Common gene expression patterns responsive to mild temperature hyperthermia in normal human fibroblastic cells

Figures & data

Table I.  The top 40 genes in the cluster Up-I.

Gene symbol	Hs68 (fold change)			OUMS-36 (fold change)			Gene title
	0 h	1 h	3 h	0 h	1 h	3 h
HSPA6	12.4	20.3	1.4	1.5	2.9	0.3	Heat shock 70 kDa protein 6 (HSP70B')
LIF	1.5	16.1	3	0.9	1.5	1.7	Leukaemia inhibitory factor
BHLHE40	1.9	8	2.2	1	4	1.4	Basic helix-loop-helix family, member e40
DLX2	2.5	6.9	2	1	2.9	1.6	Distal-less homeobox 2
EDN1	0.7	6.4	1.4	0.9	4.6	1.4	Endothelin 1
HSPA1A/B	4.8	5.6	3.9	1.1	2.2	1	Heat shock 70 kDa protein 1 A/B
HES1	5.1	5.1	0.5	1	2.4	0.2	Hairy and enhancer of split 1
C6orf62	1.4	3.9	1	1.2	3.8	1.5	Chromosome 6 open reading frame 62
HSPA1L	2.7	3.6	0.9	1.2	2.5	0.4	Heat shock 70 kDa protein 1-like
GADD45B	1.6	3.5	1.3	1.1	1.8	0.8	Growth arrest and DNA damage inducible, beta
HBEGF	1.3	3.5	2.8	1	1.4	1.7	Heparin-binding EGF-like growth factor
ADSSL1	1.6	3.1	1.1	0.6	2.2	0.9	Adenylosuccinate synthase like 1
HMOX1	1.2	3.1	2.5	1	2.4	2.8	Haem oxygenase (decycling) 1
HSPA5	1.2	3	2.6	1.1	2.1	1.8	Heat shock 70 kDa protein 5
HERPUD1	1	2.9	1.1	1	2.8	1.4	Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1
DNAJB1	2.7	2.9	1.3	1.2	2	1.4	DnaJ (Hsp40) homologue, subfamily B, member 1
DNAJB9	1.3	2.8	2.6	0.9	2	1.9	DnaJ (Hsp40) homologue, subfamily B, member 9
CSRNP1	1.6	2.8	0.9	1.1	2.4	0.9	Cysteine-serine-rich nuclear protein 1
SERTAD1	1.6	2.7	1.1	1.1	2	1	SERTA domain containing 1
TUFT1	1.1	2.7	1.7	1	2	1.8	Tuftelin 1
FAM83G	1.4	2.7	1.7	1	1.6	1.2	Family with sequence similarity 83, member G
JMJD6	2.5	2.7	1.6	0.9	2	1.4	Jumonji domain containing 6
DDIT3	0.5	2.6	0.7	0.9	4.7	3.2	DNA damage inducible transcript 3
ZFAND2A	1.4	2.5	1.5	1.1	1.8	1.3	Zinc finger, AN1-type domain 2 A
SDF2L1	0.9	2.5	1.8	1.1	2.2	1.7	Stromal cell-derived factor 2-like 1
ENC1	1.3	2.4	2.6	1	1.7	2.4	Ectodermal–neural cortex 1 (with BTB-like domain)
KLF10	1.4	2.4	1.3	0.9	1.6	0.9	Kruppel-like factor 10
SLC35C2	1.4	2.4	1.1	1.6	1.8	1.6	Solute carrier family 35, member C2
CALD1	0.9	2.4	1.1	1.8	4.6	1.1	Caldesmon 1
STMN1	3.9	2.3	1.5	0.6	2	0.7	Stathmin 1
HSPH1	2	2.3	2.3	1	1.5	1.4	Heat shock 105kDa/110kDa protein 1
NEDD9	0.7	2.3	1.5	1	2.7	1.4	Neural precursor cell expressed, developmentally down-regulated 9
C6orfl45	1.2	2.3	1.7	1.1	2.2	1.4	Chromosome 6 open reading frame 145
EFNB2	1.3	2.3	1.9	1	1.7	1.9	Ephrin-B2
PIP4K2A	0.9	2.2	1	1.6	3.2	1.7	Phosphatidylinositol-5-phosphate 4-kinase, type II, alpha
SMAD7	1.4	2.2	1.3	1	1.8	1	SMAD family member 7
DACT1	1	2.1	1.5	1.1	1.9	1	Dapper, antagonist of beta-catenin, homologue 1
SCHIP1	1.1	2.1	1.1	0.9	1.8	1.1	Schwannomin interacting protein 1
AKAP17A	1.5	2.1	1	0.9	1.8	1	Akinase (PRKA) anchor protein 17A
ANP32E	1.1	2.1	1	0.8	2.1	1.2	Acidic (leucine-rich) nuclear phosphoprotein 32 family member E

Table II.  The top 20 genes in the cluster Down-I.

Gene symbol	Hs68 (fold change)			OUMS-36 (fold change)			Gene title
	0 h	1 h	3 h	0 h	1 h	3 h
PNRC1	0.53	0.21	0.23	0.77	0.34	0.54	Proline-rich nuclear receptor coactivator 1
EGR1	0.87	0.22	0.11	0.88	0.51	0.25	Early growth response 1
NACAP1	0.73	0.25	0.32	0.66	0.51	0.75	Nascent polypeptide-associated complex alpha polypeptide pseudogene 1
KPNB1	0.46	0.3	0.42	0.77	0.61	0.63	Karyopherin (importin) beta 1
HERPUD2	0.77	0.32	0.61	0.87	0.44	0.67	HERPUD family member 2
GAS1	0.77	0.33	0.44	0.93	0.55	0.74	Growth arrest-specific 1
PLAG1	0.87	0.34	0.49	0.81	0.48	0.81	Pleiomorphic adenoma gene 1
TAF1B	0.77	0.37	0.62	0.89	0.54	0.62	TATA box binding protein (TBP)-associated factor, RNA polymerase I, B, 63 kDa
KDM4A	0.9	0.38	0.73	1.28	0.61	0.86	Lysine (K)-specific demethylase 4 A
MSRB3	0.82	0.38	0.83	0.79	0.38	0.69	Methionine sulphoxide reductase B3
PXK	0.86	0.38	0.73	0.78	0.5	0.9	PX domain containing serine/threonine kinase
KLF6	0.68	0.39	1.03	0.95	0.46	1.1	Kruppel-like factor 6
NR2F2	0.55	0.39	0.8	0.94	0.52	1.52	Nuclear receptor subfamily 2, group F, member 2
LMLN	0.87	0.39	0.79	0.8	0.4	0.73	Leishmanolysin-like (metallopeptidase M8 family)
PLDN	0.77	0.4	0.79	0.95	0.41	0.86	Pallidin homologue (mouse)
HEATR6	0.84	0.41	0.77	0.77	0.43	0.84	HEAT repeat containing 6
THSD4	0.96	0.41	0.78	0.84	0.44	0.7	Thrombospondin, type I, domain containing 4
AK3	0.91	0.41	0.79	0.9	0.44	0.83	Adenylate kinase 3
BTG1	0.82	0.42	0.96	1.06	0.61	1.27	B-cell translocation gene 1, anti-proliferative
PHACTR2	0.89	0.42	0.75	0.84	0.51	0.67	Phosphatase and actin regulator 2

Figure 1. The effects of heat on cell viability and the cell cycle in two NHF cell lines, Hs68 and OUMS-36. Cells were exposed to MHT at 41°C for 30 min or HT at 44°C for 30 min. After heat treatment, the cells were incubated for 24 h at 37°C. The cell viability (A, B) and cell cycle (C, D) were measured. (A, C) Hs68 cells. (B, D) OUMS-36 cells. Data indicate the means ± SD for four different experiments. *P < 0.05 versus the control (37°C treatment).

Figure 2. Gene expression analysis. Gene expression analysis of the probe sets that were differentially expressed by a factor of 1.5 or greater in Hs68 and OUMS-36 cells treated with MHT at 41°C for 30 min was performed using bioinformatics analysis tools. (A) Venn diagram of the probe sets that were differentially expressed in Hs68 and OUMS-36 cells. The numbers of probe sets are shown. (B) K-means clustering of commonly and differentially expressed probe sets (1,196 probe sets). Probe sets were grouped in three clusters with distinct expression profiles, Up-I, Down-I and Down-II. The numbers of commonly expressed probe sets and genes are shown. C, control.

Table III.  The top 20 of genes in the cluster Down-II.

Gene symbol	Hs68 (fold change)			OUMS-36 (fold change)			Gene title
	0 h	1 h	3 h	0 h	1 h	3 h
CCDC88A	1.65	0.09	1. 01	0.69	0.06	0.39	Coiled coil domain containing 88 A
AGGF1	0.74	0.14	0.44	0.36	0.22	0.69	Angiogenic factor with G patch and FHA domains 1
ANKRD12	0.4	0.14	0.31	0.64	0.13	0.48	Ankyrin repeat domain 12
CHD9	1.74	0.15	1.11	1.02	0.12	0.55	Chromodomain helicase DNA binding protein 9
PHACTR2	1.28	0.15	1.18	0.93	0.15	0.59	Phosphatase and actin regulator 2
ATRX	1.06	0.16	0.86	0.81	0.14	0.63	Alpha thalassemia/mental retardation syndrome X-linked
C10orf118	1.26	0.16	1.05	1.02	0.13	0.88	Chromosome 10 open reading frame 118
SMC3	1.03	0.17	0.88	0.81	0.14	0.71	Structural maintenance of chromosomes 3
THRAP3	1.03	0.17	0.99	0.93	0.14	0.69	Thyroid hormone receptor associated protein 3
MALAT1	1.08	0.17	0.96	0.51	0.13	0.49	Metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)
CEP290	0.99	0.18	0.74	0.56	0.15	0.46	Centrosomal protein 290 kDa
ASPM	1.52	0.19	1.03	0.66	0.14	0.51	Asp (abnormal spindle) homolog, microcephaly associated
SLFN5	1.05	0.19	0.77	0.9	0.19	0.76	Schlafen family member 5
SFRP1	1.22	0.2	1.04	0.86	0.18	0.98	Secreted frizzled-related protein 1
OSBPL8	1.88	0.2	2.3	0.7	0.08	0.73	Oxysterol binding protein-like 8
LUZP1	1.2	0.2	1.37	0.85	0.21	0.89	Leucine zipper protein 1
VEZF1	0.73	0.21	0.59	0.95	0.26	0.87	Vascular endothelial zinc finger 1
SPAG9	1.07	0.21	1.08	0.85	0.19	0.77	Sperm associated antigen 9
ABHD2	1.07	0.21	1.02	0.9	0.27	0.62	Abhydrolase domain containing 2
CASC5	1.16	0.21	0.84	0.9	0.16	0.54	Cancer susceptibility candidate 5

Figure 3. Gene networks. Up-regulated genes in the cluster Up-I were analysed by Ingenuity® Pathways Analysis tools. Gene networks E (A) and S (B) associated with ER stress and cell survival, respectively, were identified. The network is displayed graphically as nodes (genes) and edges (the biological relationships between the nodes). The node colour of genes indicates the expression level of genes. Nodes and edges are displayed using various shapes and labels that present the functional class of genes and the nature of the relationship between the nodes, respectively.

Figure 4. Effects of MHT on the time-course of changes in the expression levels of genes in the gene network E. Cells of the NHF lines Hs68 (Hs), KD, NTI-4 (NTI) and OUMS-36 (OUMS) were incubated at 41°C for 30 min and then cultured at 37°C for 0, 1 and 3 h. Real-time qPCR was carried out. DDIT3 (A), DNAJB1 (B), HERPUD1 (C), HMOX1 (D), HSPA1A/B (E), HSPA1L (F), HSPA5 (G), HSPA6 (H), HSPH1 (I) and PPP1R15A (J) were included in the gene network E. The expression level of each mRNA was normalised to the GAPDH expression level. Data are presented as the mean ± SD (n = 4). *P < 0.05 versus the control.

Figure 5. Effects of MHT on the time-course of changes in the expression levels of genes in the gene network S. Cells of the NHF cells Hs68 (Hs), KD, NTI-4 (NTI) and OUMS-36 (OUMS) were incubated at 41°C for 30 min and then cultured at 37°C for 0, 1 and 3 h. Real-time qPCR was carried out. BHLHE40 (A), EDN1 (B), GADD45A (C), GADD45B (D), HBEGF (E), HES1 (F), ID2 (G), KLF10 (H), LIF (I), NEDD9 (J) and SMAD7 (K) were included in the gene network S. The expression level of each mRNA was normalised to the GAPDH expression level. Data are presented as the mean ± SD (n = 4). *P < 0.05 versus the control.