The TRIM25 Gene in Ducks: Cloning, Characterization and Antiviral Immune Response

Simple Summary TRIM25, as an important member of the TRIM family, has been frequently demonstrated in regulating the host’s antiviral response by activating innate immunity. Ducks are a natural reservoir and often serve as asymptomatic carriers of influenza A viruses, but the beneficial roles of TRIM25 in modulating the immune response remain largely unknown in ducks. On this basis, this study characterized duTRIM25 and explored the role of different domains of duTRIM25 in duck antiviral immune response. The results displayed that the overexpression of duTRIM25 resulted in the IFN-β production after 5′ppp dsRNA infection and the SPRY domain of duTRIM25 partially enhanced duTRIM25’s antiviral activity. These data might reveal the functions of duTRIM25 in antiviral infections, providing new insights for understanding the protective immune responses of ducks. Abstract TRIM25, as a significant member of the TRIM family, has been frequently demonstrated in regulating the host’s antiviral response by activating innate immunity. Ducks are often asymptomatic carriers of influenza A viruses, but the beneficial roles of TRIM25 in modulating the immune response remain largely unknown in ducks. In this study, we characterized the TRIM25, which contains a 16 bp 5′-UTR, a 279 bp 3′-UTR and a 2052 bp ORF that encodes 683 amino acid residues. In addition, we found that duTRIM25 transcripts were widely expressed in the 10 tissues tested, with higher expression levels in the kidney, liver, muscle and spleen and lower expression levels in the duodenum and blood. In addition, the six kinds of virus- or bacteria-mimicking stimuli were transfected into DEFs, and duTRIM25 was induced significantly with 5′ppp dsRNA stimulation. Furthermore, overexpression of duTRIM25 followed by treatment with 5′ppp dsRNA resulted in an increase in IFN-β. The SPRY domain of duTRIM25 contributed to promoting IFN-β activity in DEFs challenged with 5′ppp dsRNA. Taken together, our findings suggest that duck TRIM25 can induce the production of IFN-β against double-stranded RNA virus stimuli and that the SPRY domain of duTRIM25 was critical for the infection.

Introduction
More than 70 distinct protein members collectively form the tripartite motif (TRIM) protein family. Previous studies have demonstrated that many members of the TRIM family have antiviral effects. As early as 2004, scientists discovered that TRIM5α in African green monkeys and rhesus monkeys restricts the replication of not only the human immunodeficiency virus type 1, but also the N-tropic murine leukemia virus [1,2]. TRIM19 had also been shown to potentially interfere with the replication cycle of viruses. When TRIM19 was overexpressed, it exhibited the effect of resisting the influenza A virus and vesicular stomatitis virus [3]. Chicken TRIM25 was also found to inhibit the A avian leukosis virus replication by upregulating MDA5 receptor-mediated IFN-I response in chickens [4]. Collectively, the TRIM family is extensively involved in interferon production and viral replication by activating innate immunity.
The proteins of the TRIM family have a highly conserved N-terminal domain, including a RING domain, one or two B-box domains and a coiled-coil domain, which is often referred to as the RBBC [5,6]. On the other hand, they have a variable C-terminus binding to target proteins. PRY-SPRY motifs are the most common C-terminal TRIM domain and were first identified in TRIM27. However, these domains play different roles in different tissues or cells. Many studies have confirmed that RING domain-containing proteins can target specific proteins for degradation to activate specific signaling pathways, thereby depending on E3 ubiquitin ligase activity to regulate antiviral immune responses. Kallijarvi et al. showed that when the two conserved cysteines of the TRIM37 RING domain were converted to cysteines, the polyubiquitination was significantly reduced [7]. In addition, the RING domain of TRIM22 can inhibit long terminal repeat-driven transcription of human immunodeficiency virus 1 [8]. It has been reported that the B-box domains have zinc-binding motifs and also function as E3 ligases. Crucially, the B-box domains are unique to TRIM proteins and may be major determinants of this family [9]. TRIM15 lacking the B-box domain lost all antiviral activity. Actually, the B-box domain alone exhibited strong antiviral activity [10]. As all TRIM proteins have an RBCC motif, the capacity of the C-terminal domain to recruit specific partners plays an important role in their respective unique functions [11]. The SPRY domain was reported to participate in binding to RNA and interacting with proteins as well as interfering with viral replication. Li et al. have demonstrated that the loss of antiviral activity of TRIM62 was related to the deletion of the SPRY domain [12]. Therefore, the different members of the TRIMs have similar domains, but they rely on different domains to exert antiviral effects.
Ducks are a natural reservoir and often asymptomatic carriers of influenza A viruses. Previous studies showed that the immune system of ducks had an advantage in fighting weakly pathogenic avian influenza viruses. However, highly pathogenic influenza viruses appear to be able to escape attack by ducks' immune systems [13]. Long-term co-evolution of ducks and influenza viruses fine-tunes many interactions between immune hosts and pathogens to confer resistance to disease. TRIM25, as an important member of the TRIM family, has been frequently demonstrated in regulating the host's antiviral response by activating innate immunity. Wei et al. showed that goose TRIM25 transcription could be induced by goose IFN-α, IFN-γ, IFN-λ, poly(I:C), ODN 2006 and R848 in vitro, which means goose TRIM25 may play an active role in regulating antiviral immune responses [14]. Han et al. found that duck TRIM25 was upregulated in cells when treated with the duck Tembusu virus and inhibited viral expression [15]. These studies shed some light on duck TRIM25, but the beneficial roles of TRIM25 in modulating the immune response are still worth further exploration in ducks. In this study, we identified and characterized the duTRIM25 gene, and investigated the spatiotemporal expression profile of duTRIM25. Moreover, the six kinds of virus-or bacteria-mimicking stimuli were selected, and then the duTRIM25 transcripts were detected in DEFs challenged with the stimulus. Finally, the key domain of duTRIM25 was determined to promote IFN-β production. These data might reveal the functions of duTRIM25 in antiviral infections, providing new insights for understanding the protective immune responses of ducks.

Sample Collection and RNA Extraction
Five three-day-old, Shaoxing egg-laying ducks, having similar weight, were euthanized after anesthesia. The heart, liver, spleen, lung, kidney, duodenum, ovary, pancreas, muscle and blood of healthy five ducks were collected as samples. The samples were stored at minus 80 degrees, but blood was stored at minus 20 degrees for long-term storage until the next use. According to the instructions of TRNzol Universal Reagent (TIANGEN, Beijing, China), the total RNA was extracted from different tissues of Shaoxing egg-laying ducks. The FastKing gDNA Dispelling RT SuperMix (TIANGEN) was used for reverse transcription to generate cDNA for RT-qPCR experiments.

DuTRIM25 cDNA Cloning and Sequence Analysis
Based on the duck TRIM25 predicted sequences (GenBank accession number XM_00501 2325), the duTRIM25-FLAG-F and duTRIM25-FLAG-R primers were designed (Table 1), and the duTRIM25 cDNA was amplified from the spleen of duck by RT-PCR. Then the GeneRacer kit (L1500-01, Invitrogen, Carlsbad, CA) was used for obtaining the complete cDNA sequence of duTRIM25 by 5 and 3 RACE.
A number of pieces of software were used to perform sequence analysis of TRIM25. Through the NCBI database, the sequences of TRIM25 in different species were found. A multiple sequence alignment of TRIM25 from representative species was performed using Clustal W. MEGA 6 software was used to perform phylogenetic analysis of the amino acid sequences of TRIM25 from 17 species. The conserved domains of duTRIM25 were predicted by NCBI Conserved Domains Database Tools.
To further analyze the effect of 5 ppp dsRNA on the dynamic expression of duTRIM25, DEFs were seeded in six-well plates and raised to 70-80% confluency. The cells were transfected with 5 ppp dsRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as specified by the manufacturer and then collected after 0, 3, 6, 9, 12 and 24 h of treatment. RT-qPCR was used to calculate duTRIM25 mRNA expression at different time points.

Plasmid Construction
According to the predicted IFN-β sequence of Anas platyrhynchos (GenBank accession no. KM032183), part of the sequence of the duck IFN-β promoter region (−167 to + 63) was selected to construct duIFN-β promoter. The duIFN-β promoter was cloned into the KpnI and BglII sites of pGL3-Basic to form pGL3-IFN-β.

Real-Time Quantitative PCR (RT-qPCR)
Primers required for RT-qPCR were designed by oligo 7 and then handed over to TSINGKE Biological Company for synthesis (Table 1). RT-qPCR experiments were used to detect changes in the mRNA levels of related genes. In the quantitative PCR system used in this experiment, the total volume of each sample was 10 µL, which contained 5 µL PowerUp SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific), 0.4 µL forward or reverse primer, 2 µL diluted cDNA template and 2.2 µL RNA-free water. The quantitative PCR program for this experiment was as follows: a holding step at 95 • C for 30 s, followed by 40 cycles of 10 s at 95 • C, and 30 s at 60 • C [18]. Duck β-actin was used as an internal reference, and the gene expression of the samples was analyzed using the 2 −∆∆Ct method.

Western Blotting Analysis
Five hundred microliters of a mixed solution consisting of cell lysis buffer (Biosharp, Guangzhou, China) for Western blotting and protease inhibitor (Beyotime, Shanghai, China) was added to lyse cells. In order to collect cellular proteins in the supernatant, the cell pellet was removed by centrifugation at 12,000 rpm at 4 • C for 10 min. After that, the samples were separated in 10% SDS-PAGE gel. Then, the extracted proteins were transferred to PVDF membranes, and 5% skim milk was used to block them. After that, the membranes were incubated with primary antibodies overnight at 4 • C and further incubated with an HRP-conjugated secondary antibody.

Statistical Analysis
Data are presented as mean ± standard error. p < 0.05 was considered statistically significant. GraphPad Prism 8.0 was used for processing figures.

Sequence Analysis of Duck TRIM25
The full-length sequence of duck TRIM25 cDNA was obtained using RT-PCR and RACE technology; the measured length was 2347 bp, which included a 2052 bp open reading frame (ORF) encoding 683 amino acids, a 16 bp 5 UTR and a 279 bp 3 UTR (KY_974316), The conserved domains of duck TRIM25 were analyzed using SMART, and we found that duTRIM25 as a protein of the TRIM family has a typical structure of RBBC, including the RING domain (20-63aa), two B-box domains (114-160aa, 163-194aa) and SPRY domain (511-679aa) ( Figure 1A).

Sequence Analysis of Duck TRIM25
The full-length sequence of duck TRIM25 cDNA was obtained using RT-PCR and RACE technology; the measured length was 2347 bp, which included a 2052 bp open reading frame (ORF) encoding 683 amino acids, a 16 bp 5′ UTR and a 279 bp 3′ UTR (KY_974316), The conserved domains of duck TRIM25 were analyzed using SMART, and we found that duTRIM25 as a protein of the TRIM family has a typical structure of RBBC, including the RING domain (20-63aa), two B-box domains (114-160aa, 163-194aa) and SPRY domain (511-679aa) ( Figure 1A).
Based on the amino acid sequences of bird, mammalian and fish TRIM25, a phylogenetic tree was constructed in order to evaluate the molecular evolutionary relationship between duTRIM25 orthologs in other vertebrates ( Figure 1B). DuTRIM25 shared high protein sequence similarity with Anser cygnoides TRIM25 (83.91%) and Gallus gallus TRIM25 (70.28%) but had a distant evolutionary relationship with mammalian TRIM25 and fish TRIM25. Consistent with the sequence alignment results, TRIM25 in birds is clustered in one group, and duTRIM25 was most closely related to the goose gene.

The Response to Different Stimulations on Duck TRIM25 Transcription Levels
To explore the regulation of duTRIM25 expression in DEFs by different kinds of virus-or bacteria-mimicking stimuli, the duTRIM25 mRNA levels were measured by RT-qPCR ( Figure 2). The virus-mimicking stimuli are 5′ppp dsRNA and R848; the bacteriamimicking stimuli are LPS, Pam3CSK4, FLS-1 and FLA-ST. The TRIM25 mRNA levels Based on the amino acid sequences of bird, mammalian and fish TRIM25, a phylogenetic tree was constructed in order to evaluate the molecular evolutionary relationship between duTRIM25 orthologs in other vertebrates ( Figure 1B). DuTRIM25 shared high protein sequence similarity with Anser cygnoides TRIM25 (83.91%) and Gallus gallus TRIM25 (70.28%) but had a distant evolutionary relationship with mammalian TRIM25 and fish TRIM25. Consistent with the sequence alignment results, TRIM25 in birds is clustered in one group, and duTRIM25 was most closely related to the goose gene.

The Response to Different Stimulations on Duck TRIM25 Transcription Levels
To explore the regulation of duTRIM25 expression in DEFs by different kinds of virusor bacteria-mimicking stimuli, the duTRIM25 mRNA levels were measured by RT-qPCR ( Figure 2). The virus-mimicking stimuli are 5 ppp dsRNA and R848; the bacteria-mimicking stimuli are LPS, Pam3CSK4, FLS-1 and FLA-ST. The TRIM25 mRNA levels were the highest after 5 ppp dsRNA treatment, followed by pam3CSK4 stimulations with higher expression. 3) and zebrafish (NP_956469.1). The symbol '*' denotes 100% conserved residues, ':' and '.' represent relatively conservative residues, spaces represent non-conservative residues and '--' indicates gaps during alignment. (B) A phylogenetic tree of the TRIM25 amino acid sequences was constructed by the neighbor-joining tree method using MEGA version 6. The sequence of each species was obtained from NCBI Genbank. The bar indicates the bootstrap value (%).

The Response to Different Stimulations on Duck TRIM25 Transcription Levels
To explore the regulation of duTRIM25 expression in DEFs by different kinds of virus-or bacteria-mimicking stimuli, the duTRIM25 mRNA levels were measured by RT-qPCR ( Figure 2). The virus-mimicking stimuli are 5′ppp dsRNA and R848; the bacteriamimicking stimuli are LPS, Pam3CSK4, FLS-1 and FLA-ST. The TRIM25 mRNA levels were the highest after 5′ppp dsRNA treatment, followed by pam3CSK4 stimulations with higher expression.  (resiquimod, which is an imidazoquinoline compound with potent antiviral activity). Asterisks indicate significant differences determined by a t-test between mock and virus-or bacteria-mimicking stimulus treatments, p < 0.05. Each bar represents the mean ± SE (n = 3).

The Spatiotemporal Expression Profile of TRIM25 in Ducks
In order to explore the spatial expression pattern of duTRIM25 in different tissues, total RNA was extracted from various tissues of healthy Shaoxing egg-laying ducks, and the levels of duTRIM25 transcripts were detected by RT-qPCR. As shown in Figure 3a, duTRIM25 is widely expressed in all tissues tested. Among them, duTRIM25 is prominently expressed in the kidney, liver and muscle; moderately expressed in the spleen, heart, pancreas, lung and ovary; and expressed at relatively low levels in the duodenum and blood (Figure 3a). levels were significantly increased in DEFs within 6-24 h after 5′ppp dsRNA stimulation. The peak expression was reached at 12 h (Figure 3b). The results showed that the 5′ppp dsRNA infection affected duTRIM25 mRNA expression. Specifically, duTRIM25 mRNA levels were first upregulated and then downregulated, indicating that duTRIM25 negatively regulated the 5′ppp dsRNA replication. Shaoxing egg-laying ducks. The tissues tested included heart, liver, spleen, lung, kidney, duodenum, ovary, pancreas, muscle and blood. The mRNA expression levels were normalized to the expression of duck β-actin gene. Each bar represents the mean ± SE (n = 5). (b) Relative expression levels of duTRIM25 in DEFs, treated with 5′ppp dsRNA and collected after 0, 3, 6, 9, 12 and 24 h. Each point represents the mean ± SE (n = 3). Asterisks indicate significant differences determined by a t-test between treatments 6, 9, 12 and 24 h and treatment 0 h (* p < 0.05).

Functional Analysis of the Induction of IFN-β by duTRIM25 and Its Different Domains
According to previous research results, we focused on the domains of duTRIM25, which could induce the expression of proinflammatory genes and further enhance the inflammatory response. The ORF of duTRIM25 has three different conserved domains, namely the RING domain, B-box domain and SPRY domain. Different domain segments are shown in Figure 4a. To explore the functions of duTRIM25 and its different domains, we cloned different duTRIM25 domains and constructed the expression plasmids TRIM25(RING), TRIM25(B-Box) and TRIM25(SPRY). Then, these recombinant plasmids were transfected into DEFs, and pcDNA3.1 + -FLAG was transfected as a negative control. Western blot was used to detect the expression of different domains. The results showed that FLAG fusion proteins of different domains were all expressed in DEFs, as shown in Figure 4b.
To determine the effect of viral or bacterial irritation on IFN-β induction by duTRIM25, duTRIM25 was overexpressed in DEFs and then stimulated with 5′ppp To further determine the temporal changes in the expression of duTRIM25 after 5 ppp dsRNA stimulation, DEFs were collected after 0, 3, 6, 9, 12 and 24 h of treatment with 5 ppp dsRNA and then detected by RT-qPCR. It turned out that the duTRIM25 expression levels were significantly increased in DEFs within 6-24 h after 5 ppp dsRNA stimulation. The peak expression was reached at 12 h (Figure 3b). The results showed that the 5 ppp dsRNA infection affected duTRIM25 mRNA expression. Specifically, duTRIM25 mRNA levels were first upregulated and then downregulated, indicating that duTRIM25 negatively regulated the 5 ppp dsRNA replication.

Functional Analysis of the Induction of IFN-β by duTRIM25 and Its Different Domains
According to previous research results, we focused on the domains of duTRIM25, which could induce the expression of proinflammatory genes and further enhance the inflammatory response. The ORF of duTRIM25 has three different conserved domains, namely the RING domain, B-box domain and SPRY domain. Different domain segments are shown in Figure 4a. To explore the functions of duTRIM25 and its different domains, we cloned different duTRIM25 domains and constructed the expression plasmids TRIM25(RING), TRIM25(B-Box) and TRIM25(SPRY). Then, these recombinant plasmids were transfected into DEFs, and pcDNA3.1 + -FLAG was transfected as a negative control. Western blot was used to detect the expression of different domains. The results showed that FLAG fusion proteins of different domains were all expressed in DEFs, as shown in Figure 4b. ize the role of different duTRIM25 domains in the activation of the duck IFN-I signaling pathway, a dual-luciferase reporter assay was conducted to measure IFN-β promoter activity. The results indicated that TRIM25(SPRY) promoted IFN-β activity in DEFs challenged with 5′ppp dsRNA (Figure 4d). In general, SPRY domain overexpression partially enhanced duTRIM25′s antiviral activity; therefore, the SPRY domain may be an important structure of TRIM25 involved in the duck antiviral response. The induction of IFN-β by 5′ppp dsRNA or LPS was further determined. DEFs were co-transfected with pcDNA3.1 + -FLAG-TRIM25 or vector together with pRL-TK Renilla luciferase plasmids and IFN-β luciferase reporter genes for 24 h, and stimulated with 5′ppp dsRNA, LPS or normal saline as a control for 12 h. Luciferase activity was then measured by a dual-luciferase reporter assay system. (d) Characterization of the effect of different domains of duTRIM25 on the induction of IFN-β. DEFs were co-transfected with expression plasmids TRIM25(RING), TRIM25(B-Box) and TRIM25(SPRY) together with pRL-TK Renilla luciferase plasmids and IFN-β luciferase reporter genes for 24 h and stimulated with 5′ppp dsRNA for 12 h. A dual-luciferase reporter assay system was used to measure luciferase activity. All data are presented as means ± SE (n = 3). Significance was analyzed by one-way ANOVA with Duncan method (* p < 0.05). The induction of IFN-β by 5 ppp dsRNA or LPS was further determined. DEFs were co-transfected with pcDNA3.1 + -FLAG-TRIM25 or vector together with pRL-TK Renilla luciferase plasmids and IFN-β luciferase reporter genes for 24 h, and stimulated with 5 ppp dsRNA, LPS or normal saline as a control for 12 h. Luciferase activity was then measured by a dual-luciferase reporter assay system. (d) Characterization of the effect of different domains of duTRIM25 on the induction of IFN-β. DEFs were co-transfected with expression plasmids TRIM25(RING), TRIM25(B-Box) and TRIM25(SPRY) together with pRL-TK Renilla luciferase plasmids and IFN-β luciferase reporter genes for 24 h and stimulated with 5 ppp dsRNA for 12 h. A dualluciferase reporter assay system was used to measure luciferase activity. All data are presented as means ± SE (n = 3). Significance was analyzed by one-way ANOVA with Duncan method (* p < 0.05).
To determine the effect of viral or bacterial irritation on IFN-β induction by duTRIM25, duTRIM25 was overexpressed in DEFs and then stimulated with 5 ppp dsRNA or LPS, respectively. The results showed that overexpression of duTRIM25 resulted in IFN-β production after 5 ppp dsRNA infection (Figure 4c). To further characterize the role of different duTRIM25 domains in the activation of the duck IFN-I signaling pathway, a dual-luciferase reporter assay was conducted to measure IFN-β promoter activity. The results indicated that TRIM25(SPRY) promoted IFN-β activity in DEFs challenged with 5 ppp dsRNA (Figure 4d). In general, SPRY domain overexpression partially enhanced duTRIM25 s antiviral activity; therefore, the SPRY domain may be an important structure of TRIM25 involved in the duck antiviral response.

Discussion
As an important member of the TRIM family, TRIM25 has been repeatedly reported to play important roles in mammalian immune responses to both viral and bacterial infections [19,20], which can induce the production of type I interferon and the upregulation of IFN-stimulated genes. In fact, TRIM25 is also an important gene in duck antiviral immune response. The present findings support the hypothesis that TRIM25 can regulate the host's antiviral response by activating innate immunity in ducks. In the study, we found that duck TRIM25 can induce the production of IFN-β against double-stranded RNA virus stimuli and that the SPRY domain of duTRIM25 was critical for the infection. These data provide references for understanding the mechanism of duTRIM25 exerting antiviral immune regulation.
We identified duTRIM25 from Shaoxing egg-laying ducks. The full-length cDNA of duck TRIM25 was cloned and sequenced; it contained a 2052 bp ORF, a 16 bp 5 -UTR and a 279 bp 3 -UTR. Duck TRIM25 conserved domains predicted by the CCD tool indicate that the duTRIM25 protein possesses typical features of TRIM family proteins, namely the RBCC domain, including one RING domain and two B-box-type zinc finger domains in the N-terminus and one SPRY domain in the C-terminus [21]. This is consistent with the results reported by Han et al. [15]. Previous studies have shown that TRIM22 relies on the RING domain to inhibit human immunodeficiency virus 1 long terminal repeat-driven transcription for antiviral effects [8]. However, the deletion of the B-box domain resulted in the loss of the antiviral activity of TRIM15 [10]. In addition, the antiviral activity of TRIM62 depends on the SPRY domain [12], which implies that the protein domain of the TRIM family may play a major role in recognizing pathogenic microorganisms and viruses and exerting antiviral immune responses.
The spatial expression profile of TRIM25 in healthy duck tissues was evaluated in this study. TRIM25 expression was monitored in all tissues tested, although expression levels differ across tissues (Figure 3a). The expression patterns of TRIM25 in different tissues have been previously studied in poultry, including in chickens, ducks and geese. This study was consistent with the results showing that chicken, duck and goose TRIM25 is widely expressed in a broad range of tissues and cells. Here, we summarized and analyzed the previous expression profiles of chickens, ducks and geese and the results of this study. Chicken TRIM25 was highly expressed in the lung, thymus and spleen [22]. The highest goose TRIM25 expression was detected in blood, proventriculus, Harderian gland and kidney [14]. Han et al. showed that duTRIM25 was highly expressed in the spleen, liver, trachea and muscle [15]. Because of the presence of IFN-stimulated response elements, TRIM25 expression was higher in immune-associated tissues. In the present study, we showed that duTRIM25 was also expressed at higher levels in immune-related tissues such as the kidney, spleen and pancreas, suggesting that TRIM25 might play an important role in the innate immune response in ducks. Although the kidney is not an immune organ in ducks, it also has many immune functions. Sun et al. proved that when ducks were infected with a highly pathogenic avian influenza virus, the virus titer in the kidney of ducks was significantly increased [23]. Ou et al. showed that when duck kidney was attacked early by the avian hepatotropic virus, cytokine storms that included type I and type II IFNs were generated [24]. Interestingly, in addition to being highly expressed in immune-related tissues, duTRIM25 was also dramatically expressed in the liver and muscle. TRIM59 has been confirmed to be highly expressed in the liver of mice, and it may be a potential therapeutic target for inflammatory diseases such as atherosclerosis [25]. Some studies suggest that TRIM25 is an E3 ligase that interacts with and degrades MTA-1 protein, which is one of the important mediators of metastatic progression in hepatocellular carcinoma [26]. Therefore, the high expression of duTRIM25 in the liver may imply that it also plays an important role in liver-related diseases. TRIM55 had been reported to act as a transient adapter between microtubules, actin and nascent myosin filaments and is involved in signaling from sarcomeres to the nucleus [27]. However, the role of TRIM25 in muscle has not been studied. We can only speculate that the high expression of duTRIM25 in muscle may be due to its potential regulatory effect on muscle sarcomere assembly. The innate immune system is the host's first line of defense against invading pathogens [28]. An essential part of the innate immune system is the host cells expressing pattern recog-nition receptors [29], which can detect pathogen-associated molecular patterns (PAMPs) and trigger the production of type I IFNs [30]. 5 ppp dsRNA is a virus mimic [31]; avian influenza virus infection can be simulated in vitro through it. It contains three phosphate groups on the 5' end as its key element and thus can specifically activate the RLR signaling pathway to induce type I interferon production [32]. However, we must admit that there are still differences between the transfected virus mimics and avian influenza infection. Our research results provide a reference for the immune procedure in ducks after the simulation of avian influenza infection, but the findings in the actual infection situation have yet to be determined. In the next experiment, we will also use avian influenza infection for verification. Duck embryo fibroblasts are often used as a model for in vitro simulated virus infection experiments in ducks [33,34]; in this study, they also provided a preferred model for exploring the mechanisms of duTRIM25 function in avian innate immunity. However, DEFs also have limitations. In the present experiment, we found that the R848 treatment of DEFs failed to induce significant expression of duTRIM25. However, Wei et al. showed that the expression of goose TRIM25 was increased after R848 treatment in goose PBMCs. The reason for this may be that goose PBMCs are immune cells; compared to GEFs, they are more sensitive to immune response.
In the current study, we showed that 5 ppp dsRNA infection affected duTRIM25 mRNA expression, first upregulating (at 12 h post-infection) and then downregulating (at 24 h post-infection) it in DEF cells, indicating that the host cells rapidly trigger antiviral defense to control infection within 12 h. Further, this shows the potential antiviral immune effect of duTRIM25 in RLR signaling pathways in duck cells.
As said before, the antiviral functions of TRIM proteins are closely related to their protein domains. To further explore the roles of different domains on IFN-β production, we detected the IFN-β dual-luciferase activity in DEFs transfected with the overexpression of different duTRIM25 domains. For full-length duTRIM25, a comparison of the luciferase levels driven after 5 ppp dsRNA treatment showed that the activity of IFN-β was increased significantly. Before and after treatment with 5 ppp dsRNA, the expression of the SPRY domain promoted IFN-β production. However, the luciferase levels of RING and Bbox domains were always low. In summary, overexpression of the duTRIM25 SPRY domain in vitro can inhibit the replication of viruses by promoting the expression of IFN-β, illustrating that SPRY domain overexpression has the main impact on antiviral activity. As mentioned above, TRIM22 relies on the RING domain to inhibit HIV transcription [8]. However, the B-box domain is important for the antiviral activity of TRIM15 [10], while the deletion of the SPRY domain will make TRIM62 lose its antiviral activity [12]. From this, it can be concluded that though the TRIM family has the same conserved domains, different TRIM proteins rely on different domains to provide antiviral immunity. The SPRY domain is a prerequisite for the antiviral activity of duTRIM25. Further study is needed to explain the mechanism of the function of the duTRIM25 domains in the restriction of viral infection.

Conclusions
In conclusion, duTRIM25 transcripts were higher in the kidney, liver, muscle and spleen as well as lower in the duodenum and blood. DuTRIM25 was induced significantly with 5 ppp dsRNA stimulation compared with the other five kinds of virus-or bacteriamimicking stimuli. Furthermore, overexpression of duTRIM25 followed by treatment with 5 ppp dsRNA resulted in increased IFN-β. The SPRY domain of duTRIM25 contributed to promoting IFN-β activity in DEFs challenged with 5 ppp dsRNA. These data might reveal the functions of duTRIM25 in antiviral infections, providing new insights into the protective immune responses of ducks.

Institutional Review Board Statement:
All animal experiments performed in this study were approved by the Animal Ethics Committee of Yangzhou University.


The Roles of TP53 and FGFR2 in Progress Made Treating Endometrial Cancer

The morbidity and mortality caused by endometrial cancer (EC) is still rising worldwide. In recent years, a new system of tumor stratification has been proposed based on POLE-mutational status, TP53, and microsatellite stability status. The aim of the study was to analyze a vast panel on the genes potentially involved in the genesis of endometrial cancer in the Polish population. One hundred and three white female patients with confirmed endometrial cancer were enrolled on the study. We performed sequencing using the Hot Spot Illumina panel and microsatellite stability with immunohistochemistry. We confirmed a key role of the TP53 mutation in progress to high-grade EC and parallelly some role of FGFR2 mutation. Moreover, our data present a vast landscape of mutations in EC and their polymorphism. We reported the meaning of FGFR2 mutation and TP53 (high copy number) in high-grade ECs. Our observation in MSI contribution is comparable with other studies. Finally, we see a strong need for the implementation of the TCGA classification.

Introduction
Endometrial cancer (EC) is considered the sixth most common malignant neoplasm in women. According to global statistical data, it is the second most common cancer of the female genital organs after cervical cancer. The highest incidence of EC is observed in well-developed countries in North America and northern and eastern Europe [1,2]. In the United States and Canada, EC is the most common gynecological cancer, while in China, EC is in the second position after cervical cancer [2][3][4].
The morbidity ratio of EC is increasing in 26 countries worldwide, with the largest increase in Asia and Africa, largely due to their rapid socio-economic changes. In the United States, EC is one of the few malignant neoplasms with increasing incidence and mortality. Between 1999 and 2016, the incidence of EC increased by 0.7% per year, while the mortality rate increased by 1.1% per year [5]. Breast cancer is the most common female malignancy in Poland, and EC frequency still raised over the past four decades. According to the statistical data for 2010-2017, the highest number of cases of endometrial cancer in Poland involved women in the 60-64 age group, and the highest number of deaths caused by this cancer was recorded in the 75-79 age group [6]. That EC overall survival dropped from 86.9% to 82.7% (p < 0.05), and from 2019 to present to 81.2% [3,7]. Endometrial cancer mainly affects older women, but there are cases in women younger than 40 years old. In most cases, it is diagnosed in the early stage of clinical advancement, for whom the five-year survival ratio is about 95%, in contrast to endometrial cancer detected in late-stage, where survival ranges from 16% to 45% [8,9]. The classical division of endometrial cancer includes two distinct pathogenetic subtypes that differ in histological and molecular characteristics, as defined by Bokhman [8].
Type I is based on the previous atypical endometrial hyperplasia showing estrogendependent proliferation and a high ratio of PTEN mutation [2,10,11]. These tumors are considered as less aggressive, often found in the early stage according to FIGO in stage I and II, with high positive steroid receptor status as an indicator of a favorable prognosis [12].
Many studies have identified PTEN mutation as the most frequent molecular event causing the initiation of intraepithelial neoplasia. Animal models of endometrial cancer have demonstrated that biallelic PTEN loss leads to the development of atypical hyperplasia, whereas biallelic PTEN loss, together with the PIK3CA mutation, leads to cancer. These findings confirmed the fact that PTEN mutation commonly co-occurs with PIK3CA and PIK3R1 mutation. A deep molecular insight into causative pathways allowed to separate the EC subgroups according to molecular pathway and clinical outcome. The first subgroup covers the POLE-mutated tumors. Patients in this subgroup presented the best disease-free survival (DFS). In the second subgroup are tumors which additionally presents microsatellite instability (MSI) or MLH1 promoter hypermethylation. The third subgroup includes low copy number (CNV)/and microsatellite stable (MSS) tumors with frequent CTNNB1 (Wnt-pathway) mutation. Finally, women in the fourth subgroup were characterized by high CNV via frequent TP53 mutation. In this subgroup, overall survival (OS) and DFS are poor [14].
The molecular subgroup proposed by the Cancer Genome Atlas (TCGA) has evolved the two-tier Bokhman's model into a precise molecular-based system [15]. This has resulted in the creation of a new diagnostic algorithm and clinically meaningful EC classification. The pathology reports should include information about the mutational status of the POLE gene and perform immunohistochemical p53, MSH6 and PMS2 to provide an integrated 'histo-molecular' diagnosis [16]. Recent papers confirm the diagnostic value of immunohistochemistry in MSI screening. In addition, the MSI status is now recognized as the very promising therapeutic target in advanced EC given the initial results of immune checkpoint inhibitors. Clinical trials evaluating these drugs in the early stage will start soon [17,18].
We aimed to analyse a vast panel on genes potentially involved in the genesis of endometrial cancer in the Polish population.

Study Population
One hundred and three white female patients with confirmed endometrial cancer were enrolled in the study. All patients were operated on in Province Hospital, Kielce, between 2005 and 2017. A total hysterectomy with pelvic lymphadenectomy was performed. The collective data for evaluation of follow-up were tabulated and successively enhanced annually in all cases. (Table 1) A computer tomography examination of the abdomen and chest, a typical blood test, and an endoscopic examination were performed. Each case was re-diagnosed according to the Eighth Edition of TNM Classification [17]. Importantly, all of the studied participants underwent typical surgical treatment without previous radio-chemotherapy. This resulted in a credible comparative analysis of tumor characteristics in the scope of treatment and an unchanged molecular profile.

Microsatellite Stability
Microsatellite stability status was assessed by immunohistochemistry. The immunohistochemical assays were performed using the automated IHC/ISH slide staining system BenchMark Ultra (Ventana Medical Systems; Roche Group, Tucson, AZ, USA). A complete diffuse lack of positive nuclear reaction of MLH-1, MSH2, MSH6, and PMS2 was coded as positive MSI.
Library preparation for NGS analysis: The analysis of genes involved in cancer was performed using the amplicon-based method AmpliSeq Cancer HotSpot Panel v2 for Illumina (San Diego, CA, USA). List of analyzed genes is presented in Supplementary Material Table S1 Amplicon-based gene panel protocol: Amplification of the hotspot regions of 50 oncogenes and tumour suppressor genes was carried out using AmpliSeq™Library PLUS for Illumina ® assay Kit (San Diego, CA, USA) according to the manufacturer's instructions (AmpliSeq for Illumina Cancer HotSpot Panel v2 Reference Guide). Briefly, the assay generated a library of 207 gene-specific amplicons and targets~2800 clinically relevant mutations. The amplification was performed in two rounds of PCR: Initially, 72 ng of DNA was used for each sample to perform multiplex PCR reactions that amplified the entire target regions. The adapters ligation was performed using AmpliSeq™ CD Indexes Set A for Illumina ® . The small residual DNA fragments were removed by use of a magneticbased DNA purification approach. The product of each sample was used as a template for the second amplification step, which amplifies the product with sequencing primers. Finally, each tagged amplicon library purification was performed using NucleoMag ® NGS Clean-up and Size select beads (Machery-Nagel GmbH& Co., Düren, Germany). Each library was qualified using QuantiFluo ® ONE dsDNA System (Promega, USA) to allow for the equimolar pooling of all sample libraries for subsequent sequencing.
Sequencing: Products were analysed by Next Generation Sequencing (NGS) using the Illumina platform MiSeq Dx. Briefly, NGS was performed using a MiSeq Reagent Micro Kit v2 (300-cycle) (Illumina, San Diego, CA, USA). Indexed DNA library concentrations were quantified as described above and normalised to 2 nM using Low TE. The library was denatured using 5 µL of 2 nM library and 5 µL 0.2 N NaOH. The library was diluted using pre-chilled HT1 buffer at a final concentration of 20 pM. Finally, the 8 pM library was spiked in 5% of PhiX Control v2 (Illumina, San Diego, CA, USA), which provides quality control for cluster generation, sequencing, and alignment.
Data analysis: Analysis of NGS data was performed using the GALAXY platform (usegalaxy.org). Sequencing reads (FASTQ files) were aligned to the human reference genome hg19 using the Bowtie2 tool. Variant calling was performed using the Varscan2 tool. The parameters used for data analysis were minimum allele frequency, 0.05; minimum quality, 20; and minimum coverage ×80. All called variants were annotated using wANNOVAR (https://wannovar.wglab.org (accessed on 10 February 2019)). Results were visualized using the R Bioconductor package Maftools (http://bioconductor.org/ (accessed on 10 February 2019)).

Statistical Methods
Quantitative data were reported as mean and standard deviation or median and range. Categorical data were expressed as number and percentage distributions. The Chisquare test or Fisher's exact test was applied to compare proportions, and a multivariable logistic regression model was used for assessing the relationship between the targeted genes. Follow-up time was calculated as the number of years from surgery to disease recurrence, death from reasons other than cancer, or the last contact with the patient. The univariate associations between disease-free survival and selected patient and tumor characteristics were evaluated by the univariate Cox proportional hazard model, and for those analyses, continuous variables were dichotomized according to the median. To identify the independent prognostic factor for disease-free survival, a multivariate Cox proportional hazard model with backward selection (with cut-off 0.05) was performed on only those statistically significant variables in univariate analysis.

Clinical Characteristics of the Study Population
A total of 103 woman aged from 48 to 99 years old with different type of endometrial cancer and full clinical history were included in this study. It was observed that the largest group were woman between 61-80 years old (62%). It was also noticed that 97% of women in the study group were obese or overweight (BMI > 30). The average time of the overall survival (OS) was 8.23 years. The time of relapse-free survival (RFS) was recorded only in six cases, and the average time was 4.6 years. The detailed description of the study group is presented in Table 1 and Supplementary Material Table S2. Additionally the characterization of Histopathological types of EC is presented in Supplementary Material Table S3.

Microsatellite Stability Status
In the studied cohort, we noted a 28% frequency of microsatellite instability. The lack of MLH-1 was observed 13.5% cases, MSH-2 in 1 case, MSH-6 in two cases, and PMS-2 in 8.7% of cases. MSI and especially MLH-1 silencing positively correlated with RFS (p < 0.01). Moreover, all MLH-1 positive tumors lacked MLH-1 mutation in the used molecular panel, and moreover were connected with high grade EC (p < 0.01). Interestingly, in a multifactorial analysis, MLH-1 followed the FGFR-2 mutation and led to higher tumour grade (p < 0.01).

Genotyping
All results were analyzed in terms of quality. All nucleotides were covered with at least the minimum amount of reads necessary for reliable variant detection with the modified targeted capture-based approach in 67 cases. Thirty-seven cases were considered low-covered following the generally applied minimum read count for nucleotides in the targeted capture-based approach. These cases were excluded from the analysis. Generally, in the studied cohort, mutations in 14 analyzed genes were detected. Figure 1 depicts a characteristic of the mutational spectrum. Additionally the mutation spectrum according to histopathological type and grade is presented in Supplementary Material Table S4. survival (OS) was 8.23 years. The time of relapse-free survival (RFS) was recorded only in six cases, and the average time was 4.6 years. The detailed description of the study group is presented in Table 1 and Supplementary Material Table S2. Additionally the characterization of Histopathological types of EC is presented in Supplementary Material Table S3 3

.2. Microsatellite Stability Status
In the studied cohort, we noted a 28% frequency of microsatellite instability. The lack of MLH-1 was observed 13.5% cases, MSH-2 in 1 case, MSH-6 in two cases, and PMS-2 in 8.7% of cases. MSI and especially MLH-1 silencing positively correlated with RFS (p < 0.01). Moreover, all MLH-1 positive tumors lacked MLH-1 mutation in the used molecular panel, and moreover were connected with high grade EC (p < 0.01). Interestingly, in a multifactorial analysis, MLH-1 followed the FGFR-2 mutation and led to higher tumour grade (p < 0.01).

Genotyping
All results were analyzed in terms of quality. All nucleotides were covered with at least the minimum amount of reads necessary for reliable variant detection with the modified targeted capture-based approach in 67 cases. Thirty-seven cases were considered low-covered following the generally applied minimum read count for nucleotides in the targeted capture-based approach. These cases were excluded from the analysis. Generally, in the studied cohort, mutations in 14 analyzed genes were detected. Figure 1 depicts a characteristic of the mutational spectrum. Additionally the mutation spectrum according to histopathological type and grade is presented in Supplementary Material Table S4. We noted the missense mutation as the most frequent single nucleotide polymorphism (SNP). PTEN mutation was found in 49% of cases, and interestingly, here, we observed a marked heterogeneity of mutation variants. Exon 5 codon 130 (c.C388G) was the most frequently altered. As was presented in Figure 2, we noted that many cases harbor more than one mutation. We noted the missense mutation as the most frequent single nucleotide polymorphism (SNP). PTEN mutation was found in 49% of cases, and interestingly, here, we observed a marked heterogeneity of mutation variants. Exon 5 codon 130 (c.C388G) was the most frequently altered. As was presented in Figure 2, we noted that many cases harbor more than one mutation. The occurrence and overlapping of additional molecular events. A picture illustrates the main pathways involved in EC genesis. We observed the PIK3CA-PTEN pathway with a frequency close to 50%. TP53 silencing was observed in 20% and the Wnt-pathway in 12%.

Genetic Background and Clinical Implications
The statistical analysis did not reveal a clear dependence of mutation type with tumor grade, RFS, and OS (p > 0.05). One case of clear cell carcinoma presented no mutation in the user panel. Multivariate analysis showed the dependence of the tumor dedifferentiation with FGFR2 and TP53 (p < 0.01). Moreover, the FGFR2 mutation was more frequently observed in more advanced tumors (p < 0.01). We performed the analysis covering the number of SNP in aspect of RFS, OS, and age. The correlation was not statistically significant. However, the TNM strongly correlate with the TP53 SNP number (p < 0.01).
It is worthy of note that the FGFR2 and TP 53 mutations correlated well with tumor grade progress. Tumor stage progress depends on the FGFR2 mutation and the mutation number in TP53. Interestingly, one case of 59-year-old female (endometrioid carcinoma G2, pT1b) revealed six mutated genes (including KRAS, TP53, APC and CTNNB1) in 11 SNP, and after 14-years of observation, the patient is still alive. In our study, we found 10 cases of EC G1 with excellent follow up (up to 15 years)   The occurrence and overlapping of additional molecular events. A picture illustrates the main pathways involved in EC genesis. We observed the PIK3CA-PTEN pathway with a frequency close to 50%. TP53 silencing was observed in 20% and the Wnt-pathway in 12%.

Genetic Background and Clinical Implications
The statistical analysis did not reveal a clear dependence of mutation type with tumor grade, RFS, and OS (p > 0.05). One case of clear cell carcinoma presented no mutation in the user panel. Multivariate analysis showed the dependence of the tumor dedifferentiation with FGFR2 and TP53 (p < 0.01). Moreover, the FGFR2 mutation was more frequently observed in more advanced tumors (p < 0.01). We performed the analysis covering the number of SNP in aspect of RFS, OS, and age. The correlation was not statistically significant. However, the TNM strongly correlate with the TP53 SNP number (p < 0.01).
It is worthy of note that the FGFR2 and TP 53 mutations correlated well with tumor grade progress. Tumor stage progress depends on the FGFR2 mutation and the mutation number in TP53. Interestingly, one case of 59-year-old female (endometrioid carcinoma G2, pT1b) revealed six mutated genes (including KRAS, TP53, APC and CTNNB1) in 11 SNP, and after 14-years of observation, the patient is still alive. In our study, we found 10 cases of EC G1 with excellent follow up (up to 15 years) with no mutation in the user panel. The Kaplan-Meier analysis concerning TP53 (HR = 2.26; 95% Cl: 0.222-23.074; p = 0.067), MLH-1 (HR = 2.34; 95% Cl: 0.465-11.730; p = 0.303), MSI (HR = 0.55; 95% Cl: 0.155-1.933; p = 0.350), and FGFR-2 (HR = 0.40; 95% Cl: 0.142-1.144; p = 0.975) showed no significant results, but the impact of FGFR-2 to OS was more significant than TP53 (Figure 3). Figure 4 presents a main molecular pathways involved in EC genesis and progression. Not surprisingly, PTEN mutation dominates, leading to AKT-mTOR activation, but in parallel, in 14% of cases, we observed an additional FGFR2 booster. A spectrum of TP53 mutations leads to an excess of misfolded p53 or a total lack of p53. Overstimulation via the Wnt-pathway and the β-catenin excess contributes to 12% of cases. G2, pT1b) revealed six mutated genes (including KRAS, TP53, APC and CTNNB1) in 11 SNP, and after 14-years of observation, the patient is still alive. In our study, we found 10 cases of EC G1 with excellent follow up (up to 15 years) with no mutation in the user panel. The Kaplan-Meier analysis concerning TP53 (HR = 2.26; 95% Cl: 0.222-23.074; p = 0.067), MLH-1 (HR = 2.34; 95% Cl: 0.465-11.730; p = 0.303), MSI (HR = 0.55; 95% Cl: 0.155-1.933; p = 0.350), and FGFR-2 (HR = 0.40; 95% Cl: 0.142-1.144; p = 0.975) showed no significant results, but the impact of FGFR-2 to OS was more significant than TP53 (Figure 3).  Figure 4 presents a main molecular pathways involved in EC genesis and progression. Not surprisingly, PTEN mutation dominates, leading to AKT-mTOR activation, but in parallel, in 14% of cases, we observed an additional FGFR2 booster. A spectrum of TP53 mutations leads to an excess of misfolded p53 or a total lack of p53. Overstimulation via the Wnt-pathway and the β-catenin excess contributes to 12% of cases.

Discussion
A current recommendation in triaging EC is focused on testing the TP53, POLE, and MSI mutational status. Our study points out that the FGFR-2 mutation has no less meaning than TP53.
To the present day, the risk stratification systems of EC use the conventional rules of TNM classification as the depth of myometrial invasion, lymph node metastases, histology, and tumor grade. Genomic factors are not yet in standard clinical use for the assessment of prognosis. The subtypes proposed by Bokhman in 1983 are still widely accepted and used. The Cancer Genome Atlas funded by the National Cancer Institute provided us with a modern insight into the EC driving pathway and tumor biology [19]. However, the  Figure 4 presents a main molecular pathways involved in EC genesis and progression. Not surprisingly, PTEN mutation dominates, leading to AKT-mTOR activation, but in parallel, in 14% of cases, we observed an additional FGFR2 booster. A spectrum of TP53 mutations leads to an excess of misfolded p53 or a total lack of p53. Overstimulation via the Wnt-pathway and the β-catenin excess contributes to 12% of cases.

Discussion
A current recommendation in triaging EC is focused on testing the TP53, POLE, and MSI mutational status. Our study points out that the FGFR-2 mutation has no less meaning than TP53.
To the present day, the risk stratification systems of EC use the conventional rules of TNM classification as the depth of myometrial invasion, lymph node metastases, histology, and tumor grade. Genomic factors are not yet in standard clinical use for the assessment of prognosis. The subtypes proposed by Bokhman in 1983 are still widely accepted and used. The Cancer Genome Atlas funded by the National Cancer Institute provided us with a modern insight into the EC driving pathway and tumor biology [19]. However, the molecular panel we used lacks the POLE gene; we noted similar results. In our group, tumor grade correlated well with TP53 and FGFR2 mutation (p < 0.01). Interestingly, the FGFR2 mutation accelerated the stage progress. Moreover, in a separate analysis covering the number of a point mutation in a singular gene, we observed a marked impact of hypermutated TP53 (copy number high) on the clinical stage (p < 0.01).
The majority of EC can be classified into one of the four molecular subgroups. However, in a small subset (3-5%) of patients, the molecular analysis will show more than one

Discussion
A current recommendation in triaging EC is focused on testing the TP53, POLE, and MSI mutational status. Our study points out that the FGFR-2 mutation has no less meaning than TP53.
To the present day, the risk stratification systems of EC use the conventional rules of TNM classification as the depth of myometrial invasion, lymph node metastases, histology, and tumor grade. Genomic factors are not yet in standard clinical use for the assessment of prognosis. The subtypes proposed by Bokhman in 1983 are still widely accepted and used. The Cancer Genome Atlas funded by the National Cancer Institute provided us with a modern insight into the EC driving pathway and tumor biology [19]. However, the molecular panel we used lacks the POLE gene; we noted similar results. In our group, tumor grade correlated well with TP53 and FGFR2 mutation (p < 0.01). Interestingly, the FGFR2 mutation accelerated the stage progress. Moreover, in a separate analysis covering the number of a point mutation in a singular gene, we observed a marked impact of hypermutated TP53 (copy number high) on the clinical stage (p < 0.01).
The majority of EC can be classified into one of the four molecular subgroups. However, in a small subset (3-5%) of patients, the molecular analysis will show more than one classifying alteration (e.g., POLEmut-MMRd EC, POLEmut-p53abn EC, MMRd-p53abn EC, or POLEmut-MMRd-p53abn EC). As there are distinct prognostic differences between the four molecular subgroups, the question arises as to which biological behavior these multiple classifiers follow. This dilemma is most pronounced in the combination of POLEmut-p53abn EC. The tumor exhibits a favorable pathogenic mutation in the POLE exonuclease domain and the unfavorable aberrant p53 IHC expression [15,20,21]. It seems that the proposed four-tiered classification will evolve in the future. In our cohort, we noted 28% MSI frequency with a dominance of MLH-1 silencing. Microsatellite instability, in general, affected RFS (p < 0.01), but MLH-1 aberration to tumor stage and grade (p < 0.01). According to the mixed molecular subgroups, our study showed a coincidence of MSI with TP53 mutation in four cases, which means that about 20% of MSI cases were additionally TP53 mutated. Imboden et al. reported a large cohort analysis in aspect POLE mutated EC. They reported a significant POLE mutation heterogeneity with hotspots in c.857C > G and c.1231G > C and better overall survival POLE-mutated patients [22]. We mentioned ten EC cases with negative mutation results. These may be only POLE-mutated, and this needs further testing.
The most important clinical aspect is how to correctly segregate EC according to survival. The traditional three-tiered FIGO classification separates EC into three grades (G1, G2, and G3). The binary FIGO grading system was proposed close to 20 years ago, being in line with gastrointestinal pathology [23,24]. For repeatable classification, FIGO grade 3 endometrioid, serous, and clear cell carcinomas will be considered "high-grade endometrial carcinomas" (HGECs) and G1-G2 endometrioid carcinoma as low-grade endometrioid carcinomas (LGECs). The overwhelming number of LGECs belongs to the copy number low and MSI-H categories. Grade 3 endometrioid carcinomas are highly heterogenous, as they are found in every genomic category and are least represented in the copy number low group. Endometrioid carcinomas mostly have an endometrioid genomic profile, with or without TP53 mutation/high copy number alterations, whereas serous carcinomas have TP53 mutations/high copy number alterations without additional mutations characteristic of endometrioid carcinomas [25][26][27]. In our study, the TP53 mutation frequency was 20%, but some clinical significance we observed in the case of FGFR2 mutation (frequency 14%).
Going forward with the International Society of Gynecological Pathologists recommendation, we agree that TCGA classification should replace Bokhman's. Unfortunately, there are unresolved financing issues be updated by FGFR-2, in our opinion with no less meaning. At this moment, the immunohistochemistry of p53 and MSI is recommended together with POLE-gene sequencing.
Assuming 6.3% frequency of the POLE hotspot mutation with an excellent prognosis the molecular testing is helpful in correct patients triage [22]. Moreover, the research based on the cell cultures confirms our results concerning the contribution of stromal fibroblasts in epithelial-mesenchymal transition and following poor outcomes [26]. Being aware of serious restrictions of immunohistochemistry, especially p53, broad testing is only one way to triage patients to different treatment algorithms [25][26][27]. We recommend a larger genes panel and NGS as a first choice method.

Conclusions
Our research confirmed a substantial value of EC molecular sub-grouping. The present study unveiled a number mutated genes contributing to cancer development and progress. It seems that the original TCGA rules should be replaced by new ones, in our opinion with no less meaning. FGFR-2 plays an important role in cancer progress even if via the epithelial-mesenchymal transition. Looking from a clinical point of view, to apply better chemotherapy regiment, and finally to modify the patient surveillance [18,19,28,29].
Future studies in larger populations with a vast panel of genes are necessary to develop evidence-based recommendations. Although there are limited evidences of FGFR-2 contribution in EC genesis, we recommend continuing along those lines.
This study was limited by the select population of one region in Poland, and the focused follow-up time varied amid the same participants. In future research we plan to add a control group. Moreover, our genes panel did not cover the POLE gene. The strengths of this work include the long-term observation and the modern methods of genetic testing. Our results unveiled other driving mutations to compare with the recommended panel.