Background
Ovarian cancer is a gynecological malignant tumor that has the highest mortality rate. At present, surgery and chemotherapy are still the main treatment methods for ovarian cancer. However, as 70% of the patients are in the advanced stage at the time of diagnosis, their extensive pelvic and abdominal metastases and chemotherapy resistance often result in poor prognosis and a low survival rate. The five-year survival rate for ovarian cancer patients is 48.6% [
1]. Although progress has been made in the diagnosis and treatment of ovarian cancer, further research is needed to clarify the causative factors and develop effective measures for early detection and treatment [
2,
3].
Zinc finger proteins are transcription factors (TFs) widely found in eukaryotes; they can regulate cancer cell proliferation, differentiation, apoptosis, invasion, and migration. ZNF703 is a member of the NET/Nlz family of transcription factors with a “finger” domain [
4], located in the cytoplasm and nucleus. Studies have shown that ZNF703 regulated embryonic development processes, such as the differentiation of the vertebrate hindbrain into rhomboids, and the formation of the Xenopus neural crest, under physiological conditions [
5,
6]. ZNF703 was originally found from the abnormal amplification of the 8p11–12 region of the human chromosome, which led to irregular gene expression and the occurrence and progression of breast cancer [
7,
8]. Recent studies confirmed that the expression of ZNF703 promoted tumor progression and was abnormally increased in breast [
9], lung [
10], and gastric cancers [
11].
An anti-apoptotic factor composed of 131 amino acids, PEA15 was originally found in primary cultured astrocytes [
12]. The first 80 amino acids form a standardized “death effect zone (DED)”, whereas the latter 51 amino acids form an irregularly structured C-terminal tail (containing phosphorylation sites) [
12]. PEA15 is primarily involved in cell proliferation, differentiation, cell signal transduction, apoptosis regulation, the cell cycle, and glucose transport processes [
13,
14]. It is closely related to tumor occurrence, development, and metastasis, and its expression is up-regulated in various tumors [
15‐
17].
We initially discovered ZNF703 in the sequencing results we obtained while investigating HE4’s protein-protein interactions using the yeast two-hybrid (Y2H) assay. The aim of the present study was to investigate the epigenetic regulation of ZNF703 in ovarian cancer, reveal the pathways involved, the underlying mechanism, and determine how this affects ovarian cancer progression and the survival time of patients.
Materials and methods
Specimen source and clinical data
The specimens included 98 ovarian epithelial malignant tumor samples (ovarian cancer group), 15 ovarian epithelial borderline tumor samples (borderline group), 14 ovarian epithelial benign tumor samples (benign group), and 12 normal ovarian tissue samples (normal group). The average age of all the patients included in this study was 53.24 years (19–84 years). The ovarian tissue samples we analyzed were selected from the archived wax blocks of the surgical specimens of inpatients in our hospital from 2008 to 2014. The patients the samples were derived from had not undergone chemotherapy, radiotherapy or hormone therapy before surgery, and their complete clinical information was available. The clinical surgical pathological staging was carried out according to the standards set by the International Federation of Obstetrics and Gynecology (FIGO) in 2009.
Immunohistochemistry and immunocytochemistry
Continuous 5 μm-thick paraffin sections were used for immunohistochemistry. The concentration of the monoclonal antibody used against ZNF703 was 1:50 (Santa Cruz, sc-271,896). After dewaxing, the samples were permeated using immunostaining permeation solution (Triton X-100, P0096, Beyotime), and the staining method was carried out using the kit for the streptavidin-peroxidase connection (SP) method according to the instructions of the manufacturer. A positive result was indicated by the presence of brown particles in the nucleus and cytoplasm. According to the coloring intensity, the samples were divided into noncolored, light yellow, brown yellow and brown, and graded with 0, 1, 2 and 3 points, respectively. The percentage of the visual field occupied by colored cells, < 5, 6% ~ 25, 26% ~ 50, 51% ~ 75 and > 75%, was graded with 0, 1, 2, 3 and 4 points, respectively. To calculate the final score, the two items were multiplied: 0 ~ 2 points (−), 3 ~ 4 points (+) 5 ~ 8 points (++) 9 ~ 12 points (+++). Two observers who are blinded read the results separately.
Cell culture
The ovarian cancer cell lines (CAOV3, SKOV3, OVCAR3, and ES-2) were purchased from the Shanghai Cell Collection Center. The CAOV3, SKOV3, and OVCAR3 cells were cultured in conventional RPMI 1640 medium (BI, USA) containing 10% fetal bovine serum (BI, USA), whereas the ES-2 cells were cultured in McCoy’s 5A medium (BI, USA) containing 10% fetal bovine serum. The cells were incubated at 37 °C, 5% CO2 with saturated humidity.
Cell transfection and construction of stably transfected cell lines
The ZNF703 small interfering (si) RNA was transfected into the CAOV3 and SKOV3 cell lines using the liposome method (Lipo 3000 transfection kit, GIBCO, Invitrogen). The cells were collected 48 h after transfection, and the effect of ZNF703 siRNA transfection on ZNF703 expression was determined using RT-PCR and western blotting. Within 48 h of transfection, the cells were used for functional experiments and flow cytometric analysis. The siRNA sequences of ZNF703 and HE4 and that of the negative control (GenePharma, Shanghai, China) are shown in Additional file
3: Table S1. The OVCAR3 and ES-2 cell lines were transfected using virus-mediated transfection and oncogene overexpression. The day before transfection, the cells were inoculated into 24-well cell culture plates at a density of 2500 cells/well. After overnight culture, RPMI 1640 culture medium, without serum, containing the lentivirus (MOI = 50) was added to the cells at 37 °C. After 8 ~ 12 h, complete medium containing serum was added.
RT-PCR
The total RNA of the cells was extracted using Trizol according to the manufacturer’s instructions, and the purity and concentration of the RNA were determined using a UV spectrophotometer. The RT-PCR kit TAKARA047A (Takara Bio, Inc., Shiga, Japan) of the Super Script III First-Strand Synthesis System was used to reverse transcribe RNA into cDNA. The amplification conditions were: denaturation at 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s, for a total of 40 cycles. Real-time PCR amplification was performed using the 7500 Fast Real-Time PCR system. The primers used for ZNF703, PEA15, internal reference glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and actin amplification are shown in Additional file
3: Table S2.
Western blot
The RIPA cell lysis buffer was used to lyse the cells for 30 min at 4 °C, followed by centrifugation at 12000×g for 30 min at 4 °C. The supernatant was collected, and the protein concentration was determined using the BCA method. After denaturation, the protein was subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred and blotted on a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was subsequently blocked for 2 h using 5% milk or bovine serum albumin (BSA) and incubated with the primary antibodies at 4 °C overnight. After washing with tris-buffered saline (TBST), the membrane was incubated with the secondary antibody (1:2000, Zhongshan Jinqiao, China) for 2 h and then washed with TBST. Proteins were visualized using the ECL reagent (Thermo Scientific ECL). The experiment was repeated three times. ECL (Thermo, USA) and the gel electrophoresis image analyzer GDS8000 were used for color development. The primary antibodies were shown in Additional file
3: Table S3.
MTT cell proliferation assay
A total of 2000 cells per well were inoculated in a 96-well plate, and the cells were counted at 0 h and after adherence for 6 h, and 20 μL of 5 μg/mL MTT (5 mg/ml, Solarbio, Beijing, China) solution was added to each well. After incubation at 37 °C for 4 h, the medium was aspirated, 150 μl of dimethyl sulfoxide (DMSO) was added to each well, and after shaking for 5 min, the optical density (OD) values was determined.
Apoptosis detection using flow cytometry
The Annexin-V-APC/7AAD (BD Biosciences, New York, USA) double staining method was used to detect apoptosis in ovarian cancer cell lines overexpressing ZNF703. Here, the Annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (KeyGen Biotech, Nanjing, China) double staining method to detect apoptosis when ZNF703 expression was inhibited by siRNA. The staining methods were carried out according to the instructions of the manufacturer.
Cell cycle
Collect the cells in each group and fix with 70% ethanol was used to fix the cells overnight at 4 °C. A staining working solution of 500 μl PI/RNase (PI: RNaseA is prepared according to 9:1) (KeyGen Biotech, Nanjing, China) was added when the ethanol was removed using centrifugation in the following day. The cells were stained at 4 °C in the dark for 30 min. A cytometer (US BD company) was used for fluorescence detection.
Wound healing test
Cells in the logarithmic growth phase were collected to prepare a single-cell suspension and seeded in 6-well plates. After the cell fusion reached 90%, the plate was gently scratched using a 100 μl pipette tip, then gently rinsed with PBS twice to replace the serum-free medium, and the width of the scratch was observed under a microscope. After incubation in serum-free medium for 24 h, the width of the scratch was again observed under a microscope.
Invasion test
A total of 70 μl Matrigel glue (BD Corporation) was spread in the upper chamber of the Transwell chamber (Corning Coster) and placed in a 37 °C incubator to dry overnight. Then, 500 μl of medium containing 20% fetal bovine serum was added in the lower chamber, and 200 μl of the cell suspension (2 × 105 cells) in serum-free medium was added in the upper chamber. After incubation at 37 °C for 48 h, the chamber was removed. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with crystal violet for 30 min. The upper chamber surface was gently wiped clean with a cotton swab, and the number of tumor cells infiltrated by the lower chamber surface filter membrane was counted under a microscope.
Co-immunoprecipitation
A total of 2 μg of anti-HE4 antibody (abcam: ab200828) or anti-rabbit IgG antibody (Zhongshan Jinqiao) was added to 500 μg protein lysate and incubated at 4 °C overnight. The next day, 40 μl protein A/G PLUS-Agarose beads (Santa Cruz: sc-2003) was added at 4 °C. After 6 h, the immunoprecipitated protein complex was added with 2 × loading buffer and boiled to denature the protein and separate it from the protein-G beads. The same method was performed using 2 μg of the anti-ZNF703 primary antibody to precipitate HE4, and the precipitated protein was detected using western blotting with the anti-HE4 primary antibody.
Immunofluorescence and immunofluorescence co-localization
The cells on glass coverslips were fixed in 4% paraformaldehyde at room temperature for 20 min. After permeation with immunostaining permeation solution (Triton X-100, P0096, Beyotime) for 15 min, cells were blocked with PBS containing 5% BSA for 30 min. Subsequently, the cells were incubated with the primary antibody against ZNF703 (1:30) diluted in blocking buffer at 4 °C overnight and then incubated with Alexa Fluor 488-goat anti-mouse IgG (1:100) secondary antibody for 2 h. Next, the cells were rinsed with PBS for 15 min, and the nuclei were stained using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 5 min. For detecting co-localization, the anti-HE4 primary antibody (1:200, DF8160, Affinity) and the anti-ZNF703 primary antibody (1:30, Santa Cruz) were incubated at the same time. Alexa Fluor 488-goat anti-mouse and Alexa Fluor 594-goat anti-mouse were used together as secondary antibodies.
Nuclear cytoplasmic fractionation
Nuclear and cytoplasmic proteins were extracted and separated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The HE4 overexpression plasmid was constructed by GeneChem (Shanghai, China).
Chromatin immunoprecipitation-sequencing (ChIP-seq)
The OVCAR3 cells were normally cultured in 10% FBS. Approximately 5 × 10
7 cells were used in each ChIP-seq assay. After the cells were cross-linked, they were subjected to nuclear lysate extraction. Following sonication, monoclonal antibodies against ZNF703 were used for chromatin immunoprecipitation, followed by deep sequencing (ChIP-seq) (SeqHealth Tech, China). For ChIP-seq results, after obtaining the raw sequencing data (raw data), they were filtered, and high-quality sequencing data (clean data) were compared to the human genome (hg19). Then, the results were compared to the whole genome de novo peak calling, to study the protein’s binding preference in the genome, and to conduct a motif analysis of the binding site. Additional file
3: Table S4.
ChIP-PCR
The ovarian cancer cells OVCAR3 were collected in the exponential growth phase and cross-linked in a medium containing 1% formaldehyde at room temperature for 15 min. Then, the cells were incubated with glycine at room temperature for 5 min to terminate cross-linking, followed by washing twice with PBS, and collection by centrifugation using a protease inhibitor in PBS. The rest of the procedure was performed according to the manufacturer’s instructions (SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004S, CST), using 10 μl of the anti-ZNF703 antibody (sc-271,896X, Santa Cruz) and 2 μg of the IgG antibody (5415S, [Mouse], CST). The primer used for the peak 150 corresponding to PEA15, is shown in Additional file
3: Table S5. The experiment was performed in two cell lines and repeated three times.
Dual luciferase reporter gene assay
The plasmids employed in the luciferase experiments were synthesized by GeneChem (Shanghai, China). The PEA15 promoter fragments (2 kb upstream of TSS) were digested with restriction endonucleases KpnI and XhoI (Thermo Fisher Scientific). It recognizes the sequences GGTACC^CTCGAG. The enhancer-WT or enhancer-Mut sequences were digested with restriction endonucleases XbaI and XbaI. They recognize the sequences TCTAGA^TCTAGA. Then, they were ligated into the pGL3-basic plasmid (Promega). The HEK293T cells were divided into 24-well plates, co-transfected 500 ng each of the luciferase reporter gene construct of pGL3-Basic, PEA15 enhancer-wt, the mutant and the promoter region (2 kb upstream of TSS) plasmids with a total of 500 ng ZNF703 overexpression or control plasmid, and 2 μl transfection reagent lipo 3000 were added per well. Renilla luciferase plasmids was used as internal control transfected with 50 ng per well. After 48 h, the whole cell lysate was collected and used in the dual luciferase reporter assay (Promega, Madison, WI, USA), which was performed according to the manufacturer’s protocol (Promega). The data were calculated using Renilla luciferase as a control.
Nude mouse xenograft model
Twelve 4-week-old female BALB/cA-nu nude mice, purchased from Beijing Huafukang Biosciences (Beijing, China), were maintained in specific pathogen-free conditions. Control vector/ZNF703 -overexpressed OVCAR3 cells (5 × 106) cells were suspended in 150 μL of phosphate buffered saline (PBS) and injected subcutaneously into the axilla of mice (n = 6). The changes in tumor occurrence time, tumor formation rate, tumor formation number, tumor body diameter, mass and mouse body weight were recorded in each group every 4 days. The calculation method used for the tumor volume was V = 1/2 × a × b2 (a is the long axis and b is the short axis). All the mice were sacrificed after 37 days. The tumor samples were then fixed in 4% paraformaldehyde and embedded in paraffin. Continuous 4 μm-thick sections were cut and analyzed using hematoxylin and eosin (HE) or immunohistochemical staining. The animal study was approved by the Institutional Animal Research Committee of China Medical University.
Functional and pathway enrichment analysis of genes co-expressed with ZNF703 were performed using DAVID (
https://david.ncifcrf.gov), which integrates biological data and analysis tools to provide a systematic and comprehensive annotation of biological function. Genomic enrichment analysis was performed using the gene set enrichment analysis (GSEA) 3.0 software. The Oncomine database (
http://www.oncomine.org), and GEPIA (
http://gepia.cancer-pku.cn/), was used to analyze the mRNA expression level of PEA15 in the ovary.
Statistical analysis
The data were counted using the x2 test and Fisher’s exact probability tests, and measurements of the data were performed using single factor analysis of variance. Statistical differences between two groups were carried out by using the t test, and one-way analysis of variance analysis was used for the comparison of more than two groups. A two-tailed P value of < 0.05 was considered statistically significant, *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Discussion
Ovarian cancer is the most malignant gynecological tumor. It is often detected at an advanced stage with a short survival time and poor prognosis. ZNF703 is a TF belonging to the NET/NlZ family; it is located at 8p11.23 and is mainly distributed in the nucleus. Studies have demonstrated that ZNF703 and its homologs have six evolutionarily conserved domains, among which the three known domains are the spacer (SP) domain, the round head box (BTD) domain, and the cysteine2–histidine2 (C2H2) zinc finger domain [
19]. Among the SP family proteins, these specific domains are necessary for the correct subcellular distribution and transcriptional inhibition of the NET/NlZ protein. The three newly discovered domains are unique to the NET protein family. Castro et al. [
20], named these three specific domains as LP, PY, and YL based on the most abundant conserved amino acids in each of these domains. The NET protein lacks nuclear localization sequences and it is speculated that ZNF703 may interact with other proteins through the unique PY and YL domains to transport the NET protein into the nucleus [
20]. This is consistent with the opinion that the C-terminal residue of the zebrafish Nlz1 protein was essential for the protein nuclear localization proposed by Runko et al. [
21] and Sager strom et al. [
22] Janesick A et al. [
23] found that ZNF703 had different and separate functions in early mesoderm, neural crest, and substrate development. Other researchers have found that ZNF703, as a target of TFs Pax3 and Zic1, promoted the formation of the Xenopus neural crest [
6]. Several studies have shown that the expression of ZNF703 was increased in malignant tumors and promoted the development of tumors [
10,
24,
25]. In ovarian cancer, there is only one study showing that ZNF703 was upregulated and regulated the expression and role of LINC00460 in ovarian cancer tissues [
26], but unfortunately we did not detect this binding during sequencing, which might be different from the cell lines or antibodies or kits we use, or even experimental deviations. This is worth thinking about.
Sircolomb et al. [
27] found that ZNF703 inhibited the activity of transforming growth factor beta (TGFβ) receptor and the expression of E-cadherin and up-regulated the expression of pro-migratory P120 catenin, thereby promoting cell proliferation and migration and reducing intercellular adhesion. Furthermore, ZNF703-overexpressing cells altered retinoblastoma-associated protein (RB1) phosphorylation and downregulated P27kip1 protein, thereby up-regulating the expression of E2F1 and cyclin E1 (CCNE1) (G1/S-specific cyclin E1), inducing cells to escape from the R restriction point of the G1 phase and enter S phase [
27]. This study found that ZNF703 was overexpressed in ovarian cancer, and the survival time of patients with high expression of ZNF703 was significantly shortened. But ZNF703 was not an independent prognostic factor. In the cells overexpressing ZNF703, it was found that their proliferation was accelerated and apoptosis was reduced; thus, more cells entered the S and G2/M phases. However, when siRNA was used to down-regulate the expression of ZNF703 in the ovarian cancer cell lines, cell proliferation slowed down and apoptosis increased; moreover, cell cycle arrest at the G1 phase was observed. Regarding cell invasion and migration, the ability of ovarian cancer cells to invade and migrate decreased after the expression of ZNF703 was inhibited, and increased after ZNF703 overexpression. In vivo experiments also proved that ZNF703 overexpression promoted tumor formation and growth. This was consistent with the reported role of ZNF703 as an oncogene [
10,
28,
29]. As the PI3K/AKT pathway is a key coordinator of intracellular signal transduction that regulates multiple cellular processes [
18], it is often excessively activated in human malignancies and plays a key role in cancer progression [
30]. The results in ovarian cancer cell lines also confirmed that ZNF703 could activate the PI3K/AKT pathway to promote the malignant biological behaviors of ovarian cancer cells. In fact, ZNF703 is also a protein with phosphoserine at position 252. We guessed whether it was involved in the phosphorylation process of PI3K/AKT so as to directly participate in its activation. On the other hand, we speculated whether ZNF703 regulated the upstream activators of PI3K/AKT pathway, such as RTKs, FAK, JAK [
31]. Considering that ZNF703 is a transcription factor, whether it regulated the transcription of PI3K/AKT pathway or its upstream, although this was not found in our results until now.
We discovered ZNF703 by yeast two-hybrid of HE4. Subsequent verification experiments also confirmed that there was an interaction between ZNF703 and HE4, but the functional impact of HE4 on ZNF703 remains unknown. On the one hand, Castro et al. [
20] transfected the human HEK293T cell line using an overexpression vector encoding MYC markers, and proved that ZNF703/NLZ1 was mainly located in the nucleus, however the NLS (nuclear transfer mechanism) pathway of ZNF703 was not identified. Furthermore, it has been found that the central ZNF703 PY domain and the C-terminal YL domain were very important for the nuclear localization of proteins [
20]. This conclusion was similar to that proposed by other researchers studied in zebrafish [
21,
22]. Therefore, it was speculated that ZNF703 may associate via its LY and PY domains with other proteins to participate in a known nuclear localization pathway and enter the nucleus. On the other hand, HE4 is a glycoprotein that has been used clinically as a tumor marker for ovarian cancer. Many studies have focused on the role of HE4 as a secreted protein, but one study reported that HE4 could interact with importin-4 for nuclear translocation [
32]. Based on the current research conclusions, it is speculated that ZNF703 and HE4 may interact through the LY and PY domains of ZNF703, and that HE4 may promote the nuclear translocation of ZNF703. Furthermore, it has been found that after inhibiting the expression of HE4, ZNF703 accumulated in the cytoplasm. Moreover, when HE4 was overexpressed, the nuclear expression of ZNF703 increased, but this was not obvious in the immunofluorescence images, probably because ZNF703 was already highly expressed in the nucleus, irrespective of the levels of HE4, making it difficult to detect changes in fluorescence intensity. However, whether ZNF703 and HE4 interact via the LY and PY domains, and whether the nuclear translocation of ZNF703 depends on the interaction between the two remains to be confirmed. This is one of our future research aims. So we speculated that ZNF703 could interact with HE4 in the cytoplasm and ZNF703 could be brought into the nucleus by HE4, or with the help of HE4, when the nuclear pore is opened. The correlation between HE4 and ZNF703 was not significant in cell lines, but it was significant in clinical specimens, although the correlation coefficient was very low. This might be because HE4 was highly expressed in cell lines, but for clinical patients, the level of HE4 was quite different. But it also showed that the correlation between the two was not high.
In recent years, with the development of gene sequencing technology, genomic analysis has provided the prospect of revealing the characteristics of tumors, and an opportunity to explore the underlying mechanism of ovarian cancer development. At present, there is little evidence about the transcription function and epigenetic regulation of ZNF703. Nakamura et al. [
5] demonstrated that ZNF703 could suppress gene expression by recruiting the histone deacetylation complex (HDAC) and regulate the embryonic development process, such as the differentiation of vertebrate hindbrain into rhomboids. Sircoulomb et al. [
27] proposed that ZNF703 inhibited the transcription of related genes by forming a nuclear transcription complex with DNA damage-binding protein 1 (DDB1) and cullin 4 (CUL4) associated factor 7 (DCAF7), CUL4, prohibitin-2 (PHB2), and nuclear receptor corepressor 2 (NCOR2) and exerted its regulatory effects on cell proliferation, differentiation, apoptosis, and cell cycle. In terms of promoter binding, Holland et al. [
29] claimed that ZNF703 overexpression can lead to ZNF703 binding to the promoter site of the TGFβ receptor II (TGFβR2), preventing TGFβ from inhibiting cell proliferation. ChIP-sequencing technology was used to detect the binding of ZNF703 on chromatin in ovarian cancer cells. This study was the first to conduct epigenetic studies on ZNF703 in vitro. However, whether the transcriptional activity of TFs differs in different tissues, and whether it is related to the formation of transcription complexes with other proteins and the target genes that are bound, should be further investigated. Furthermore, whether the binding of ZNF703 to chromatin is related to the structure of ZNF703 requires further study.
An inhibitor of apoptosis (IAP) family member, PEA15 was cloned from astrocytes [
33]. It is a small molecule protein with a broad-spectrum anti-apoptotic function. Studies have shown that p-PEA15(Ser116) can bind to other proteins through its DED region, thereby inhibiting the formation of the death-inducing complex (DISC), inactivating caspase-8 and caspase-10 and preventing the activation of the caspase cascade. This results in the inhibition of apoptosis in tumor cells, and the occurrence, development and metastasis of tumors [
15‐
17,
34,
35]. PEA15 has two phosphorylation sites, Ser104 and Ser116. Ser104 can be phosphorylated by protein kinase C (PKC) [
36], while Ser116 can be phosphorylated by calmodulin-dependent protein kinase II (CaM kinase II) or AKT [
37,
38]. Phosphorylation at Ser116 enhances the binding of Fas-related proteins to the death domain (FADD) and to caspase-8, inhibiting apoptosis [
36,
37]. PEA15 appears to play a role in promoting or suppressing tumors. For example, the expression of PEA15 was found to be increased in non-small cell lung cancer [
39], chronic lymphocytic leukemia [
40], thyroid cancer [
41], prostate cancer [
42] and liver cancer cells [
43], where it inhibited apoptosis and promoted tumor growth. In contrast, increased PEA15 expression was also found to inhibit extracellular signal-regulated kinase (ERK)-dependent functional transcription and proliferation in ovarian cancer [
44] and breast cancer cells [
45]. But there is also evidences in ovarian cancer that PEA15 was positively correlated with the FIGO stage and cell proliferation was obstructed by knockdown of PEA15 [
46].
We also found that HE4 affected the transcription and protein expression levels of PEA15, but had no effect on the phosphorylation of PEA15, and PEA15 had an anti-apoptotic effect after phosphorylation. This was also consistent with our observation of the effects of HE4 knockdown on ZNF703 roles. It showed that HE4 affected the nuclear translocation of ZNF703, but it was not enough to cause significant changes in the functions of ZNF703. Of course, we only tested these biological behaviors. As for other aspects, such as metabolism, chemotherapy resistance, angiogenesis, etc., this will be our next research direction.
The results of this study demonstrated that ZNF703 promotes the expression of PEA15 by binding to its enhancer region. Furthermore, ZNF703 played a significant role in promoting cancer gene expression, but this seemed inconsistent with previous studies in which PEA15 was found to inhibit ovarian cancer cell proliferation. Moreover, this study determined the protein expression levels of PEA15, p-PEA15(Ser116) and p-PEA15(Ser104). The results showed that after overexpression of ZNF703, the phosphorylation of p-PEA15(Ser116)/PEA15 increased, consistent with the increased phosphorylation level of AKT found in this study. The results of Lee J et al. [
47] also confirmed that PEA15 in ovarian cancer mostly exists in the phosphorylated form. Fiory F et al. [
14] also suggested that some differences in PEA15-mediated effects may depend on the phosphorylation status or cellular environment, which might be important in determining whether PEA15 regulates cell survival or apoptosis.
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