A universal activator of microRNAs identified from photoreaction products

Article abstract of DOI:10.1039/c2cc32157b

A small molecule activator of microRNAs has been identified from photoreaction products of naphthalene-1,4-dione with acetylenes and the universal activation on miRNAs was realized through significant promotion on the processing of precursor microRNAs by the compound.

Full text of DOI:10.1039/c2cc32157b

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Supplementary Information
Universal activator of microRNAs identified from
photoreaction products
Xuejiao Chen, Chengmei Huang, Wenjie Zhang, Yihan Wu, Xi Chen, Chen-yu Zhang and Yan Zhang
Contents
Description
Page number
General
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Experimental procedures
Scheme 1. Photoreactions leading to compound 3-5 in the compound
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S5
S5
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block
Figure 1: MTT assay on the cytotoxicity of 3a in C2C12, HepG2 and
MCF-7 cell
Table 1. Cellular assay results on 3-5 with representative structure in
the compound block
Figure 2: Dose dependence of the regulation activity on
miR-214/25/126/34a of 3a.
Figure 3. Cellular Ago2 expression level and amount of miRNAs
associated with Ago2 upon treatment of compound 3a
Figure 4. Cellular expression level of Dicer and TRBP with or without
treatment of compound 3a
The preparation of 3-5 with representative structure and compound
S7
characterization data
Copies of 1H NMR and 13C NMR spectra
S10
General
Nuclear magnetic resonance spectra were recorded on a Bruker DPX 300 at 300MHz with CDCl3 as
solvent. Chemical shifts are reported in δ (ppm) with the solvent resonance as the internal standard
(chloroform, H, 7.263 ppm; C, 77.66, 77.23, 76.81 ppm). Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and br s = broad), coupling
constant in Hz, integration, and assignment. Elemental analyses were done on a Elementar Vario
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MICRO analyzer. MTT assay was tested on Bio-Rad (680) Micro plate spectrophotometer. Modulus
Luminometer used for luciferase assay was from Turner BioSystems (9200-002). Quantitative RT-PCR
was carried out on an Applied Biosystems 7300 Real-Time PCR System. Benzene (AR grade) was
dried with sodium and distilled. MTT powder was purchased from Sigma-Aldrich. TRIzol Reagent and
Lipofectamine 2000 reagent were purchased from Invitrogen. TaqMan miRNA probes, AMV reverse
transcriptase, dNTP mixture, Taq transcriptase, RRI and oligo (dT) were purchased from TaKaRa. Eva
green dye was purchased from Invitrogen. The stem-loop RT primers were synthesized by Invitrogen.
Other materials were commercially available.
Experimental procedures
Cells, and Antibodies: Mouse myoblast cell C2C12, human hepatocellular carcinoma cell HepG2,
human breast cancer MCF-7 were cultured with DMEM (Invitrogen) supplemented with 10% fetal
bovine serum (FBS, Invitrogen) and 1% penicillin and streptomycin (GIBCO) in a humidified
atmosphere with 5% CO2. Anti-Ago2 and anti-TRBP monoclonal antibody were purchased from
Abcam, anti-Dicer monoclonal antibody were purchased from Cell Signaling. Anti-GAPDH and
anti-α-tubulin were purchased from Santa Cruz Biotechnology.
Plasmid Construction: A system to screen miRNA inhibitor and activator based on the
pMIR-REPORT (Applied Biosystem) reporter plasmid was constructed. This construct consists of an
exprimental firefly luciferase reporter vector and an associated β-gal report control plasmid. The
predicted miRNA target sequence was inserted into 3’UTR of the firefly luciferase gene between the
SpeI and HindⅢrestriction sites. We selected miR-1/122/21/214/25/26b/34a/126 as the target miRNA,
The plasmid without target sequence of miRNA was taken as a control.
Transfection: Plasmid transfection into C2C12, HepG2 and MCF-7 cells using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s protocol. All transfections were performed in triplicate for
statistical analysis. The cells were incubated for 6 h followed by the replacement of transfection media
(OPTI-MEM) with normal media (DMEM with 2% FBS), and the compounds were added at the same
time. After 48 h incubation, the cells were lysed and assayed with a Luciferase assay Kit (E4550,
Invitrogen) using a turner biosystem instrument luminometer with a measurement time of 1s and a
delay time of 2s.
Cell toxicity assay: Cell toxicity was examined by MTT assay. Cells were cultured in 96-well plates
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(104 cells / well). After 12 h, replace the 10% FBS DMEM media with the 2% one. Then the cells were
treated with complexes. After incubation of cells for up to 48 h, 20μL MTT solution (5mg/mL) was
added to each well. The cells were incubated for another 4 h, the mixtures containing the medium and
MTT were removed and the formazan crystals were dissolved in 150 μL DMSO/well. The absorbance
of each well at 490 nm was determined by analysis with a micro plate Spectrophotometer, The percent
growth inhibitory rate of treated cells was calculated by (Atested -Amedia control)/(Adrug-free control- Amedia control
× 100%, where A is the mean value calculated using the data from three replicate tests.
)
RNA Isolation and Quantitative RT-PCR: After cells were incubated with compounds (10 μM) or
DMSO (1‰ final DMSO concentration) for 48 h (2% FBS DMEM, 5% CO2). The media was removed,
and total RNA was extract using TRIzol Regent (Invitrogen) according to the manufacturer’s
instruction. For analysis of mature miRNA, quantitative RT-PCR was carried out using TaqMan
miRNA probes (Applied Biosystems; Foster City, CA) according to the manufacturer’s instructions.
Briefly, 1μg of total RNA was reverse transcribed to cDNA using AMV reverse transcriptase (TaKaRa;
Dalian, China) and a stem-loop RT primer (Applied Biosystems). The procedure is 16 °C, 30 min;
42 °C, 30 min; 85 °C, 5 min. Real-time PCR was performed using a TaqMan PCR kit on an Applied
Biosystems 7300 Real-Time PCR System. The procedure is 95 °C, 5 min; followed by 40 cycles of
95 °C, 15 s; 60 °C, 60 s. After the reaction, the level of miRNA was determined using threshold cycles
(CT) and copy number. The data were then normalized to the DMSO control. For quantitative RT-PCR
analysis of pre-miRNA or some gene, 1μg total RNA was reverse to cDNA with oligo (dT) and
thermoscript, Quantitative Real-Time PCR using Eva green dye (Invitrogen). The sequence of the
sense and antisense primers were as follows: pre-miR-1 (5’-AAACATACTTCTTTATATGCCCA-3’,
5’-TACATACTTCTTTACATTCCATAGC-3’); pre-miR-122 (5’-GGAGTGTGACAATGGTGTTT
G-3’, 5’-TTTAGTGTGATAATGGCGTTTG-3’); pre-miR-21 (5’-GCTTATCAGACTGATGTTGA
CTG-3’, 5’-CAGCCCATCGACTGGTG-3’); pre-miR-214 (5’-CCTGGCTGGACAGAGTGG-3’,
5’-TACAGGTGAGCGGATGTT-3’); pre-miR-25 (5’-TGAGAGGCGGAGACTTGG-3’, 5’-TCAGAC
CGAGACAAGTGCAA-3’); pre-miR-26b (5’-TTCAAGTAATCCAGGATAGGCTGT-3’, 5’-CAAG
TAATGGAGAACAGGCTG-3’); pre-miR-34a (5’-TGGCAGTGTCTTAGCTGGTTG-3’, 5’-GGCAG
TATACTTGCTGATTGCTT-3’) pre-miR-126 (5’-TATTACTTTTGGTACGCGCTG-3’,5’-GCGCAT
TATTACTCACGGTAC-3’)
Western blotting: Samples of cultured cells after treatment with compound were lysed in a buffer
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containing cell lysis buffer (Biocam), 1×protease inhibitor (Complete, Roche) and 1×PMSF
(Phenylmethanesulfonyl fluoride, Amresco) . The cell extract was used in western blot analysis with
anti-Ago2, anti-Dicer, anti-TRBP anti-GAPDH and α-tubulin monoclonal antibody.
Immunoprecipitation Analysis: Immunoprecipitation assays were performed according to the
manufacturer’s instructions. After treatment with compound for 48 h, Cells were resuspended in IP
buffer (20 mM Tri-HCl (PH=7.5), 150 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 0.5 mM
dithiothreitol (DTT), 1 mM NaF, 1 × protease inhibitor and 1 × PMSF). Mouse monoclonal anti-AGO2
antibody (2 μg) was used to immunoprecipitate RNA-binding proteins. After purification,
immunoprecipitated RNA was analyzed by quantitative RT-PCR using TaqMan miRNA probes.
Scheme 1. Photoreactions leading to compound 3-5 in the compound block.
O
+
O
hv
16h
2a
2b
2c
O
O
4a (24.3%)
O
3a (37.4%)
N
N
N
N
O
hv
+
48h
N
O
O
O
N
O
+
4b (19.8%)
3b (40.1%)
1
N
O
N
N
N
N
hv
O
+
18h
N
O
O
3c (29.5%)
5 (19.5%)
N
O
O
N
O
hv
+
48h
2d
N
O
3d (28.3%)
4d (21.1%)
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Figure 1. MTT assay on the cytotoxicity of 3a in C2C12, HepG2 and MCF-7 cell. Cells were
incubated with 3a (10, 20 and 30μM) for 48 h, then the cell viability was tested by MTT assay. Values
are mean ± s.d. for triplicate samples after normalization to DMSO control. Compound 3a showed no
obvious toxicity at concentrations under 10μM. Due to solubility limit, cytotoxicity of 3a at
concentrations higher than 30μM was not measured.
120
100
80
60
40
20
0
10 μM
20 μM
30 μM
C2C12
HepG2
MCF-7
Table 1. Cellular assay results on 3-5 with representative structure in the compound block. The
concentration of the compounds used to treat the cells was the highest concentration under which the
compound could dissolve well and showed no obvious cytotoxicity on cells. Relative luciferase level
on C2C12 cells transfected with luciferase reporter for miR-1 and empty vector, relative luciferase
level on HepG2 cells transfected with luciferase reporter for miR-122 and empty vector.
Relative Fluc.
Relative Fluc.
Concn.
Compound
(μM)
3
empty vector
miR-1
empty vector
miR-122
0.64 ± 0.04
0.97 ± 0.08
0.53 ± 0.08
2.11 ± 0.22
1.48 ± 0.06
2.0 ± 0.01
0.62 ± 0.04
1.18 ± 0.07
0.47 ± 0.11
2.03 ± 0.21
1.75 ± 0.24
1.83 ± 0.36
1.24 ± 0.15
0.94 ± 0.01
0.90 ± 0.06
1.04 ± 0.18
1.01 ± 0.21
2.19 ± 0.26
0.99 ± 0.12
1.17 ± 0.23
0.87 ± 0.07
0.86 ± 0.04
1.05 ± 0.19
1.00 ± 0.11
1.80 ± 0.26
1.15 ± 0.14
1.27 ± 0.01
3b
3c
3d
4a
4b
4d
5
1
5
10
10
10
5
1.30 ± 0.11
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Figure 2. Dose dependence of the regulation activity on miR-214/25/126/34a of 3a (Determined by
luciferase assay on MCF-7 reporter cells).
Figure 3. Cellular Ago2 expression level and amount of miRNAs associated with Ago2 upon treatment
of compound 3a (10μM). (A): Western blot assay was used to detect the Ago2 protein level after
treatment with 3a in MCF-7 cell. (B): The quantification of the Ago2 protein expression in (A). (C): IP
assay was used to detect the miRNA level after treatment with 3a. Values are mean ± s.d. for triplicate
samples. The results indicatd that 3a showed no effect on Ago2 protein expression and did not change
the amount of Ago2-associated miRNAs.
1.6
1.2
0.8
0.4
0.0
(B)
(C)
(A)
DMSO
3a
DMSO
3a
0.9
0.6
0.3
0.0
DMSO
3a
Ago2
GAPDH
AGO2
miR-122 miR-21 miR-25
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Figure 4. Cellular expression level of Dicer and TRBP with or without treatment of compound 3a
(10μM). (A) Western blot assay was used to test the protein level of Dicer and TRBP in MCF-7 cell
after treatment with 3a. (B) The quantification of the protein leve in (B). Values are mean ± s.d. for
triplicate samples. Compound 3a showed no influence on Dicer expression, but significant
upregulation on TRBP protein level was observed in cells treated with 3a.
(B)
(A)
2.0
1.6
1.2
0.8
0.4
0.0
DMSO
3a
Dicer
TRBP
GAPDH
Dicer
TRBP
The preparation of 3-5 with representative structure and compound characterization data
Photolysis of 1 with 2a. A solution of 1 (158 mg, 1 mmol) and 2a (356 mg, 2 mmol) in benzene (100 mL) was
irradiated with light of wavelength >400 nm under Ar atmosphere for 16 h to reach a 64.1% conversion of 1
Solvent was removed under reduced pressure, the residue was separated by Flash column chromatograph on silica
gel (petroleum ether/ethyl acetate) to give pure analytic samples of 3a (37.4%) and 4a (24.3%).
7-benzoyl-4H-benzo[de]anthracen-4-one (3a): yellow crystal; m.p.: 208 ˚C; 1H NMR (300MHz, CDCl3): δ 9.05
(d, J = 7.9 Hz, 1H), 8.80 (d, J = 8.5 Hz, 1H), 8.70 (dd, J = 7.5, 0.7 Hz, 1H), 7.98 (t, J = 7.9 Hz, 1H), 7.92 (d, J =
7.4 Hz, 2H), 7.82–7.76 (m, 1H), 7.72–7.54 (m, 4H), 7.48 (t, J = 7.7 Hz, 2H), 6.69 (d, J = 10.0 Hz, 1H)；13C NMR
(75 MHz, CDCl3): δ 198.2, 185.1, 141.9, 138.3, 137.4, 134.8, 131.3, 130.3, 130.1 (2C), 129.8, 129.7, 129.5, 129.2
(2C), 129.1, 128.9, 128.4, 128.1(2C), 127.9, 126.5, 123.4, 123.1; MS m/z (EI): 334 (100), 306 (27), 305 (38), 257
(38), 201 (29), 200 (46), 105 (41), 77(35); Anal. Calcd. for: C24H14O2: C 86.21, H 4.22; Found: C 86.04, H 4.32%.
1,2-diphenylcyclobuta[b]naphthalene-3,8(2aH,8aH)-dione (4a): pale yellow crystal; m.p.: 170–171 ˚C; 1H
NMR (300MHz, CDCl3): δ 8.04–8.00 (m, 2H),7.74–7.71 (m, 2H), 7.63–7.60 (m, 4H), 7.36–7.30 (m, 6H), 4.48 (s,
2H)；13C NMR (75 MHz, CDCl3): δ 195.7 (2C), 140.9, 134.5 (3C), 134.1, 133.2, 129.0 (3C), 128.6 (4C), 127.6
(3C), 127.0 (4C), 50.7 (2C); MS m/z (EI): 336 (100), 308 (34), 307 (89), 231 (22). 202 (27), 178 (66); Anal. Calcd.
for: C24H16O2: C 85.69, H 4.79; Found: C 85.75, H 4.94%.
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Photolysis of 1 with 2b. A solution of 1 (158 mg, 1 mmol) and 2b (288 mg, 2 mmol) in benzene (100 mL) was
irradiated with light of wavelength >400 nm under Ar atmosphere for 48 h to reach a 55.4% conversion of 1
Solvent was removed under reduced pressure, the residue was separated by Flash column chromatograph on silica
gel (petroleum ether/ethyl acetate) to give pure analytic samples of 3b (40.1%) and 4b (19.8%).
7-(cyclopropanecarbonyl)-4H-naphtho[1,β-gh]quinazolin-4-one (3b): pale yellow crystal; m.p.: 235–236 ˚C;
1H NMR (300MHz, CDCl3): δ 9.60–9.56 (m, 2H), 9.41 (s, 1H), 8.79 (dd, J =7.4, 1.6 Hz, 1H), 8.03 (td, J = 7.7, 1.9
Hz, 1H), 7.87 (dd, J = 10.1, 1.2 Hz 1H), 6.84 (dd, J =10.1, 1.2 Hz, 1H), 2.53–2.45 (m, 1H), 1.69–1.65 (m, 2H),
1.42–1.36 (m, 2H)；13C NMR (75 MHz, CDCl3): δ 205.2, 184.2, 157.6, 157.1, 151.0, 140.6, 136.9, 132.5, 131.2,
130.8, 129.7, 129.6, 129.1, 128.8, 123.7, 119.9, 25.7, 15.1 (2C); MS m/z (EI): 300 (100), 272 (35), 259 (78). 231
(48), 204 (67), 176 (37); Anal. Calcd. for: C19H12N2O2: C 75.99, H 4.03, N 9.33%; found: C 76.78, H 4.11, N
9.14%.
1-cyclopropyl-2-(pyrimidin-5-yl)cyclobuta[b]naphthalene-3,8(2aH,8aH)-dione (4b): brown crystal; m.p.:
134–135 ˚C; 1H NMR (300MHz, CDCl3): δ 9.01 (s, 1H), 8.90 (s, 2H), 8.01–7.94 (m, 2H), 7.75–7.67 (m, 2H), 4.29
(d, J = 3.8 Hz, 1H), 3.99 (d, J = 3.8 Hz, 1H), 1.80–1.73 (m, 1H), 1.08–0.76 (m, 4H)；13C NMR (75 MHz, CDCl3):
δ 195.4, 194.8, 157.0, 154.2 (2C), 150.6, 134.8, 134.7, 134.0, 133.8, 133.7, 127.8, 127.5, 127.3, 50.7, 48.2, 12.5,
7.0, 6.5; MS m/z (EI): 302 (64), 274 (22), 273 (25), 259 (44), 159 (58), 144 (100), 76 (23); Anal. Calcd. for:
C19H14N2O2: C 75.48, H 4.67, N 9.27%; found: C 75.61, H 4.64, N 9.24%.
Photolysis of 1 with 2c. A solution of 1 (158 mg, 1 mmol) and 2c (288 mg, 2 mmol) in benzene (100 mL) was
irradiated with light of wavelength >400 nm under Ar atmosphere for 48 h to reach a 45.2% conversion of 1
Solvent was removed under reduced pressure, the residue was separated by Flash column chromatograph on silica
gel (petroleum ether/ethyl acetate) to give pure analytic samples of 3c (29.5%) and 5 (19.5%).
7-(cyclopropanecarbonyl)-4H-naphtho[1,8-fg]quinoxalin-4-one (3c): pale yellow crystal; m.p.: 246–247 ˚C; 1H
NMR (300MHz, CDCl3): δ 9.52 (dd, J = 8.1, 0.9 Hz, 1H), 9.00 (d, J = 3.2 Hz, 2H), 8.77 (dd, J = 7.4, 0.9 Hz, 1H),
8.04 (t, J = 7.8 Hz, 1H), 7.82 (d, J = 10.1 Hz, 1H), 6.86 (d, J = 10.1 Hz, 1H), 2.52–2.44 (m, 1H), 1.64–1.55 (m,
2H), 1.30–1.23 (m, 2H)；13C NMR (75 MHz, CDCl3): δ 205.9, 184.6, 145.3, 145.2, 143.2, 142.3, 140.3, 137.5,
131.4, 131.1, 130.4, 129.7, 129.6, 128.8, 127.9, 125.8, 25.3, 13.4 (2C); MS m/z (EI): 272 (100), 244 (37), 231 (28),
176 (29); Anal. Calcd. for: C19H12N2O2: C 75.99, H 4.03, N 9.33%; found: C 76.16, H 3.81, N 9.17%.
4-(1-cyclopropyl-2-oxo-2-(pyrimidin-2-yl)ethylidene)naphthalen-1(4H)-one (5): brown crystal; m.p.: 132–133
1
˚C; H NMR (300MHz, CDCl3): δ 9.37 (s, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.67–8.66 (m, 1H), 8.56 (d, J = 8.1 Hz,
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1H), 8.30 (d, J = 7.7 Hz, 1H), 7.68(td, J = 7.6, 1.3 Hz, 1H ), 7.57 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 10.3 Hz, 1H),
13
6.30 (d, J = 10.2 Hz, 1H), 2.40–2.30 (m, 1H), 1.18–1.11 (m, 2H), 0.80–0.74 (m, 2H)； C NMR (75 MHz, CDCl3):
δ 196.4, 184.4, 151.5, 148.4, 147.9, 144.8, 144.6, 144.3, 140.5, 132.6, 131.4, 130.5, 128.8, 128.2, 127.1, 126.6,
15.6, 10.4 (2C); MS m/z (EI): 302 (76), 273 (58), 195 (100), 167 (35), 165 (86), 152 (64), 139 (23), 79 (31); Anal.
Calcd. for: C19H14N2O2: C 75.48, H 4.67, N 9.27%; found: C 75.31, H 4.47, N 9.04%.
Photolysis of 1 with 2d. A solution of 1 (158 mg, 1 mmol) and 2d (286 mg, 2 mmol) in benzene (100 mL) was
irradiated with light of wavelength >400 nm under Ar atmosphere for 48 h to reach a 78.9% conversion of 1
Solvent was removed under reduced pressure, the residue was separated by Flash column chromatograph on silica
gel (petroleum ether/ethyl acetate) to give pure analytic samples of 3d (28.3%) and 4d (21.1%).
o
7-(cyclopropanecarbonyl)-4H-naphtho[1,8-fg]isoquinolin-4-one (3d): pale yellow crystal; m.p.: 184–185 C;
1H NMR (300MHz, CDCl3): δ 9.65 (d, J = 8.1 Hz, 1H), 9.10 (d, J = 3.0 Hz, 1H), 8.75 (d, J = 7.4 Hz, 1H), 8.23 (d,
J = 8.4 Hz, 1H), 8.02 (t, J = 7.8 Hz, 1H), 7.85 (d, J = 10.1 Hz, 1H), 7.63 (dd, J = 8.3, 4.4 Hz, 1H), 6.84 (d, J = 10.7
Hz, 1H), 2.51–2.43 (m, 1H) 1.64–1.61 (m, 2H), 1.38–1.32 (m, 2H)；13C NMR (75 MHz, CDCl3): δ 206.8, 184.9,
151.2, 147.2, 142.8, 137.6, 134.6, 130.8, 130.6(2C), 130.5, 129.7, 128.5, 128.0, 122.9, 122.6, 122.5, 25.4, 14.5
(2C); MS m/z (EI): 299 (100), 271 (92), 230 (85), 202 (48), 201 (22), 175 (23); Anal. Calcd. for: C20H13NO2: C
80.25, H 4.38, N 4.68%; found: C 82.42, H 4.54, N 4.84%.
1-cyclopropyl-2-(pyridin-3-yl)cyclobuta[b]naphthalene-3,8(2aH,8aH)-dione (4d): brown crystal; m.p.:
154–155 ˚C; 1H NMR (300 MHz, CDCl3): δ 8.86 (s, 1H), 8.47 (d, J = 4.6 Hz, 1H), 8.04 (dd, J = 7.3, 1.4 Hz, 1H),
8.00–7.96 (m, 1H), 7.89–7.85 (m, 1H), 7.80–7.69 (m, 2H), 7.31–7.26 (m, 1H), 4.31 (d, J = 3.8 Hz, 1H), 3.99 (d, J
= 3.8 Hz, 1H), 1.89–1.80 (m, 1H), 1.07–0.75(m, 4H)；13C NMR (75 MHz, CDCl3): δ 196.1, 195.5, 148.5, 147.9,
147.7, 139.2, 137.4, 134.6, 134.5, 133.8, 133.4, 129.1, 127.7, 127.4, 123.4, 50.2, 48.5, 12.2, 6.5, 6.2; MS m/z (EI):
301 (76), 272 (45), 258 (36), 244 (23), 159 (31), 143 (100), 142 (34), 76 (29); Anal. Calcd. for: C20H15NO2: C
79.72, H 5.02, N 4.65%; found: C 80.02, H 4.88, N 4.75%.
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Copies of 1H NMR and 13C NMR spectra
1H NMR and 13C NMR of 3a:
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1H NMR and 13C NMR of 4a:
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1H NMR and 13C NMR of 3b:
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1H NMR and 13C NMR of 4b:
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1H NMR and 13C NMR of 3c:
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1H NMR and 13C NMR of 5:
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1H NMR and 13C NMR of 3d:
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1H NMR and 13C NMR of 4d:
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