± S.E.M of three independent experiments performed in triplicate. ** P < 0.01. NC, negative control. 48 4
49 50 51 Supplemental Fig. S2. RBMS1 is a target of mir-383. A, Schematic diagram of the putative binding sites for mouse (mmu) mir-383 and human (hsa) mir-383 in the RBMS1 3 UTR. The mutated residues 52 of mir-383 binding sites are shown in red. RBMS1 3 UTR MT, mutation-type RBMS1 3 UTR. B-D, 5
53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 Hsa-miR-383 reduces RBMS1 protein (B and C) and mrna (D) expression in MCF-7 cells. MCF-7 cells were transfected with 120 nm each of mir-383 mimics and mimics NC, or 150 nm each of mir-383 inhibitors and inhibitors NC. The expression levels of RBMS1 mrna and protein were determined by real-time PCR and Western blotting analysis, respectively. Representative Western blot for RBMS1 (upper bands) and GAPDH (lower bands) (B) and the corresponding densitometric analysis (C) are shown. The ratio of the RBMS1 band intensity over the GAPDH band intensity in controls was arbitrarily set at 1.0. E, mir-383 inhibits RBMS1 3 UTR luciferase activity. Luciferase reporters containing wild-type RBMS1 3 UTR were cotransfected with 10, 20, 30, 40, 60 nm mir-383 mimics or mimics NC. Luciferase activity was measured 30 h after transfection. F, Efficacy of RBMS1 overexpression and RBMS1 sirna. Protein extracts from GCs transfected with 1μg/ml each of pegfp-c1 or pegfp-rbms1 vector or sirna negative control (si.nc) and RBMS1 sirna (si-rbms1), were immunoblotted with anti-rbms1 antibodies. G, RBMS1 attenuated E 2 release from GCs. GCs were transfected with different concentrations (0.5, 1.0, 1.5μg/ml) of pegfp-c1 and pegfp-rbms1 vector for 48h. After culture, media were collected for measurement of E 2 levels. H-I, Enforced RBMS1 expression and knockdown of RBMS1 (si-rbms1) did not alter GC proliferation and apoptosis. GCs were transfected with EGFP- RBMS1 vector/ control vector pegfp-c1 or si-rbms1/si.nc for 24, 48, 72h. Cell viability was measured by the cell counting kit-8 assay after transfection. The apoptosis rate was measured using flow cytometry with Annexin V and PI staining after transfection either with EGFP- RBMS1 vector/ control vector pegfp-c1 or si-rbms1/si.nc for 48 h. NC, negative control. In C-E and G-I, data shown represent the mean ± S.E.M of three independent experiments performed in triplicate. ** 73 P < 0.01. 6
74 75 76 77 78 79 80 81 Supplemental Fig. S3. c-myc is not a target of mir-383. A and B, mir-383 did not target 3 UTR of c-myc. HEK 293T cells were cotransfected with luciferase reporters containing the c-myc 3 UTR and 10, 20, 30nM of mir-383 mimics/ mimics NC or 30nM of mir-383 inhibitors and inhibitors NC. C and D, Effects of RBMS1 on c-myc protein expression in GCs. c-myc was determined by Western blotting after transfection of si-rbms1/si.nc or pegfp-rbms1 /control into GCs. Representative Western blot for c-myc (upper bands) and GAPDH (lower bands) (C) and the corresponding densitometric analysis (D) are shown. The ratio of the c-myc band intensity over the GAPDH band intensity in controls was arbitrarily 7
82 83 84 85 set at 1.0. E, Efficacy of c-myc overexpression and c-myc sirna. Protein extracts from GCs transfected either with EGFP-c-Myc vector/ control vector pegfp-c1 or si-rbms1/si.nc for 48 h, were immunoblotted with anti-c-myc antibodies. In A, B and D, data shown represent the mean ± S.E.M of three independent experiments performed in triplicate. ** P < 0.01. 86 8
87 9
88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 Supplemental Fig. S4. SF-1 transactivates mir-383 expression. A, Schematic representation of the mir-383 genomic locus hosted in the intron 1 of SGCZ. B, Overexpression of SF-1 enhances the RNA levels of SGCZ, pri-mir-383, pre-mir-383 and mature mir-383. NIH3T3 cells were transfected with pegfp-rbms1 vector /control vector, and the RNA levels were determined after 48 h transfection by real-time PCR. C, FSH promotes the expression of SGCZ mrna, mir-383 and SF-1 mrna in GCs. mgcs were treated with FSH (100ng/ml) for 6 h and subjected to real-time PCR analysis. D and E, Expression patterns of SF-1 and mir-383 in the ovary. D, Total RNA was extracted from the PND 1, PND 4, PND 9, PND 14, and PND 21 mouse ovaries and then subjected to real-time PCR. The SF-1 mrna and mir-383 levels (D) were normalized to GAPDH and U6 for each sample, respectively. Data obtained from the PND 1 mouse ovary were arbitrarily set at 1.0. E, SF-1 protein expression levels were determined by Western blotting analysis in the PND 1, PND 4, PND 9, PND 14, PND 21 mouse ovaries. Representative Western blot for SF-1 (upper bands) and GAPDH (lower bands) (left panel of Fig. S4E) and the corresponding densitometric analysis (right panel of Fig. S4E) are shown. The ratio of the SF-1 band intensity over the GAPDH band intensity in PND 1 ovaries was arbitrarily set at 1.0. F, SF-1 mrna expression levels in various stages of follicle development: preantral, antral and ovulated. Total RNA was extracted from the different stage follicles and then subjected to real-time PCR. Data obtained from the preantral follicles was arbitrarily set at 1.0. G, Changes in SF-1 mrna expression in ovaries of PMSG/hCG-treated immature mice. Total RNA was extracted from ovaries of PMSG-treated mice at 24 h and PMSG/hCG-treated mice at indicated post-hcg time points, and then subjected to real-time PCR. H, mir-383 had no effect on the expression level of SF-1 protein in GCs. GCs were transfected with 120 nm each of mir-383 mimics and mimics NC, or 150 nm each of mir-383 inhibitors and inhibitors NC. SF-1 protein expression levels were determined by Western blotting analysis. I, Mutational analysis of SGCZ promoter-luciferase reporter constructs. Left panel, schematic representation of 5 - or 3 - mutant fragments of the mouse SGCZ promoter in conjunction with the luciferase gene (LUC) in pgl3-basic vector. The nucleotides are numbered from the transcriptional starting site that was assigned +1. Right panel, luciferase activity of the mutant promoter constructs. SGCZ (-1489/+56), SGCZ (-1489/+56) 10
114 115 116 117 118 119 120 121 mutant 1 (mutated site at -980 bp) and SGCZ (-1489/+56) mutant 2 (mutated site at -419 bp) promoter constructs were cloned into the luciferase reporter vector pgl3-basic. Relative luciferase activity was calculated as a ratio of luciferase activity of SGCZ mutant constructs driven by SF-1 over the pgl3-basic empty vector, which was arbitrarily set at 1. J, ChIP analysis reveals in vivo binding of SF-1 to the SGCZ promoter. ChIP assays were performed on NIH3T3 cell extracts using antibodies against IgG and SF-1, followed by PCR amplification with primers sequences corresponding to the mouse SGCZ promoter region between nucleotides -200 and -8. In B-G, I, data were presented as means ± S.E.M for at least 3 independent experiments. * P < 0.05, ** P < 0.01. NC, negative control. 122 11
123 124 125 126 127 128 Supplemental Fig. S5. SF-1 mediates the effects of mir-383/ RBMS1/c-Myc on the Cyp19a1 mrna expression in GCs. A, C and D, SF-1 overexpression in mgcs increased the suppressed Cyp19a1 mrna levels by mir-383 inhibitors (A), overexpression of RBMS1 (C) or of c-myc (D). B, si-sf-1 partially abrogated mir-383-induced Cyp19a1 expression in mgcs. E, mir-383 inhibitors-induced decrease of Cyp19a1 mrna levels in GCs was further suppressed by si-sf-1. GCs were co-transfected with 12
129 130 131 pegfp-sf-1 and indicated expression vectors or mir-383 inhibitors, or with si-sf-1 and mir-383 mimics/inhibitors for 48 h. The culture medium was collected for hormone analysis. Data were presented as means ± S.E.M for at least 3 independent experiments. * P < 0.05, ** P < 0.01. NC, negative control. 13
Supplementary Table S1. PCR and RT-PCR primers used in this study Gene Forward primer sequence (5-3 ) RBMS1 GCTAGAATTCTATGGGCAAAGTGTGGAAACAAC c-myc GTACGAATTCTCTGGATTTCCTTTGGGCGTT SF-1 GTATCCTCGAGCTATGGACTATTCGTACGACGAG RBMS1 3'UTR CTACTCTCGAGCTGTGAGATGTACCGAAGGGAGT c-myc 3'UTR GTTATGCGGCCGCACTGACCTAACTCGAGGA SGCZ promoter(-1489/+56) GCATCGGTACCGGCCCACAAATTATGGATTATT SGCZ promoter(-1489/+56) mutant1 TGATATCAGTTCCGCATACCATTTATTGAAGAAG SGCZ promoter(-1489/+56) mutant 2GATGTAGACTGGAGTTAGAATGAATTCAAAGAA SGCZ promoter(-703/+56) GTATCGGTACCAGCTCAGCAGTGCAGAAATATG SGCZ promoter(-524/+56) GTGTCGGTACCACTGAAGCCTTAACATAAATCA SGCZ promoter(-230/+56) GTATCGGTACCTGATACAGGCAAGCCTGTGTCT SGCZ promoter(-150/+56) GTATCGGTACCCCTAGTAGATTCCGGACAGAGG SGCZ promoter(-128/+56) GTATCGGTACCGCAGTCTTTAAGACCCAGCG SGCZ promoter(-100/+56) GTATCGGTACCTGTCTGGAGAGGAAATGTTGCT SGCZ promoter(-128/+56) mutant fogtatcggtaccgctgacataatgtcgctgcc Supplementary Table S2. Sequence of oligonucleotides Name Sequence (5' to 3') Inhibitor NC CAGUACUUUUGUGUAGUACAA mmu-mir-383 inhibitors AGCCACAGUCACCUUCUGAUCU mimics NC UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT mmu-mir-383 mimics AGAUCAGAAGGUGACUGUGGCU CCACAGUCACCUUCUGAUCUUU LNA scramble-mir GTGTAACACGTCTATACGCCCA LNA mmu-mir-383 AGCCACAGTCACCTTCTGATCT Supplementary Table S3. qrt-pcr primers used in this study Gene Forward primer sequence (5-3 ) Cyp19a1 TGTGTTGACCCTCATGAGACA Cyp11a1 TCCCTGTAAATGGGGCCATAC GAPDH TGTGTCCGTCGTGGATCTGA RBMS1 CTGAGCAAGACAAACCTCTACAT Pri-miR-383 TCGACCACTTCAGTGACTGA Pre-miR-383 CTCAGATCAGAAGGTGACTG c-myc CAGAGGAGGAACGAGCTGAAGCGC FAS GATGCACACTCTGCGATGAAG SGCZ TGAGAGTCACCAAGAAGGGA SF-1 ATGGCCGACCAGACCTTTATCTC
Reverse primer sequence (5-3 ) GCATGTCGACTTACTTATTGGGTGGAAAGGTAT CTAGGTCGACTTATGCACCAGAGTTTCGAAG GACATGAATTCTCAAGTCTGCTTGGCCTGCAGC GTGTAGCGGCCGCACTTTACATTGCATCATACAGCAG GCAGCGTTTAAACTTCAAGGCCCTATTTACATGGG GGTATGCGGAACTGATATCAGACCAGTCTTATTT TTCTAACTCCAGTCTACATCGCACTTGTAATCTG Reverse primer sequence (5-3 ) CTTGACGGATCGTTCATACTTTC AGGTCCTTCAATGAGATCCCTT TTGCTGTTGAAGTCGCAGGAG GGCCTTATCCAAAATCGCCTT CTCTTTCTGACCAGGCAGTG CTGACCAGGCAGTGCTGTGG TTATGCACCAGAGTTTCGAAGCTGTTCG CAGTGTTCACAGCCAGGAGAAT GTTCCTTGCATTCACTGTTA TGGTCCAACACCAGCAGCTC