Expressão de Fra-1, c-Jun e c-Fos em OKCs

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doi: 10.1111/his.13705 
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DR BING LIU (Orcid ID : 0000-0002-2103-5494) 
Article type : Original Article 
 
Overexpression of Fra-1, c-Jun and c-Fos in odontogenic 
keratocysts: potential correlation with proliferative and 
anti-apoptotic activity 
 
Lin-Zhou Zhang1*, Qi-Wen Man1*, Jin-Yuan Liu1, Wen-Qun Zhong2, Yue-Yu 
Zheng2, Yi-Fang Zhao2, Bing-Liu2 
1The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key 
Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of 
Stomatology, Wuhan University, Wuhan, China. 
2Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, 
Wuhan University, Wuhan, China. 
 
*Authors contribute equally to this work. 
 
Corresponding author: Bing-Liu, School & Hospital of Stomatology, Wuhan 
University, Wuhan 430079, China. Tel: +86 27 87686125; Fax: +86 27 87873260; 
E-mails:liubing9909@whu.edu.cn(Bing-Liu); 
 
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Keywords: AP-1; proliferation; apoptosis; odontogenic keratocysts 
 
Abstract 
Aims: The purpose of this study was to explore the potential involvement of Fra-1, 
c-Jun and c-Fos, three vital members of the AP-1 complex, in the pathogenesis of 
odontogenic keratocysts (OKCs). 
Methods and results: Tissue samples, containing 10 normal oral mucosa (OM), 10 
dentigerous cysts (DC) and 32 OKC specimens, were applied to investigate the 
expression levels of Fra-1, c-Jun and c-Fos by immunohistochemistry and real-time 
quantitative polymerase chain reaction (RT-qPCR). The association between Fra-1, 
c-Jun and c-Fos expression levels and markers of proliferation [Ki-67, proliferating 
cell nuclear antigen (PCNA)], anti-apoptosis [B cell lymphoma 2 (Bcl-2)] was then 
investigated in the OKC serial tissue sections. The results showed that Fra-1, c-Jun 
and c-Fos expression levels were significantly increased in OKCs compared to these 
in OM and DC tissue samples. Meanwhile, the expression levels of Fra-1, c-Jun and 
c-Fos were positively associated with the expression levels of Ki-67, PCNA and Bcl-2, 
as confirmed further by double-labelling immunofluorescence analysis and 
hierarchical analysis. 
Conclusions: This study revealed for the first time that Fra-1, c-Jun and c-Fos were 
overexpressed in OKCs and had a close correlation with proliferation and 
anti-apoptosis potential of OKCs. 
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Introduction 
Odontogenic keratocysts (OKCs), also known as keratocystic odontogenic tumors, 
are defined as jaw developmental abnormalities [1]. After being defined as benign 
tumors many years [1, 2], keratocystic odontogenic tumors have been reclassified as 
the odontogenic keratocysts by the World Health Organization (WHO) in 2017 [3]. 
Compared with other cysts for example dentigerous cysts (DCs) and radicular 
cysts, OKCs have distinct clinicopathologic features: parakeratinised stratified 
squamous epithelium, higher locally aggressive behavior, higher recurrence rates 
and so forth [4, 5]. 
Since cell population and turnover are controlled by a balance between cell 
proliferation and programmed cell death, cell proliferation and apoptosis are critical 
for the development and tissue homeostasis [6]. OKCs epithelial lining is unique and 
characterized by increased mitotic activity and high proliferative rates [7-9]. 
Previously, we have employed immunohistochemical staining of cellular proliferation 
markers [Ki-67, proliferating cell nuclear antigen (PCNA), cyclin D1] and cellular 
anti-apoptosis markers [B cell lymphoma 2 (Bcl-2)] to evaluate the proliferative and 
anti-apoptotic features of OKCs [10-12]. However, the precise mechanism that 
underlies the unique clinical behaviors of OKC, remains to be elucidated. 
The AP-1 transcription factors are involved in activation of many genes concerning 
regulation of proliferation, growth, survival and apoptosis [13, 14]. AP-1 complex is 
comprised of members with a basic leucine zipper domain, including Jun family 
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(c-Jun, Jun B, Jun D) and Fos family (c-Fos, Fos B, Fra-1, Fra-2) [15]. Forming stable 
homodimers or heterodimers among AP-1 proteins are critical for their functions in 
transcription and in binding to conserved cis-elements, such as, the 
12-O-tetradecanoylphorbol-13-acetate, responsive element (TRE, 5′-TGAG/CTCA-3′) 
and cAMP responsive element (CRE, 5′-TGACGTCA-3′). All these genes are 
involved in cellular proliferation and apoptosis [16]. A large number of studies have 
demonstrated that the up-regulation of Fra-1, c-Jun and c-Fos could result in aberrant 
proliferation, abnormal differentiation and enhanced anti-apoptotic potential in various 
tumors [17-20] . However, the expression levels of Fra-1, c-Jun and c-Fos and their 
potential significance in OKCs remain to be determined. 
In the present study, therefore, we evaluated and compared the expression levels of 
Fra-1, c-Jun and c-Fos in OM, DC and OKC samples by immunohistochemistry and 
real-time quantitative polymerase chain reaction (RT-qPCR) analysis. In addition, to 
investigate the possible association of Fra-1, c-Jun and c-Fos with proliferative and 
anti-apoptotic behaviors in OKCs, the expression levels of Fra-1, c-Jun and c-Fos 
and those of Ki-67, PCNA, and Bcl-2 were explored by double-labelling 
immunofluorescence staining and hierarchical analysis. 
 
 
 
 
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Materials and Methods 
Experimental samples 
We collected 10 OM, 10 DC, and 32 OKC samples who were underwent surgical 
curettage at the Hospital of Stomatology, Wuhan University. Diagnosis was 
determined by 2 pathologists according to the World Health Organization (WHO) 
2017 Classification of Head and Neck Tumors [3]. Representative hematoxylin and 
eosin (H&E) was shown in Figure S1. This study was approved by the review board of 
the Ethics Committee of Hospital of Stomatology, Wuhan University and all patients 
signed the informed consents. All specimens were fixed in 4 % formalin and routinely 
embedded in paraffin material. Importantly, paraffin blocks were suitable for 
immunohistochemistry. 
 
Immunohistochemistry analysis 
5 μm sections were deparaffinized and analyzed as previously described [21]. Briefly, 
StreptAvidin–Biotin Complex (SABC) immunohistochemical kit (Boster Biological 
Technology Ltd) was used to evaluate the Fra-1, c-Jun, c-Fos, Ki-67, PCNA and Bcl-2 
expression in OM, DC and OKC specimens. Briefly, the sections from the OM, DC 
and OKC samples were deparaffinized in xylol and hydrated in decreasing 
concentrations of ethyl alcohol. The histological sections were incubated with 3 % 
H2O2 for 20 min at room temperature to block internal peroxidase activity. Antigen 
retrieval was carried out through incubating the sections in 10mM citrate buffer with 
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0.1 % Triton under high temperature and pressure for 90 s each. The slides were 
incubated with normal goat serum for 20 min at 37 ℃. The sections were then 
incubated with primary antibodies were as follows: Fra-1 (1:150, Santa Cruz 
Biotechnology, USA), c-Jun (1:200, Cell Signaling Technology, USA), c-Fos (1:150, 
Santa Cruz Biotechnology, USA), Ki-67 (1:200, Zhong-Shan Golden-bridge 
Biotechnology,China), PCNA (1:200, Zhong-Shan Golden-bridge Biotechnology, 
China) and Bcl-2 (1:200, Zhong-Shan Golden-bridge Biotechnology, China) overnight 
at 4 °C. After primary antibody incubation, the sections were washed with PBS, and 
were incubated with a biotinylated secondary antibody, SABC. To visualize the 
reaction, 3, 30-diaminobenzidine (DAB Chromogen, DakoCytomation) was used and 
then sections were counterstained by Mayer’s hematoxylin for 12 s each. Negative 
controls consisted of sections in which the primary antibodies were replaced by PBS, 
Representative negative staining was shown in Figure S2. Cytoplasmic reaction with 
brown staining was deemed as indicate immunopositivity for Bcl-2. Overlapping 
nuclei sections were deemed as positive immunostaining for Fra-1, c-Jun, c-Fos, 
PCNA and Ki-67. Five random fields of each section were taken by two investigators 
at a magnification of 200 ×. The images were then analyzed using the Image-Pro Plus 
version 6.0 software as described previously [22]. Briefly, a cell number-counting 
program was conducted using the ‘count/size’ command. The basic steps were 
performed as follows: (a) define the area of interest (AOI), (b) set the reference value 
that identifies the positive staining and (c) use the ‘pathology’ command to count the 
objects automatically. These procedures generated sum integrated optical density 
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(IOD) and sum area (area). The quantitation was expressed as a mean density 
(IOD/area). 
 
RNA extraction and RT-qPCR analysis 
Total RNA was extracted from 4 cases of OM, 4 cases of DC and 4 cases of OKC 
using an RNeasy® kit (Qiagen, Carlsbad, CA, USA). The total RNA samples (2μg) 
were transcribed into cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit 
(Takara, Otsu, Japan). And then one fifth of cDNA was used for polymerase chain 
reaction (PCR) by FastStart Universal SYBR Green Master (Roche, Switzerland) in a 
7900HT Real-time PCR System (Applied Biosystems). The primer sequences used 
for RT-qPCR were as follows: Fra-1: 5’ -AGGAAGGAACTGACCGAC- 3’ and 5′ 
-GAAGGGGAGGAGACATTG- 3′; c-Jun: 5′ -GCCTACAGATGAACTCTTTCTGGC- 3′ 
and 5′ -CCTGAAACATCGCACTATCCTTTG- 3′; c-Fos: 5′ 
–TCTTCCTTCGTCTTCACC- 3′ and 5′ -AATCAGAACACACTATTGCC- 3′; Ki-67: 5′ 
-CACCACCAGAGCCAATAGATAC- 3′ and 5′ –CTGTGTCCAATTTCCGCTTTAC- 3′; 
PCNA: 5′ -TGGAGAACT TGG AAATGG AAA- 3′ and 5′ 
-GAACTGGTTCATTCATCTCTATGG-3′; Bcl-2: 5′ 
-TCCGATCAGGAAGGCTAGAGTT- 3′ and 5′ -TCGGTCTCCTAAAAGCAGGC- 3′ 
β-actin: 5′ -TACCACTGGCATCGTGATGGACT- 3′ and 5′ 
-TCCTTCTGCATCCTGTCGGCAAT- 3′. β-actin was selected as the internal control 
for each experiment. The expression for mRNA was assessed using evaluated 
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threshold cycle (CT) values. The CT values were normalized with the expression 
levels of β-actin. We used the 2−ΔΔCT method to calculate the relative quantity of 
mRNA specific to each of the tested genes [23, 24]. 
 
Hierarchical clustering, visualization of data, and statistical analysis 
According to previous studies [25], Cluster 3.0 with average linkage based on 
Pearson’s correlation coefficient was used for hierarchical analysis, and the results 
were visualized using Java TreeView1.0.5. 
Student’s t test and Spearman’s rank correlation test were used for statistical 
analysis. Statistical significance was determined if pc-Fos, Ki-67, PCNA and Bcl-2 were rarely detected in the 
epithelium of OM and DC tissues. These resluts showed the possible close 
relationship between the AP-1 complex related proteins and proliferative and 
anti-apoptotic characteristics of OKCs. These results showed the Fra-1, c-Jun and 
c-Fos possible close association with proliferative and anti-apoptotic characteristics of 
OKCs. 
 
Discussion 
AP-1 family transcription factors are critical for various cellular processes, and display 
both distinct and overlapping functions among family members, such as Fra-1, c-Jun 
and c-Fos [26]. The Ras-AP-1 signaling pathway plays a critical role in regulating cell 
growth and their mutation can lead to abnormal proliferation [27, 28]. Among AP-1 
family transcription factors, Fra-1 is encoded by the fos-like-1 gene (fosl1) and also 
induced by mitogenic stimuli [29]. In exponentially growing cells, Fra-1 expression is 
elevated [30] and knockdown of Fra-1 causes a proliferative block and apoptosis [31]. 
c-Fos, another important member of the Fos family, is also described as an 
immediate early response gene [32]. c-Fos can interfere the dynamic equilibrium of 
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cell proliferation and induce cell transformation and tumor formation [33]. c-Jun is the 
founding member and the most potent transcriptional activator of the AP-1 family. It is 
also the downstream effector of many pathways, like mitogen activated protein kinase 
(MAPK) and p38 MAPK-AP-1, regulating development and differentiation during 
embryogenesis, cellular proliferation and apoptosis [28]. High levels of c-Jun are also 
found in multiple tumors [34-36]. However, the activated status of Fra-1, c-Jun and 
c-Fos in OKCs, a benign odontogenic lesion with potentially proliferative behavior and 
high recurrence, remains unknown. 
AP-1 family transcription factors are regulated in a given cell by a large number of 
physiological and pathological stimuli, including cytokines, growth factors, stress 
signals and oncogenic stimuli and so on [26]. Oral soft tissues are complex biologic 
systems with the components of their extracellular matrix (ECM) responding 
differentially to physiologic forces, which include hydrodynamic stresses, 
compression, elongation, friction, and shear generated during saliva flow, 
mastication, speech, toothbrushing, and so forth [37-39]. In our present study, we 
found that a few Fra-1, c-Jun and c-Fos expressed in the epithelium of oral mucosa, 
which might be a kind of explanation for Fra-1, c-Jun and c-Fos activity induced by 
various physiological environmental stresses. Similarly, in the oral and maxillofacial 
diseases such as jaw cysts, the cyst wall was also exposed to hydrostatic pressures, 
osmotic pressures, and so on [40]. It has been widely demonstrated that OKCs own a 
distinct growth behavior compared with the other jaw cysts and normal tissues [4]. 
OKCs have a characteristic histologic appearance with palisading of the basal 
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epithelial cells and a corrugated, parakeratotic stratified squamous epithelial layer [5]. 
Previous studies have demonstrated that overexpression of Fra-1, c-Jun and c-Fos 
were possibly associated with the increased expression of tumor cell’s proliferative 
and anti-apoptotic markers [17, 20, 41, 42]. In our present study, the 
immunohistochemical analysis first showed that the expression levels of Fra-1, c-Jun 
and c-Fos are significantly up-regulated in OKCs compared with those in OM and DC 
samples. In addition, the mRNA expression levels of Fra-1, c-Jun and c-Fos were 
also increased in OKCs. Importantly, Spearman’s rank correlation test and 
double-labelling immunofluorescence staining revealed that the expression levels of 
Fra-1, c-Jun and c-Fos have a meaningful relationship with Ki-67, PCNA and Bcl-2 in 
OKCs, which is also reflected intuitively in the clustering analysis. Collectively, these 
results provide the first convincing evidence for the potential tumor promoting roles of 
Fra-1, c-Jun and c-Fos in OKCs, and the results indicate that dysfunction of these 
tested proteins may function as a possible route of pathogenesis in OKCs. 
 
Acknowledgments 
This work was supported by National Natural Science Foundation of China 
(81570994). We would like to thank the patient and their families for their participation 
in this study. 
 
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Conflicts of Interest 
The authors declared that they had no conflicts of interest. 
 
Author contributions 
Lin-Zhou Zhang and Qi-Wen Man contributed the study design and experimental 
studies. Jin-Yuan Liu, Wen-Qun Zhong and Yue-Yu Zheng participated in sample 
collection and data analysis. Yi-Fang Zhao and Bing-Liu revised manuscript. All 
contributors approved the final version of the manuscript before submission. 
 
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Figure legends 
Figure 1. Increased expression of Fra-1, c-Jun and c-Fos in human odontogenic 
keratocysts (OKCs). (A) Representative immunohistochemical staining of Fra-1, 
c-Jun and c-Fos in OKC tissues compared with normal oral mucosa (OM), 
dentigerous cyst (DC) samples. Scale bar 50 μm. (B) Quantification the expression 
levels of Fra-1, c-Jun and c-Fos in OM (n=10) (green bars), DC (n=10) (blue bars) 
and OKC (n=32) (red bars) specimens. (C) The relative mRNA expression levels of 
Fra-1, c-Jun and c-Fos in samples from OM (green bars), DC (blue bars) and OKC 
(red bars) were evaluated by real-time qPCR analysis. All data are presented as the 
means ±SEM. ***pnuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2) in normal oral mucosa 
(OM), dentigerous cyst (DC) and odontogenic keratocyst (OKC) tissues. The white 
arrows indicated the colocalizations of c-Fos with Ki-67, PCNA and Bcl-2 signals in 
OKCs. Scale bar 50 μm. 
Figure S1. Representative hematoxylin and eosin (H&E) of normal oral mucosa 
(OM), dentigerous cyst (DC) and odontogenic keratocyst (OKC) tissues. Scale bar 50 
μm. 
Figure S2. Representative negative staining of normal oral mucosa (OM), 
dentigerous cyst (DC) and odontogenic keratocyst (OKC) samples. Scale bar 50 μm. 
 
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