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COMPARATIVE ANALYSIS OF THE EXPRESSION PROFILES OF GENES RELATED TO THE GADD45α SIGNALING PATHWAY IN FOUR KINDS OF

LIVER DISEASES

Authors: Xianguang Yang, Xuelin Chen, Cong Xia, Shuaihong Li, Lin Zhu and Cunshuan Xu DOI: 10.14670/HH-18-218 Article type: ORIGINAL ARTICLE Accepted: 2020-04-16 Epub ahead of print: 2020-04-16

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Comparative analysis of the expression profiles of genes related to the

Gadd45α signaling pathway in four kinds of liver diseases

Xianguang Yangab*, Xuelin Chenab, Cong Xiaab,Shuaihong Liab, Lin Zhuab

and Cunshuan Xuab

a College of Life Science, Henan Normal University, Xinxiang 453007, Henan

Province, China;

b State Key Laboratory Cultivation Base for Cell Differentiation Regulation and Henan

Bioengineering Key Laboratory, Henan Normal University, Xinxiang 453007, Henan

Province, China;

*Corresponding author: Xianguang Yang, College of Life Science, Henan Normal

University, No. 46, Construction East Road, Xinxiang 453007, Henan Province, China.

Tel., +86-373-3326340; fax: +86-373-3329102, E-mail: yangxg@htu.cn

Running Head:

Gadd45α signaling pathway in liver diseases

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Comparative analysis of the expression profiles of genes related to the

Gadd45α signaling pathway in four kinds of liver diseases

Abstract

Gadd45α (growth arrest and DNA damage inducible alpha) is a member of a

group of genes whose transcript levels are increased following stressful

conditions that lead to growth arrest and treatment with agents that lead to DNA

damage. Gadd45α is upregulated in liver cirrhosis (LC), hepatic cancer (HC),

acute liver failure (AHF) and non-alcoholic fatty liver disease(NAFLD). Here,

we investigated the essential differences in the Gadd45α signaling pathway in

these diseases at the transcriptional level. The results showed that 44, 46, 71 and

27 genes significant changes in these diseases, and the H-cluster showed that the

expression of the Gadd45α signaling-related genes was significantly different in

the four liver diseases. DAVID functional analysis showed that the Gadd45α

signaling pathway-related genes were mainly involved in cell adhesion and

migration, cell proliferation, apoptosis, stress and inflammatory responses, etc.

Ingenuity pathway analysis (IPA) software was used to predict the functions of

the Gadd45α signaling-related genes, and the results indicated that there were

significant changes in cell differentiation, DNA damage repair, autophagy,

apoptosis and necrosis. Gadd45α signaling pathway is involved in four kinds of

liver disease and regulates a variety of activities via P38 MAPK, NF-κB,

mTOR/STAT3, P21, PCNA, PI3K/Akt and other signaling pathways. Modulation

of Gadd45α may be exploited to prevent the progression of liver disease, and to

identify specific treatments for different stages of liver disease. In summary, the

Gadd45α signaling pathway is involved in four kinds of liver disease and

regulates a variety of physiological activities through various signaling pathways.

Keywords: Gadd45α signaling pathway; liver cirrhosis; hepatic tumor; acute

hepatic failure; non-alcoholic fatty liver disease

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1. Introduction

The liver is the largest digestive gland in the body. The liver has a wide range of

functions, including detoxification, protein synthesis, and biochemical production,

which is necessary for digestion. Liver cirrhosis (LC), the end stage of many forms of

chronic hepatitis of different etiologies, is a widespread condition characterized by

active fibrosis and the conversion of normal liver architecture into structurally abnormal

nodules surrounded by annular fibrosis;LC is predominantly related to chronic viral

infection, alcohol consumption, and autoimmune and metabolic etiologies (Wang et al.,

2007; Soresi et al., 2014). Hepatic cancer (HC) is a primary malignancy of the liver

what frequently arises in patients with liver cirrhosis and is the result of a complex

pathogenesis involving chronic hepatitis, necrosis, and regenerative and active fibrosis.

Acute hepatic failure (AHF) is a syndrome caused by multiple factors, such as toxic or

infectious agents, and is characterized by massive hepatocyte degeneration and necrosis

as well as inflammatory cell infiltration shortly after injury (Reddy et al., 2016).

Nonalcoholic fatty fiver disease (NAFLD) is defined as fat content exceeding 5 % of the

liver weight in the absence of excessive alcohol intake or other causes of steatosis, such

as viral hepatitis, autoimmune hepatitis, Wilson disease, alpha-1-antitrypsin deficiency,

or other metabolic causes (Uppal et al., 2016) .

Gadd45α is a stress-induced gene that is constitutively expressed at low levels

but is transcriptionally activated by a variety of stress stimuli, including ultraviolet and

ionizing radiation; Gadd45α interacts with a number of intracellular signaling molecules

involved in cell cycle regulation, cell death and senescence, apoptosis, genomic stability

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maintenance, immune responses, and in DNA damage repair. In general, Gadd45α

provides cellular stability either by arresting cell cycle progression until DNA damage

is repaired or, in cases of fatal damage, by inducing apoptosis. As a stress-induced gene,

Gadd45α has been found to be abundantly expressed in various liver diseases, and is

closely related to the development and prognosis of liver cancer, liver fibrosis and fatty

liver. Increased levels of the GADD45α transcript and protein are seen after the

treatment of cells with ionizing radiation and other agents and treatments that damage

DNA. In dimethylbenzanthracene (DMBA)- induced liver cancer in mice, the lack of

basal and DMBA-induced Gadd45α may result in enhanced tumorigenesis because of

decreased DNA repair and increased mutation frequency (Hollander et al., 2001). In our

study, the expression of the Gadd45α gene and protein was significantly in the

hepatocytes of rats undergoing; in addition, the high expression of Gadd45α was

involved in the acute stress response of hepatocytes and may be involved in the process

of hepatocyte apoptosis during acute liver failure (Wang, 2016). Bae et al. demonstrated

that KR62776 increased the levels of GADD45 in hepatic stellate cells (HSCs) and may

have potential in the chemoprevention of liver fibrosis/cirrhosis (Bae et al., 2010). The

role of Gadd45α in non-alcoholic fatty liver disedse has not been reported.

Gadd45 exerts a protective effect on CCl4-induced liver fibrosis by inhibiting

TGF-TGF / smad-mediated liver fibrosis gene expression, regulating HSC activation

and ROS production (Hong et al., 2016). BRCA1 regulates the stability of downstream

genes by promoting the negative regulation of carcinogenesis and tumor malignancy by

Gadd45α (Li et al., 2015). Gadd45α activates p38 MAPK through H-ras thereby

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inhibiting tumorigenesis (Bulavin et al., 2003). Gadd45α modulates the innate immune

functions of granulocytes and macrophages by differentially regulating p38 and JNK

signaling, including reactive oxygen species production, phagocytosis, and adhesion

(Salerno et al., 2012). Gadd45α suppresses tumor angiogenesis via the inhibition of the

mTOR/STAT3 protein pathway (Yang et al., 2013). The study by Meyer NJ revealed

that Gadd45α exerts its function in inflammatory lung injury induced by

lipopolysaccharide (LPS)-and ventilators by influencing Akt signalling (Meyer et al.,

2009). GADD45Α and p21 bind to CDK1, resulting in the dissociation of the cyclin

B/CDK1 complex and the eventual induction of cell cycle arrest at G2/M (Katagi et al.,

2016). The study performed by Jung et al. proved of APE1 enzyme activity via the

GADD45α, PCNA and APE1 protein complex regulates p53-mediated base excision

repair by APEX1 (Jung et al., 2013). Gadd45α disruption strengthens the association

between mTOR and STAT3 and induces the phosphorylation of STAT3 on Ser-727 to

enhance its transcriptional activity and inhibit tumor angiogenesis (Yang et al., 2013).

Zhang et al. found that GADD45Α inhibits autophagy by impairing BECN1-PIK3C3

complex formation.GADD45Α is able to induce cancer cell death through 2 important

mechanisms: apoptosis and autophagy (Zhang et al., 2015).

Therefore, to further understand the role of Gadd45α in the pathogenesis of liver

diseases, we established a network of the Gadd45α signaling pathway that illustrates the

main downstream signaling pathways that are possibly activated by Gadd45α in liver

cirrhosis (LC), hepatic cancer (HC), acute liver failure (AHF) and nonalcoholic fatty

liver disease. Therefore, to further comprehensively and systematically understand the

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role of the Gadd45α signaling pathway in this liver diseases, established models of LC,

HC ,AHF, and NAFLD and used a rat whole genome gene expression profiling chip, the

Rat Genome 230 2.0 Array, to identify the expression profiles of Gadd45α signaling

pathway-related genes at transcriptional level.Then, the role of Gadd45α in the

occurrence and development of the four liver diseases listed above was analyzed by

bioinformatics and systems biology methods, including ingenuity pathway analysis

(IPA) software; this analysis will be helpful in exploring the molecular role of Gadd45α

in various liver diseases.

2. Materials and methods

2.1. Preparation of rat liver disease models

Adult healthy Sprague–Dawley (SD) rats (200 ± 10 g), which were supplied by the

Experimental Animal Center of Henan Normal University, were housed in a room with

a controlled temperature (22 ± 1°C) with a 12:12 h light–dark cycle. All the rats were

fed a standard rodent chow diet and allowed free access to food and distilled water. All

the handling procedures were carried out in accordance with the current Animal

Protection Law of China. All the procedures in this study that involved were performed

in accordance with the standard protocols approved by the Ethical Committee of Henan

Normal University.

LC model: Rats were randomly divided into a model group with 18 rats and a

control group with 6 rats. The rats in the LC model group were fed normal food and

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0.35g/L phenobarbital sodium solution in the first week. A dose of 0.5ml/100g CCl4

diluted 2:3 in colza oil was injected into the abdominal cavity twice per week in the

2nd-4th weeks, and the only drinking water given to these rats contained 10% alcohol.

The drinking water was exchanged with water containing 30% alcohol in the 5th-8th

weeks. The livers were harvested for analysis at 3 w, 6 wand 9 w after CCl4

administration. The rats in the control group received injections of the same volume of

physiological saline at the corresponding timepoints.

HC model: A total of 60 male rats were randomly divided into 6 groups,

including a model group with 50 rats and a control group with 10 rats. The rats in the

model group underwent intragastric administration of diethyl nitrosamine (DENA) (7

mg/100 g) once a week for 20 consecutive weeks, while the saline control group

received intragastric administration of the same volume of physiological saline. The

DENA solution was prepared by mixing 99.75 ml of distilled water and 0.25 ml of

DENA concentrate. The rats were anesthetized by ether and sacrificed with cervical

dislocation at the end of 0, 5, 8, 12, 16 and 18 weeks after DENA-treatment.

AHF model: A total of 36 male rats were randomly divided into a model group

with 24 rats and a control group with 12 rats. Carbon tetrachloride (CCl4) was diluted

2:3 in sesame oil under sterile conditions. The rats were fed a single dose of 4 ml/kg

diluted CCl4(Li et al., 2011). The rat livers were harvested for analysis at each of the

following time points: 3, 6, 12, 24, 48 and 72 h after CCl4 treatment.

NAFLD model: Rats were randomly divided into a normal control group (NC

group, 10 rats) and a high-fat emulsion model group (HF group, 30 rats). All the rats

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were fed a standard rodent chow diet and allowed free access to an aqueous solution

containing 18% sucrose. The model rats were orally treated with the high-fat emulsion

(10 ml/kg) via gavage once per day for 6 weeks. The compositions of the high-fat

emulsion diet were as follows: corn oil 400 g, saccharose 150 g, milk powder 80 g,

cholesterol 100 g, sodium deoxycholate 10 g, Tween-80 36.4 g, propylene glycol 31.1

g, vitamin mixture 2.5 g, cooking salt 10 g, mineral mixture 1.5 g, and distilled water

300 ml (Lai, 2005). The rats were sacrificed at 2, 4, and 6 weeks after high-fat emulsion

feeding. The control group was fed the same volume of physiological saline at the same

time.

The rats were sacrificed by cervical dislocation at the abovementioned time

points in each of the liver disease model. Four small cuboids of approximately

5mm×5mm× (2-3) mm were excised from the middle part of the right liver lobe for

histopathology. The remaining liver tissue from the middle part of the right liver lobe

were collected in RNase-free tubes and stored at﹣80°C until use. All the operations

and handling procedures were carried out in accordance with the current Animal

Protection Law of China.

2.2. Construction of the Gadd45α-mediated signaling pathway network

Previous studies have reported that Gadd45α could be activated by UV / ionizing

radiation and then exert its function via the P38 MAPK,NF-κB (Hur et al., 2008),

mTOR/STAT3 (Yang et al., 2013), P21 (Chen et al., 2014a), PCNA (Kim et al., 2013)

and PI3K/Akt (Amente et al., 2011) signaling pathways. Therefore, “P38 MAPK

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signaling pathway”, “NF-κB signaling pathway”, “mTOR/STAT3 signaling pathway”,

“P21 signaling pathway”, “PCNA signaling pathway” and “PI3K/Aktsignaling

pathway” were entered into the “Pathway and Function” of the Ingenuity Pathway

Analysis (IPA) software to obtain the interaction network of these pathway and

Gadd45α. The above results were filtered and integrated by researching the signaling

pathway maps with the Gen MAPP, KEGG, BIOCARTA, QIAGEN and Biocompare

databases, and then the network of Gadd45α-mediated signaling pathway was

confirmed with relevant articles.

2.3. Rat Genome 230 2.0 microarray assessment and data analysis

The transcription profiles of the liver tissues from the abovementioned four liver disease

models were determined by Rat Genome 230 2.0 chips as previously described,

including cDNA synthesis, in vitro transcription, cRNA hybridization, microarray wash,

image scanning and data analysis (Guo and Xu, 2008). The genes were defined as

present (P<0.05), marginal (0.05<P<0.065) and absent (P>0.065) according to the

P-values of the probe signal. Then, the data of each array were initially normalized, and

the ratio at each time point was calculated by comparing the normalized signal values of

the experimental group to those the control group (0 h). To minimize the differences in

the experimental operation and microarray test, each sample was repeated three times,

and the average value was used for statistical analysis. Finally, the relative gene

expression level was obtained by dividing the normalized signal values in the

experimental groups by that in the control group. When the relative expression was≥

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1.5-fold higher than that of the control group, we considered the gene to be significantly

upregulated, when the ralative value was≤ 2/3 of that in the control group,we considered

the gene be significantly downregulated, and when the relative value was between 2/3

and 1.5, we considered the gene to have no biologically meaningful change

expression.To reduce the analytical error of the microarray, this study assessed each

sample at least, three times and considered the average of three relative values as a

credible value to bean accurate value. The coefficients of variation (CV) for the three

technical repeats of every gene were below 6%.

2.4. Real-time quantitative RT-PCR

To verify the chip data, a total of eight genes were selected. Primers were designed with

Primer Express 2.0 software according to the mRNA sequences of the eight target genes

Lcn2, Myc, Tgfb1, Alpl, Cyp1a1, Mgmt, Spc24 and A2m and the internal control gene

β-actin, and the primers were synthesized by Sangon Biotech (Shanghai) Co.Ltd .(Table

1). Prior to RT, the contaminating genomic DNA in the total RNA was removed by

DNase I (Promega, Madison, USA). Total RNA (2 μg) was reverse-transcribed using

random primers and a Reverse Transcription kit (Promega, Madison, USA). The

first-strand cDNA samples were subjected to quantitative PCR by using SYBR Green I

on a Rotor-Gene 3000A instrument (Corbett Robotics, Brisbane, Australia), and each

sample was analyzed in triplicate. As described previously, the changes in the relative

expression of the target genes were calculated according to that of the internal control

β-actin (Wang and Xu, 2010).

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2.5. Expression profiling and function prediction of the genes in

Gadd45α-mediated signaling pathway

The probe number of the Gadd45αsignaling pathway-related genes was used for

DAVID analysis by the EASE analysis software (http://david.abcc.ncifcrf.gov), and the

enriched GO categories were selected from each cluster according to the EASE scores.

The EASE score is a significance level, Smaller EASE scores indicate increasing

confidence in overrepresentation, and the GO categories with EASE scores of <0.01

represented classes of genes in which individual members occurred more often than

would be expected by a random distribution of genes.

2.6. Prediction of function of the Gadd45α signaling pathway in four kinds of

liver disease

To elucidate the function of the Gadd45αsignaling pathway in the four kinds of liver

disease the Gadd45αsignaling pathway-related genes were summarized. Then, the

ingenuity pathway analysis (IPA) software ver. 9.0 was utilized to analyze the heatmap

of the biofunctions based on the changes in the expression changes of the Gadd45α

signaling-related genes in each liver disease using comparison analysis. Briefly, the

differentially expressed genes and their extreme expression values in the four kinds of

liver diseases were uploaded to the ‘Dataset Files’ of IPA. Then, core analysis and

comparison analyses proceeded successively. Next, heatmaps of biofunctions were

obtained in the model of ‘Diseases and Functions’; each square of the heatmap can be

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clicked to display the network and the possible crucial genes in the network.

3. Results

3.1. Gadd45α-mediated signaling pathway network

The IPA software and several databases (including Gen MAPP, KEGG, BIOCARTA,

QIAGEN and Biocompare) were mainly used to construct the interaction network of the

Gadd45α signaling pathway, and articles were used to confirm to the network. The

results showed that 7 branches were involved in the Gadd45α-mediated signaling

pathway (Fig.1). In branches 1 and 2, Gadd45α promotes cell proliferation by directly

stimulating modification MAP3K4.Branch 1: GADD45α→ MAP3K4→ MKK3/6 → P

38 MAPK→MAPKMAPK2/3→IκB→NF-κB, GADD45α→ MAP3K4→ MKK3/6→

P38 MAPK→ ATF2, GADD45α→MAP3K4→MKK3/6→ P38 MAPK→HSP27;

Branch 2: GADD45α→MAP3K4→MKK4/7→JNK→JUN→FAS. In branches 3 and 4,

GADD45α directly inhibits the modification of DNA repair. Branch 3:

GADD45α→P21┤ CDK2/CCNE; Branch 4:

GADD45αAPEX1┤PCNA→CDK1/CCNB1.GADD45α promotes the G2 / M phase

transition in branch 5. Branch 5: GADD45α┤ MTOR→STAT3→MYC→CDC25A. In

branch 6, GADD45α directly inhibits AKT and promotes apoptosis. Branch 6:

GADD45α┤AKT┤ BAD┤BCL2→ BAX→ CASPASES. Taken together, GADD45α

may function through the abovementioned branches to regulate several vital biological

processes including cell proliferation, DNA repair, G2/M phase transition and apoptosis

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responses.

3.2. Global analysis of the expression profiles of Gadd45α signaling

pathway-related genes in the 4 liver diseases

The Rat Genome 230 2.0 Array was used to determine the genome-wide expression

profile of liver tissues from rat models of LC, HC, AHF and NAFLD models, and the

obtained microarray data were submitted to the Gene Expression Omnibus database

with the accession numbers GSE73499, GSE73498, GSE73494 and GSE73500,

respectively. In each liver disease model, Gadd45α signaling pathway related genes

were selected for further analysis, and the expression of levels of a total of 84 genes

were examined in the four kinds of liver disease. Among the Gadd45α signaling

pathway-related genes, 44 genes were significantly changed, including 30 upregulated,

12 down-regulated and 2 upregulated/downregulated genes at 3, 6 and 9 weeks in the

LC model. However, in HC, it was found that 46 known genes were significantly

changed, of which 38 were upregulated,7 were downregulated and 1 was

upregulated/downregulated at 5, 8, 12, 16 and 18 weeks. A total of 71 known genes

were identified in AHF, including 43 upregulated, 20 downregulated and 8

upregulated/downregulated genes at 3, 6, 12, 24, 48 and 72 h. In NAFLD, the

expression of 27 genes was significantly changed, comprising 18 upregulated, 8

downregulated and 1 upregulated/downregulated genes at 0, 2, 4 and 6 weeks (Fig. 2).

To comprehensively characterize the expression patterns of the genes related to

Gadd45α-mediated signaling pathway during LC, HC, AHF and NAFLD, we employed

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hierarchical means (H-means) to classify the significantly changed Gadd45α signaling

pathway-related genes according to gene expression similarity, and the results indicated

that the number of upregulated genes in LC, HC, AHF and NAFLD was different from

that of downregulated genes (Fig.3). Based on the expression similarity shown by the

dendrogram, 2, 4 and 6 weeks of NAFLD and 24, 48 and 72 h of AHF were clustered as

two separate groups in major branches (Fig.4). Moreover, 6 and 9 weeks of LC and 3, 6,

12 and 24 h of AHF were clustered together; 5, 8, 12, 16 and 18 weeks of HC and 48

and 72 h of AHF were clustered together; and 3 weeks of LC and 2, 4 and 6 weeks of

were clustered together. It was clear that some time points of LC, HC, AHF and

NAFLD were highly correlated, although their gene expression profiles were different

overall. Therefore, the correlation between the gene expression profiles of these time

points requires further exploration.

3.3. Real-time quantitative RT-PCR

To confirm the results of the microarray analysis, real-time quantitative RT-PCR assays

were used to measure the changes in the expression of the genes, including Lcn2 and

Myc in LC at 3, 6 and 9 weeks; Lcn2 and Tgfb1 in NAFLD at 2, 4 and 6 weeks; Alpl,

Cyp1a1, Mgmt and Myc in HC at 5, 8, 12, 16 and 18 weeks; and Spc24, A2m, Cyp1a1

and Lcn2 in AHF at 2, 6, 12, 24, 48 and 72 h. The results showed that, the expression

patterns of the genes as measured by the two methods above were generally consistent,

suggesting that the results of the array analysis were reliable; however, there were

differences between the relative degrees of upregulation as measured by RT-PCR and

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microarray(Fig.4), and these differences may be caused by the use of two different

detection methods.

3.4. Analysis of the prediction of the function of the Gadd45α signaling

pathway-related genes in the4 liver diseases

In this study, we performed EASE analysis on the gene sets in Table 2, and the

overrepresented GO categories were selected according to the EASE scores. For each

category, the number of genes identified in the category precedes the modified EASE

score (P value). The results showed that the genes related to apoptosis, migration, cell

cycle, inflammation. etc. were involved in the four liver diseases (Table 2). Obviously,

Gadd45α signaling pathway related genes were mainly associated with apoptosis,

migration, cell cycle, DNA damage checkpoints, cell proliferation, and inflammatory

response.

Furthermore, to elucidate the changes in the physiological processes, the

changes in the expression of the Gadd45α signaling pathway-related genes in the four

kinds of liver disease were determined, and their expression extremes were uploaded

into IPA. Then, core analysis and comparison analyses were performed successively.

The heatmap of the biofunctions indicated that various physiological processes were

significantly changed in LC, NAFLD and AHF, while these processes exhibited few

changes or even no change in HC (Fig.5). Among these processes, cell survival

increased in LC, HC, and AHF, and cell proliferation increased in HC and NAFLD. LC

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Cell migration was decreased at 6 w in LC, whereas it was upregulated at early time

points and downregulated at later time points in AHF and NAFLD.

4. Discussion

In this study, we used the Rat Genome 230 2.0 Array to determine the expression

profiles of Gadd45α signaling pathway-related genes in four kinds of liver disease,

including LC, HC, AHF, and NAFLD, and the results revealed that a total of 84 genes

were significantly changed at the mRNA level. Then, the expression profiles of

Gadd45α signaling pathway-related genes were analyzed by H-clustering and K-means

clustering, and the dendrogram results surprisingly indicated that 6 and 9 weeks of LC

and 3, 6, 12 and 24 h of AHF were clustered together; 5, 8, 12, 16 and 18 weeks of HC

and 48 and 72 h of AHF were clustered together; and 3 weeks of LC and 2, 4 and 6

weeks of were clustered together.

Yu et al. suggested that the deletion of GADD45α (GADD45α-/-) in mouse

embryonic fibroblasts (MEFs) resulted in an increase in the phosphorylation of

MKK4/7 and MKK3/6 and, consequently, a specific increase in the activation of

JNK/p38 and their downstream transcription factors, which are implicated in several

physiological processes, including proliferation, apoptosis and differentiation (Yu et al.,

2013). The progression of HCC is characterized by abnormal cell differentiation and

rapid infiltrating growth (Lazarevich et al., 2004). In hepatocellular carcinoma, DEN

treatment induced DNA damage in hepatocytes (Chen et al., 2015b). Our results also

showed that the physiological activities of DNA degradation were enhanced.

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Researchers have indicated that the expression of Gadd45α was upregulated in the

hepatic carcinoma cells lines Bel7402 and SMMC772 (Xiu et al., 2014), and the

DNA-damage response gene GADD45Α induced the differentiation of hematopoietic

stem the cells without inhibiting cell cycle or survival (Wingert et al., 2016). In

glioblastomamultiforme cells, transfection of GADD45Α significantly decreased cell

viability, and this effect was inhibited by cotransfection with FTL (Wang et al., 2017).

Wang et al. found that AgNPs produced a strong dose-dependent increase in the

transcriptional activation of Gadd45α and a significant decreases in cell viability. Su et

al. confirmed that overexpression of Gadd45α in human lung adenocarcinoma cells did

not affect viability or survival during exposure (Roper et al., 2005). Overall, cell

viability was not dependent on GADD45Α (Su et al., 2014). In summary, the expression

of Gadd45α was induced by DNA damage in liver cells, which regulated physiological

activities, such as cell differentiation in hepatocellular carcinoma, but had no effect on

hepatocarcinoma cell viability. In this study, we found that cell viability was

significantly enhanced, and may be regulated by other signaling pathways.

In a CCl4 - induced liver cirrhosis model, hepatocellular DNA damage

happened after CCl4 treatment (Garcia-Fernandez et al., 2005; Alkreathy et al., 2014),

and Gadd45α was highly expressed in cirrhosis. After SD rats were exposed to

trichloroethylene (TCE), the expression of Gadd45α in the skin tissue was significantly

enhanced. It was conclude that DNA damage and the immune response induced the

expression of Gadd45α. The Gadd45α and CDKN1A proteins were associated with high

repair activity and low-to-moderate proliferative capacity in cirrhotic and HCC tissues

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(Gramantieri et al., 2005). In summary, the overexpression of Gadd45α promotes the

repair of DNA damage. It is generally known that liver cells show significant apoptosis

during cirrhosis induced by diethyl nitrosamine (DEN) show (Xu et al., 2010).

Recently, Gadd45α has been shown to have a pro-apoptotic role via the activation of the

p38 and JNK kinases (Zhu et al., 2009). It was suggested that Gadd45α may play an

important role in promoting DNA repair, necrotic hepatocyte apoptosis and tissue repair

in liver cirrhosis. Wang et al. pointed out that cell proliferation is a feature of liver

fibrosis and cirrhosis, and the liver can compensate for damaged liver tissue by cell

proliferation (Chung et al., 2005; Wang, 2007); however, overexpression of Gadd45α

suppresses cell proliferation in numerous cell lines (Jin et al., 2002). In this study, cell

proliferation in liver cirrhosis increased at 6 w, but decreased at 9 w; thus,

Gadd45α-regulated cell proliferation in cirrhosis needs to be further studied.

Autophagy plays an important role in the progression of obesity-related liver

complications and the evolution of fat into hepatocellular carcinoma (Lavallard and

Gual, 2014).In skeletal muscle atrophy caused by the stress response, Gadd45α

stimulates muscle atrophy-related proteins, including autophagy and caspase -mediated

protein hydrolysis (Ebert et al., 2012). In this study, we found that autophay was

enhanced in the late stage of non-alcoholic fatty liver disease, so Gadd45α may be

involved in the regulation of autophagy in the late stage of NAFLD. Chemokine

expression on and by endothelial cells is important for immune cell recruitment,

adhesion and transmigration through the endothelium (Oo et al., 2010). CCL2 has been

shown to be particularly relevant to NAFLD, in which it is involved in the recruitment

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of inflammatory cells expressing its receptor CCR2 (McMahan et al., 2016). This study

showed that cell migration was increased in early and mid-NAFLD, and Gadd45α may

be involved in human trophoblast migration and invasion (Liu et al., 2014). Gadd45α

may be involved in cell migration and adhesion in the early and middle stages of

NAFLD. These results revealed that Gadd45α plays an essential role in gene-specific

active DNA demethylation during adult stem cell differentiation. The genes involved in

cell differentiation change significantly in the progression of NAFLD (Wang et al.,

2013), and Gadd45α plays an essential role in gene-specific active DNA demethylation

during adult stem cell differentiation (Zhang et al., 2011). Another recent study revealed

a protective role for GADD45α in non-alcoholic steatohepatitis, a common liver disease

worldwide (Tanaka et al., 2017).This study found that cell differentiation was

significantly enhanced in NAFLD, so Gadd45α may have a significant impact on cell

differentiation in NAFLD.

Bandi et al. found that DNA damage repair and inflammation-related gene

expression was upregulation in liver injury, indicating that the repair of DNA damage is

activated during the course of liver injury (Bandi et al., 2011). In this study, we found

that DNA damage repair was upregulated at 48-72 h of AHF. Chen et al. indicated that

Gadd45α has an important function in regulating HSC stress responses by preventing

excessive proliferation after 5-FU treatment or serial transplantation, promoting DNA

damage repair and eliminating damaged HSCs after IR-induced DNA damage (Chen et

al., 2014b). Above all, the Gadd45α signaling pathway may regulate DNA damage and

repair in AHF. Studies have shown that the infiltration of inflammatory cells was

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significantly enhanced in the early acute liver failure induced by D-galactosamine and

lipopolysaccharide (Chen et al., 2015a), Liver failure is accompanied by an increase in

the adhesion and migration of white blood cells, especially monocytes and macrophages

(Sole et al., 2016). During the invasion of epithelial cells by bacteria, Gadd45α was

activated which may activate apoptosis through the regulation of its signaling pathway

(Lembo-Fazio et al., 2011). This study found that DNA damage was reduced in acute

liver failure, while the inflammatory cell migration was increased. These results suggest

that Gadd45α may be closely related to the infiltration and migration of cells in acute

liver failure. Gadd45α inhibits cell migration and invasion by altering global RNA

expression; therefore, the intrinsic effect of Gadd45α on the migration of inflammatory

cells in acute hepatic failure is worthy of further study.

The results showed that the activities of various physiological processes

including cell differentiation, DNA repair, autophagy, apoptosis, and necrosis were

significantly different ROS production, cell adhesion and migration, cell proliferation

etc., were significantly changed, However, there are many differences in cell

proliferation and inflammatory cell migration among the four kinds of liver disease,

which must be verified by further studies. Furthermore, it is more important for this

study to validate the microarray results in human liver biopsy samples, and we will try

to study the expression of Gadd45α signaling pathway-related genes or proteins in the

four kinds of human liver diseases by microarray or 2-DE/MS on a large scale. In

addition, the conclusions were drawn mainly based on the microarray data, and there

was no direct cause-effect relationship between gene profiling and the conclusions.

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Therefore, to demonstrate that these changes are truly dependent upon Gadd45α,

experiments of Gadd45α overexpression or knockdown are needed to further analyze

the regulatory impact of the Gadd45α signaling pathway on various liver diseases.

In this study, the Gadd45α pathway-related genes were functionally analyzed by

DAVID, and the results showed that the Gadd45α signaling pathway was mainly

involved in apoptosis, migration, cell cycle, DNA damage checkpoints, cell

proliferation, and inflammatory response. The function of the Gadd45α-related genes in

the four kinds of liver disease was predicted by IPA. The activity of necrosis, invasion

and migration of cells was significantly enhanced. Collectively, the Gadd45α signaling

pathway was involved in a variety of physiological activities via P38 MAPK, NF-κB,

mTOR/STAT3, P21, PCNA, PI3K/Akt and other signaling pathways, and the Gadd45α

signaling pathway regulated the progression of LC, HC, AHF and NAFLD.

Funding

This study was funded by the National Natural Science Foundation Project of China

(No. U1404312, U1704182), the Science and Technology Project of Henan Province

(No. 182300410334), and the Project of Young Cadre Teacher in Henan Province (No.

2017GGJS045).

Declaration of Interests

The authors report no conflicts of interest.

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Acknowledgments

We thank Dr.Gaiping Wang and Cuifang Chang for their comments during revision of the

manuscript.

References

Alkreathy H.M., Khan R.A., Khan M.R. and Sahreen S. (2014). Ccl4 induced genotoxicity and DNA oxidative damages in rats: Hepatoprotective effect of sonchus arvensis. BMC complementary and alternative medicine. 14, 452.

Amente S., Zhang J., Lavadera M.L., Lania L., Avvedimento E.V. and Majello B. (2011). Myc and pi3k/akt signaling cooperatively repress foxo3a-dependent puma and gadd45a gene expression. Nucleic Acids Res. 39, 9498-9507.

Bae M.A., Rhee S.D., Jung W.H., Ahn J.H., Song B.J. and Cheon H.G. (2010). Selective inhibition of activated stellate cells and protection from carbon tetrachloride-induced liver injury in rats by a new ppargamma agonist kr62776. Arch. Pharm. Res. 33, 433-442.

Bandi S., Joseph B., Berishvili E., Singhania R., Wu Y.M., Cheng K. and Gupta S. (2011). Perturbations in ataxia telangiectasia mutant signaling pathways after drug-induced acute liver failure and their reversal during rescue of animals by cell therapy. Am. J. Pathol. 178, 161-174.

Bulavin D.V., Kovalsky O., Hollander M.C. and Fornace A.J., Jr. (2003). Loss of oncogenic h-ras-induced cell cycle arrest and p38 mitogen-activated protein kinase activation by disruption of gadd45a. Mol. Cell Biol. 23, 3859-3871.

Chen L.Y., Yang B., Zhou L., Ren F., Duan Z.P. and Ma Y.J. (2015a). Promotion of mitochondrial energy metabolism during hepatocyte apoptosis in a rat model of acute liver failure. Mol. Med. Rep. 12, 5035-5041.

Chen N., Nishio N., Ito S., Tanaka Y., Sun Y. and Isobe K. (2015b). Growth arrest and DNA damage-inducible protein (gadd34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (den)-treated murine model. Cancer Immunol. Immunother. 64, 777-789.

Chen Y., Yang R., Guo P. and Ju Z. (2014a). Gadd45a deletion aggravates hematopoietic stem cell dysfunction in atm-deficient mice. Protein Cell. 5, 80-89.

Chen Y., Ma X., Zhang M., Wang X., Wang C., Wang H., Guo P., Yuan W., Rudolph K.L., Zhan Q. and Ju Z. (2014b). Gadd45a regulates hematopoietic stem cell stress responses in mice. Blood. 123, 851-862.

Chung H., Hong D.P., Kim H.J., Jang K.S., Shin D.M., Ahn J.I., Lee Y.S. and Kong G. (2005). Differential gene expression profiles in the steatosis/fibrosis model of rat liver by chronic administration of carbon tetrachloride. Toxicol. Appl. Pharmacol. 208, 242-254.

Ebert S.M., Dyle M.C., Kunkel S.D., Bullard S.A., Bongers K.S., Fox D.K., Dierdorff J.M., Foster E.D. and Adams C.M. (2012). Stress-induced skeletal muscle gadd45a expression reprograms myonuclei and causes muscle atrophy. J. Biol. Chem. 287, 27290-27301.

Garcia-Fernandez M., Castilla-Cortazar I., Diaz-Sanchez M., Navarro I., Puche J.E., Castilla A., Casares A.D., Clavijo E. and Gonzalez-Baron S. (2005). Antioxidant effects of insulin-like growth factor-i (igf-i) in rats with advanced liver cirrhosis. BMC gastroenterology. 5, 7.

HISTOLO

GY AND H

ISTOPATHOLO

GY

(non-e

dited

man

uscri

pt)

23 / 28

Gramantieri L., Chieco P., Giovannini C., Lacchini M., Trere D., Grazi G.L., Venturi A. and Bolondi L. (2005). Gadd45-alpha expression in cirrhosis and hepatocellular carcinoma: Relationship with DNA repair and proliferation. Hum Pathol. 36, 1154-1162.

Guo G.B. and Xu C.S. (2008). Expression profiles of the organic acid metabolism-associated genes during rat liver regeneration. Amino Acids. 34, 597.

Hollander M.C., Kovalsky O., Salvador J.M., Kim K.E., Patterson A.D., Haines D.C. and Fornace A.J., Jr. (2001). Dimethylbenzanthracene carcinogenesis in gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency. Cancer Res. 61, 2487-2491.

Hong L., Sun Q.F., Xu T.Y., Wu Y.H., Zhang H., Fu R.Q., Cai F.J., Zhou Q.Q., Zhou K., Du Q.W., Zhang D., Xu S. and Ding J.G. (2016). New role and molecular mechanism of gadd45a in hepatic fi brosis. World J. Gastroenterol. 22, 2779-2788.

Hur J.M., Yun H.J., Yang S.H., Lee W.Y., Joe M.H. and Kim D. (2008). Gliotoxin enhances radiotherapy via inhibition of radiation-induced gadd45a, p38, and nfkappab activation. J. Cell Biochem. 104, 2174-2184.

Jin S., Tong T., Fan W., Fan F., Antinore M.J., Zhu X., Mazzacurati L., Li X., Petrik K.L., Rajasekaran B., Wu M. and Zhan Q. (2002). Gadd45-induced cell cycle g2-m arrest associates with altered subcellular distribution of cyclin b1 and is independent of p38 kinase activity. Oncogene. 21, 8696-8704.

Jung H.J., Kim H.L., Kim Y.J., Weon J.I. and Seo Y.R. (2013). A novel chemopreventive mechanism of selenomethionine: Enhancement of ape1 enzyme activity via a gadd45a, pcna and ape1 protein complex that regulates p53-mediated base excision repair. Oncol. Rep. 30, 1581-1586.

Katagi A., Sui L., Kamitori K., Suzuki T., Katayama T., Hossain A., Noguchi C., Dong Y., Yamaguchi F. and Tokuda M. (2016). Inhibitory effect of isoamericanol a from jatropha curcas seeds on the growth of mcf-7 human breast cancer cell line by g2/m cell cycle arrest. Heliyon. 2, e00055.

Kim H.L., Kim S.U. and Seo Y.R. (2013). A novel role for gadd45alpha in base excision repair: Modulation of ape1 activity by the direct interaction of gadd45alpha with pcna. Biochem. Biophys. Res. Commun. 434, 185-190.

Lai L.Y., Yang, X., Xu, X.Q., Jiang, Y.F., Tong, M. . (2005). Establishment of animal model with acute hepatic failure caused by lavage carbon tetrachloride. China Journal. of Modern Medicine. 15, 3.

Lavallard V.J. and Gual P. (2014). Autophagy and non-alcoholic fatty liver disease. Biomed Res. Int. 2014, 120179.

Lazarevich N.L., Cheremnova O.A., Varga E.V., Ovchinnikov D.A., Kudrjavtseva E.I., Morozova O.V., Fleishman D.I., Engelhardt N.V. and Duncan S.A. (2004). Progression of hcc in mice is associated with a downregulation in the expression of hepatocyte nuclear factors. Hepatology. 39, 1038-1047.

Lembo-Fazio L., Nigro G., Noel G., Rossi G., Chiara F., Tsilingiri K., Rescigno M., Rasola A. and Bernardini M.L. (2011). Gadd45alpha activity is the principal effector of shigella mitochondria-dependent epithelial cell death in vitro and ex vivo. Cell Death Dis. 2, e122.

Li D., Kang N., Ji J. and Zhan Q. (2015). Brca1 regulates transforming growth factor-beta (tgf-beta1) signaling through gadd45a by enhancing the protein stability of smad4. Mol Oncol. 9, 1655-1666.

HISTOLO

GY AND H

ISTOPATHOLO

GY

(non-e

dited

man

uscri

pt)

24 / 28

Li P., Robertson T.A., Thorling C.A., Zhang Q., Fletcher L.M., Crawford D.H. and Roberts M.S. (2011). Hepatic pharmacokinetics of cationic drugs in a high-fat emulsion-induced rat model of nonalcoholic steatohepatitis. Drug Metab Dispos. 39, 571-579.

Liu X., Mu H., Luo X., Xiao X., Ding Y., Yin N., Deng Q. and Qi H. (2014). Expression of gadd45alpha in human early placenta and its role in trophoblast invasion. Placenta. 35, 370-377.

McMahan R.H., Porsche C.E., Edwards M.G. and Rosen H.R. (2016). Correction: Free fatty acids differentially downregulate chemokines in liver sinusoidal endothelial cells: Insights into non-alcoholic fatty liver disease. PLoS One. 11, e0168301.

Meyer N.J., Huang Y., Singleton P.A., Sammani S., Moitra J., Evenoski C.L., Husain A.N., Mitra S., Moreno-Vinasco L., Jacobson J.R., Lussier Y.A. and Garcia J.G. (2009). Gadd45a is a novel candidate gene in inflammatory lung injury via influences on akt signaling. FASEB J. 23, 1325-1337.

Oo Y.H., Shetty S. and Adams D.H. (2010). The role of chemokines in the recruitment of lymphocytes to the liver. Dig Dis. 28, 31-44.

Reddy K.R., Ellerbe C., Schilsky M., Stravitz R.T., Fontana R.J., Durkalski V., Lee W.M. and Acute Liver Failure Study G. (2016). Determinants of outcome among patients with acute liver failure listed for liver transplantation in the united states. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. 22, 505-515.

Roper J.M., Gehen S.C., Staversky R.J., Hollander M.C., Fornace A.J., Jr. and O'Reilly M.A. (2005). Loss of gadd45a does not modify the pulmonary response to oxidative stress. Am. J. Physiol. Lung Cell Mol Physiol. 288, L663-671.

Salerno D.M., Tront J.S., Hoffman B. and Liebermann D.A. (2012). Gadd45a and gadd45b modulate innate immune functions of granulocytes and macrophages by differential regulation of p38 and jnk signaling. J. Cell Physiol. 227, 3613-3620.

Sole C., Sola E., Morales-Ruiz M., Fernandez G., Huelin P., Graupera I., Moreira R., de Prada G., Ariza X., Pose E., Fabrellas N., Kalko S.G., Jimenez W. and Gines P. (2016). Characterization of inflammatory response in acute-on-chronic liver failure and relationship with prognosis. Sci. Rep. 6, 32341.

Soresi M., Giannitrapani L., Cervello M., Licata A. and Montalto G. (2014). Non invasive tools for the diagnosis of liver cirrhosis. In: World J. Gastroenterol. pp 18131-18150.

Su L.Y., Xin H.Y., Liu Y.L., Zhang J.L., Xin H.W. and Su X.L. (2014). Anticancer bioactive peptide (acbp) inhibits gastric cancer cells by upregulating growth arrest and DNA damage-inducible gene 45a (gadd45a). Tumour Biol. 35, 10051-10056.

Tanaka N., Takahashi S., Hu X., Yu L., Fujimori N., Golla S., Fang Z.-Z., Aoyama T., Krausz K.W. and Gonzalez F.J. (2017). Growth arrest and DNA damage-inducible 45α protects against nonalcoholic steatohepatitis induced by methionine- and choline-deficient diet. Biochim Biophys Acta. S0925443917302995.

Uppal V., Mansoor S. and Furuya K.N. (2016). Pediatric non-alcoholic fatty liver disease. Curr. Gastroenterol. Rep. 18, 24.

Wang G., Li B., Hao Y., Zhi J., He C. and Xu C. (2013). Correlation analysis between gene expression profile of high-fat emulsion-induced non-alcoholic fatty liver and liver regeneration in rat. Cell Biol. Int. 37, 917-928.

HISTOLO

GY AND H

ISTOPATHOLO

GY

(non-e

dited

man

uscri

pt)

25 / 28

Wang G.P. and Xu C.S. (2010). Reference gene selection for real-time rt-pcr in eight kinds of rat regenerating hepatic cells. Mol. Biotechnol. 46, 49-57.

Wang J., Che B., Zhang L.W., Dong G., Luo Q. and Xin L. (2017). Comparative genotoxicity of silver nanoparticles in human liver hepg2 and lung epithelial a549 cells. J Appl Toxicol. 37, 495-501.

Wang M.G.M., D.W.; Zhang, R.Z.; Long, F.L.; Qiu, H.; Shi, Q.L.; Wang, X.F. (2016). Detoxify and stasis-dissolving particles regulate gadd45a expression in rat hepatocyte of acute hepatic failure. Lishizhen Medicine and Materia Medica Research. 27, 3.

Wang W., Chen, X.G., Xu, C.S. (2007). Correlation analysis of ccaat/enhancer-binding proteins and their regulated genes with rat liver regeneration. World Chinese J. Digestol. 15.

Wingert S., Thalheimer F.B., Haetscher N., Rehage M., Schroeder T. and Rieger M.A. (2016). DNA-damage response gene gadd45a induces differentiation in hematopoietic stem cells without inhibiting cell cycle or survival. Stem Cells. 34, 699-710.

Xiu P., Xu Z., Liu F., Li Z., Li T., Zou F., Sun X. and Li J. (2014). Downregulating sclu enhances the sensitivity of hepatocellular carcinoma cells to gemcitabine by activating the intrinsic apoptosis pathway. Dig. Dis. Sci. 59, 1798-1809.

Xu X.M., Yuan G.J., Deng J.J., Wu Y.G., Ge W. and Song Q.B. (2010). Hepatic oval cells activated by hepatocyte apoptosis in diethylnitrosamine-induced rat liver cirrhosis. Saudi Med. J. 31, 490-494.

Yang F., Zhang W., Li D. and Zhan Q. (2013). Gadd45a suppresses tumor angiogenesis via inhibition of the mtor/stat3 protein pathway. J. Biol. Chem. 288, 6552-6560.

Yu Y., Li J., Wan Y., Lu J., Gao J. and Huang C. (2013). Gadd45alpha induction by nickel negatively regulates jnks/p38 activation via promoting pp2calpha expression. PLoS One. 8, e57185.

Zhang D., Zhang W., Li D., Fu M., Chen R. and Zhan Q. (2015). Gadd45a inhibits autophagy by regulating the interaction between becn1 and pik3c3. Autophagy. 11, 2247-2258.

Zhang R.P., Shao J.Z. and Xiang L.X. (2011). Gadd45a protein plays an essential role in active DNA demethylation during terminal osteogenic differentiation of adipose-derived mesenchymal stem cells. J. Biol. Chem. 286, 41083-41094.

Zhu N., Shao Y., Xu L., Yu L. and Sun L. (2009). Gadd45-alpha and gadd45-gamma utilize p38 and jnk signaling pathways to induce cell cycle g2/m arrest in hep-g2 hepatoma cells. Mol. Biol. Reports. 36, 2075-2085.

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Table 1. Primers sequences used in real-time quantitative RT-PCR

Gene Accession

numbers Primer sequences

Amplified

products (bp)

Lcn2 NM_130741 FP：5'-CACCCTGTACGGAAGAACC-3'

164 RP：5'-CACATCCCAGTCAGCCAC-3'

Myc NM_012603 FP：5'-GAGGAGAAACGAGCTGAAGCG-3'

126 RP：5'-TGAACGGACAGGATGTAGGC-3'

Tgfb1 NM_021578 FP：5'-CTTCAGCTCCACAGAGAAGAACTGC-3'

298 RP：5'-CACGATCATGTTGGACAACTGCTCC-3'

Alpl NM_013059 FP：5'-CATCGGACCCTGCCTTACCA-3'

231 RP：5'-CGTGTCTCCTCGCCCGTGTT-3'

Cyp1a1 NM_012540 FP：5'-AGGACAGGAGGCTGGACGAGA-3'

289 RP：5'-ATGGTGAATGGGACAAAGGAT-3'

Mgmt NM_012861 FP：5'-GAAGCCTATTTCCACGAACC-3'

103 RP：5'-TCCATAACACCTGTCTGGTGAA-3'

Spc24 XM_003750448 FP：5'-AGCTTCGGGAGACTCTGGCTGTG-3'

130 RP：5'-TCCTCCTTGCTGGCCTTCTGG-3'

A2m NM_012488 FP：5'-CGAACATCCGTAAACCCAAAGTC-3'

184 RP：5'-GCTGAGTCCACCACCACCAAGTC-3'

β-actin NM_031144 FP：5'-CATCCGTAAAGACCTCTATGCCAACA-3'

109 RP：5'-GTGCTAGGAGCCAGGGCAGTAATCT-3'

FP: forward primer; RP: reverse primer.

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Table 2. Over-represented functional categories in four clusters

Cluster enriched biological process No. of genes P-Value

regulation of apoptosis 32 1.30E-19

regulation of cell death 32 2.10E-19

apoptosis 20 7.50E-13

cell death 20 5.80E-12

lipid phosphorylation 6 6.60E-09

regulation of caspase activity 8 3.10E-07

lipid modification 7 3.40E-06

cell activation 10 2.00E-05

cell cycle 13 2.70E-05

DNA damage checkpoint 5 4.00E-05

inflammatory response 9 6.30E-05

regulation of cell migration 8 7.70E-05

regulation of lipid metabolic process 7 1.00E-04

G1/S transition of mitotic cell cycle 4 1.60E-03

cell cycle process 9 1.70E-03

G2/M transition of mitotic cell cycle 3 2.50E-03

positive regulation of immune system process 7 3.30E-03

cell proliferation 7 5.10E-03

regulation of DNA replication 4 6.00E-03

negative regulation of cell proliferation 7 6.20E-03

regulation of cyclin-dependent protein kinase

activity 3 8.30E-03

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Figure 1. The network of the six branches of the GADD45α-mediated signaling

pathway and their relationships are shown. The ordinal numbers in the circles represent

the P38 MAPK, JNK, P21, PCNA, MTOR and AKT signaling pathways.

Figure 2. Venn diagram showing the significantly expressed genes during LC, HC, AHF

and NAFLD. The yellow, blue, purple, green and red colors denote LC, HC, AHF and

NAFLD, respectively.

Figure 3. Global comparison of gene expression patterns in NAFLD, LC, HC and AHF.

K-means clustering of a total of 84 genes. A dendrogram was produced from the

complete array expression values of LC (3 w, 6 w and 9 w), HC (5 w, 8 w, 12 w, 16 w

and 18 w), AHF (3 h, 6 h, 12 h, 24 h, 48 h and 72 h) and NAFLD (2 w, 4 w and 6 w).

The red and green colors denote expression levels that are higher and lower than the

control, respectively.

Figure 4. Verification of the gene expression in the four kinds of liver disease by

real-time RT-PCR. The light gray represents mRNA levels measured by the Rat

Genome 230 2.0 Array, and the dark gray color represent those measured by RT-PCR.

The time points of LC are represented by 3 w, 6 w and 9 w; the time points of NAFLD

are represented by 2 w, 4 w and 6 w; the time points of HC are represented by 5 w, 8 w,

12 w, 16 w and 18 w; and the time points of AHF are represented 3 h, 6 h, 12 h, 24 h,

48 h, and 72 h. The Y-axis represents the relative mRNA abundance of the genes. The

mean values are represented in the bar graph, and the bars represent standard error (n =

3). **P < 0.01 versus the results from the gene chip.

Figure 5. Heat map of the biological of the GADD45α signaling pathway-related genes

as predicted by IPA software for the about four kinds of liver diseases. The color of the

squares in the heat map square represents the activity of biological function. The jacinth

color represents activation, and the blue color represents inhibition of biological

function.

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