Identification of genes responsive to paeoniflorin, a heat shock protein-inducing compound, in human leukemia U937 cells

Figures & data

Table I.  Nucleotide sequences of primers for target genes.

Genes	Orientation	Nucleotide sequence (position)	Genbank accession number
ATF5	Sense	5′-AGCCTCTCCTGTTGCCATCA-3′(276-295)	AB021663
	Antisense	5′-TCAGAGAAGCCATCACCTGC-3′(513 -494)
CCL2	Sense	5′-TGAAAGTCTCTGCCGCCCTT-3′(75-94)	NM_002982
	Antisense	5′-CTTTGGGACACTTGCTGCTG-3′(257-238)
EGR1	Sense	5′-GAGGAGATGATGCTGCTGAG-3′(360-379)	M62829
	Antisense	5′-TTGGCTGGGGTAACTGGTCT-3′(626-607)
F0SL1	Sense	5′-ACCCATCTGCAAAATCCCGG-3′(552-571)	BC016648
	Antisense	5′-GTTAGGGAGGGTGTGGTCAT-3′(725-706)
GAPDH	Sense	5′-AAGGCTGGGGCTCATTTGCA-3′(394-413)	NM_002046
	Antisense	5′-ATGACCTTGCCCACAGCCTT-3′(737-718)
HSPA1B	Sense	5′-AGGTGCAGGTGAGCTACAAG-3′(509-528)	NM_005346
	Antisense	5′-ATGATCCGCAGCACGTTGAG-3′(722-703)
REL	Sense	5′-CCTGGTCTCCTCGGTTCAAT-3′(1219-1238)	X75042
	Antisense	5′-TTGTATTGCCTGAGCGTGGG-3′(1453-1434)

ATF, activating transcription factor 5; CCL2, chemokine (C-C motif) ligand 2; EGR1, early growth response 1; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; FOSL1, FOS-like antigen 1; HSPA1B, heat shock 70kDa protein IB; REL, reticuloendotheliosisviral oncogene homolog (avian).

Figure 1. PF induced Hsp70 in a dose- and time-dependent manner. (a) SDS-PAGE and Western blotting were performed on U937 cells treated with PF for 30 min at different concentrations (80–640 μg/ml for dose-dependent experiments and 160 µg/ml for time-dependent experiments). After which cells were incubated at different times (0–12 h for time-dependent experiments and 3 h for dose-dependent experiment). The signals were visualized by a luminescent image analyser using an ECL system. (b,c) Bands were quantified by densitometry and normalized with β-actin. Data are presented as mean ± SD (n = 3). *p < 0.05 vs. control (Student's t test). Cont, control.

Table II.  Upregulated genes under PF treatment.

		Incubation time (h)
Name	Cont.	0	1	3	Family	Entrez gene ID
Transiently increased genes
ARHGAP26	0.9	3.0	1.1	0.5	Other	23092
ATM	1.6	3.8	0.5	0.4	Kinase	472
AZI2	0.7	2.0	1.3	0.4	Other	64343
CDC2	0.7	2.2	0.2	1.4	Kinase	983
CDKN2C	1.0	2.6	0.4	1.0	Transcription regulator	1031
EGR3	0.5	1.1	16.2	0.9	Transcription regulator	1960
F0SL1	0.3	0.6	3.6	1.5	Transcription regulator	8061
GMCL	0.9	2.0	0.3	1.1	Other	64395
IER3	1.0	1.0	6.3	0.8	Other	8870
ITPR2	1.0	0.5	3.0	1.0	Ion channel	3709
MAFF	0.4	0.8	7.1	1.3	Transcription regulator	23764
MERTK	1.0	2.1	0.4	1.0	Kinase	10461
PARG	0.9	2.8	0.1	1.1	Enzyme	8505
PDCD4	1.1	2.5	0.4	0.9	Other	27250
PIAS1	0.6	2.8	0.5	1.5	Transcription regulator	8554
POLH	0.9	0.5	2.9	1.1	Enzyme	5429
RAD 17	1.1	2.2	0.4	0.9	Other	5884
TIPARP	0.4	0.7	13.2	1.4	Other	25976
TRA2A	0.8	2.5	1.1	0.2	Other	29896
TUSC2	0.5	1.9	0.3	1.7	Other	11334
Gradually increased genes
ATF5	0.6	0.3	1.6	1.9	Transcription regulator	22809
CCL2	0.2	0.2	2.7	6.9	Cytokine	6347
CCNE2	0.9	0.4	1.1	4.8	Other	9134
CD69	0.9	1.1	0.9	8.7	Transmembrane receptor	969
CLK1	1.0	1.0	0.5	5.5	Kinase	1195
FCAR	0.2	0.3	2.3	5.9	Other	2204
HSPA1A	1.0	1.0	0.5	13	Other	3303
HSPA1B	1.2	0.7	0.9	6.8	Other	3304
IGFBP6	1.3	0.5	0.7	3.9	Other	3489
MAT 2 A	0.7	0.6	1.4	3.3	Enzyme	4144
NQO1	0.5	0.5	0.3	6.2	Enzyme	1728
SLC30A1	0.2	0.3	2.3	2.8	Transporter	7779
SLC7A11	0.5	0.6	0.6	4.4	Transporter	23657
SOX18	1.0	0.5	1.0	9.7	Transcription regulator	54345
SQSTM1	0.8	0.7	1.3	5.6	Transcription regulator	8878

The cells were treated with PF (160 µg/ml, 30 min) and harvested after 0, 1, and 3 h incubation. Cont, control. The values indicate expression level.

Table III.  Downregulated genes under PF treatment.

		Incubation time (h)
Name	Cont.	0	1	3	Family	Entrez gene ID
Transiently decreased genes
ADRB2	1.0	1.6	0.3	1.4	G-protein coupled receptor	154
APIP	1.6	1.3	0.2	0.7	Other	51074
ASAH1	0.9	1.2	0.2	1.2	Enzyme	427
CALR	1.0	1.4	0.2	1.0	Transcription regulator	811
CPOX	1.0	1.8	0.1	1.0	Enzyme	1371
ELK1	1.0	1.0	0.1	1.8	Transcription regulator	2002
ERBB2IP	1.3	0.7	0.1	2.2	Other	55914
GALNACT-2 0.9	1.1	0.2	1.8		Enzyme	55454
GCLC	0.8	1.3	0.2	2.9	Enzyme	2729
HLA-A	0.9	1.2	0.2	1.3	Transmembrane receptor	3105
IRF2	1.4	2.6	0.5	0.7	Transcription regulator	3660
MAP2K3	1.0	1.0	0.2	1.4	Kinase	5606
NCOA3	2.3	1.2	0.1	0.7	Transcription regulator	8202
PBX3	0.8	1.2	0.2	1.2	Transcription regulator	5090
PTPN11	2.0	0.2	3.3	0.4	Phosphatase	5781
RAB5B	1.2	1.2	0.1	0.8	Enzyme	5869
STAT5B	1.2	1.4	0.2	0.8	Transcription regulator	6777
TPT1	1.1	1.0	0.1	1.0	Other	7178
TM2D1	1.0	1.1	0.2	1.0	G-protein coupled receptor	83941
Gradually decreased genes
ART 3	1.7	1.7	0.3	0.5	Enzyme	419
BCL11A	1.8	1.8	0.1	0.4	Transcription regulator	53335
CD302	1.9	2.1	0.4	0.3	Transmembrane receptor	9936
EGR1	1.1	0.9	1.9	0.0	Transcription regulator	1958
FLU	1.4	1.7	0.2	0.6	Transcription regulator	2313
GRB10	1.4	1.9	0.7	0.3	Other	2887
NGLY1	1.3	1.3	0.8	0.2	Enzyme	55768
REL	1.3	2.2	0.7	0.3	Transcription regulator	5966
RGS2	1.1	1.4	0.9	0.3	Other	5997
UGDH	1.8	2.0	0.3	0.5	Enzyme	7358

The cells were treated with PF (160 µg/ml, 30 min) and harvested after 0, 1, and 3 h incubation. Cont, control. The values indicate expression level.

Figure 2. Genetic networks of genes associated with PF treatment. The cells were treated with PF (160 µg/ml, 30 min) and harvested after 3 h incubation. The genes associated with PF were analysed by the Ingenuity Pathway Analysis tool. The network is displayed graphically as nodes (genes) and edges (biological relationships between nodes).

Figure 3. Verification of microarray results with real-time quantitative PCR assay. The cells were treated with PF (160 µg/ml, 30 min) and harvested after incubation for 0, 1, and 3 h at 37°C. Each mRNA expression level was normalized with GAPDH. Data are presented as mean ± SD (n = 4). (a) ATF5 (Activating transcription factor (5); (b) EGR1 (Early growth response 1); (c) REL (Reticuloendotheliosis viral oncogene homolog); (d) FOSL1 (FOS-like antigen 1); (e) CCL2 (Chemokine (C–C motif) ligand 2); (f) HSPA1 (Heat shock 70 kDa protein B). *p < 0.05 versus control (Student's t test). Cont, control.