The overexpression of EPS8L1 upregulates TIAM2 to promote cytoskeleton remodeling by activating the Rac1/MAPK signaling pathway in the migration of ovarian cancer

The overexpression of EPS8L1 upregulates TIAM2 to promote cytoskeleton remodeling by activating the Rac1/MAPK signaling pathway in the migration of ovarian cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The overexpression of EPS8L1 upregulates TIAM2 to promote cytoskeleton remodeling by activating the Rac1/MAPK signaling pathway in the migration of ovarian cancer Yuting Wang, Lei Zhang, Xianqiang Luo, Hongmei Zhu, Meichen Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4263533/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : EPS8L1, an analog of epidermal growth factor receptor pathway substrate 8 (Eps8), was screened out in our previous work from clinical samples of patients with ovarian cancer. We also found that EPS8L1 was involved in various biological activities. In this study, the participation and mechanisms of EPS8L1 ion the migration and metastasis of ovarian cancer were investigated. Methods : In vitro scratch-healing, transwell assay, and actin-staining studies were performed in SKOV-3 ovarian cancer cells with EPS8L1 overexpression or knockdown. An ovarian cancer mouse model with lung colonization was established to evaluate the in vivo colonization and migration. To identify correlated proteins, a bioinformatics assay was conducted and verified via qRT-PCR and Western blot. Results : EPS8L1 knockdown inhibited cellular migration in vitro and reduced tumor colonization in vivo. The actin-staining and ELISA experiments suggested that EPS8L1 regulated actin formation as well as cytoskeleton remodeling. Furthermore, mRNA and protein expression confirmed that EPS8L1 regulated the downstream T-cell lymphoma invasion and metastasis 2 (TIAM2) molecule and stimulated the activation of Rac1. Additionally, the phosphorylation levels of P38, Erk, and Jnk in the MAPK pathway decreased after EPS8L1 knockdown. Conclusions : The upregulation of EPS8L1 could promote the migration and metastasis of ovarian cancer cells by regulating cytoskeleton remodeling. The mechanism underlying this might be that EPS8L1 regulates TIAM2 to induce the transformation of Rac-GDP into Rac-GTP and then activates the downstream MAPK pathway. As a regulatory factor in cell migration and metastasis, EPS8L1 could be a new prognostic biomarker and a promising therapeutic target for ovarian cancer patients. Ovarian cancer EPS8L1 cell migration and metastasis TIAM2 Rac1/MAPK cytoskeleton remodeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The incidence rate of ovarian cancer ranks third among gynecological malignant tumors in the female reproductive system; however, its mortality rate ranks at the top, being as high as 66% (Sung et al., 2021)(Cao et al., 2021). In the clinic, 85%–90% of ovarian cancer is classified as epithelial ovarian cancer, among which serous cystadenocarcinoma is the most common type, accounting for 40% (Cho, 2009). During treatment, most patients experience relapse due to the systemic metastasis featured in ovarian cancer, resulting in a less than 30% 5-year survival rate(Penny, 2020) . Therefore, the investigation of an effective target with which to inhibit the migration and metastasis of ovarian cancer is significant for reducing the mortality rate. In 1993, Fazioli et al. identified epidermal growth factor receptor kinase substrate 8 (Eps8) for the first time in fibroblasts(Fazioli et al., 1993). Eps8 consisted of an N-terminal phosphotyrosine binding (PTB) region, an intermediate Src-homology 3 (SH 3 ) domain, and a C-terminal effector region capping on the barbed end of filamentous actin to promote elongation(Tocchetti et al., 2003). Three analogs are in this family, namely epidermal growth factor receptor kinase substrate 8-like protein 1, 2, and 3 (EPS8L1, EPS8L2, and EPS8L3)(Matoskova et al., 1995) . Previous publications have reported that the overexpression of Eps8 promotes the proliferation of esophageal cancer (Bashir et al., 2010), non-small-cell lung cancer(Wen et al., 2019) , and pancreatic cancer(Tod et al., 2017) , as well as the migration and metastasis of oral squamous cell carcinoma(Yap et al., 2009) , colon cancer(Maa et al., 2007) , and breast cancer(Chen et al., 2015) . At a later point in time, Offenhauser et al. demonstrated that EPS8 participated in cell migration and metastasis via activating the Rac-GEF of Sos-1 and inducing tyrosine kinase receptor-mediated cell remodeling by binding to actin(Helfand et al., 2003) . In our previous work(Zhang et al., 2019) , we collected 31 epithelial ovarian cancer samples and 10 adjacent normal tissue samples from patients with ovarian cancer, after which we performed next-generation sequencing. The Eps8l1 gene was screened out as a differentially expressed gene with a high possibility of participating in the occurrence and development of epithelial ovarian cancer. Consistently, the bioinformatics analysis demonstrated that the clustered genes of Eps8l1 concentrated on the function of cell movement and migration; however, the mechanism of Eps8l1 and its protein, EPS8L1, in ovarian cancer, especially in metastasis, was not elucidated. To illustrate the role of EPS8L1 in the regulatory pathway, we analyzed the clinical information and outcomes of patients with ovarian cancer. Additionally, in vitro and in vivo experiments were performed to investigate the regulation of EPS8L1 in cell migration. 2. Methods 2.1. Cell Culture and Transfection Cells from the human ovarian cancer cell lines SKOV-3 and ES-2 were purchased from the cell bank of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. Cells were cultured in McCoy's 5A medium, containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin, at 37°C in a humidified environment with 5% CO2. SKOV-3 cells are typically serous ovarian cancer cells, being the most common type in epithelial ovarian cancer; therefore, the SKOV-3 cell line was selected for the following experiments, including the establish-ment of EPS8L1 knockdown (Sh-EPS8L1) and overexpression (OE-EPS8L1) cells. For the lentiviral transfection, the Sh-RNA with the designed sequence (shown in Tables S1 and S2) was applied. The designing and packaging of the above lentiviruses were completed by the Jikai Gene Company (Shanghai, China). Cells in the logarithmic growth was trypsinized, and complete medium were added to adjust the cell density of 2x10 4 /mL. Then, cells were seeded in a 24-well plate in a 500 μL/well and cultured overnight. In the preliminary experiment, the optimal MOI value for lentiviral infection of SKOV3 was determined as 10, as a result, the cor-responding virus volume was calculated as V= (MOI X number of cells)/virus titer. Then, an appropriate amount of virus solution and 5 μg/mL of Polybrene (enhancement solution) were added to 500 μL medium. Similarly, a control was constructed. After incubating for 24 hours, the virus-containing medium was replaced with complete medium for continuous culture. After 48 hours, the infection efficiency was observed under fluorescence microscopy. At the same time, SKOV3 cells were seeded in a 96-well plate with cell density of 4x10 4 /mL, and cultured overnight. Medium containing different concentrations of puromycin (1, 2, 4, 8, 10 μg/mL) was added to each well. The proportion of viable cells was observed daily, and the puromycin-containing medium was changed every other day. We screened that 2μg/mL was the lowest concentration (kill all cells within 2 days). After transfection, cells were cultured in a 24-well plate, and when the density reached 80%, culture medium containing 2 μg/mL puromycin was added. The puromycin-containing medium was changed daily or every other day according to the color and cultured until 14 days. The transfected cell line was considered to be stably established by the result more than 95% of the cells with green fluorescence were obtained under the fluorescence microscope. For the transfection of siRNA, cells were seeded in 6-well plates at a density of 105 cells per well, and transfected with relevant siRNA (80 pmol), followed by transfection with the LIPO3000 (Sigma) transfection reagent according to the manufacturer's instructions. Then, the expression ability of siRNA molecules was analyzed. 2.2. Scratch Healing Assay After cells were seeded into 6-well plates at 3 × 10 6 cells per well with 90%-100% confluency, the complete medium was discarded and treated with 4 μg/mL mitomycin C (GLPbio) for 1 h. A sterilized micropipette tip was applied to scratch the cell layer, and the floating cells were washed off. Multiple spots on the scratch were imaged using a Zeiss microscope. Images were taken at 100x magnification. Cell migration measurements were performed using ImageJ software, and the mobility ratio was calculated by the migrated area divided by the original area. 2.3. Transwell Migration Assay On a transwell migration plate (Corning, 8.0 μm pore size), 3 × 10 4 transfected SKOV-3 cells were resuspended in the upper chamber with 400 μL of serum-free McCoy’s 5A medium and the lower chamber was filled with 500 μL of McCoy's 5A medium containing 40 ng/mL EGF. After 24 h of incubation, cells on the surface of the cavity that had been invaded were stained with 0.5% crystal violet and imaged under a fluorescence microscope. The number of migrated cells was counted using ImageJ software. 2.4. In Vivo Colonization Assay The 2- to 3-week immunodeficient female Balb/c-nu mice (ASM Pharmaceutical Research Institute Co., Ltd., Hunan, China) were injected with 5 × 10 6 GFP-labeled SKOV-3 cells via a tail vein injection. After 8 weeks, the distribution of fluorescent signal was observed under a small-animal in vivo imager (Bruker in vivo FX, Germany) for whole-body imaging. After this, the mice were sacrificed and dissected organs were imaged. For the evaluation of pulmonary colonization, lungs were cut into 5 μm pieces and stained with H&E. Three sections throughout the lung per mice were screened, and the number of metastases was counted. In addition, the lungs were stained with MMP-2 and MMP-9 to quantify the expression levels of the corresponding proteins. 2.5. Immunohistochemistry (IHC) Assay After being embedded with paraffin, the tumor tissues were stored in a citrate antigen retrieval buffer (pH = 6.0). Next, the tissue was blocked with 3% H 2 O 2 and covered with 3% BSA at room temperature for 30 min. Then, primary antibody MMP-9 (1:200, Cell Signaling Technology, 13667) and MMP-2 (1:200, Cell Signaling Technology, 40994S) were added and incubated overnight at 4°C. After washing tissues with PBS, the corresponding secondary antibodies (HRP-labeled) were added and incubated at room temperature for 50 min. Then, freshly prepared diaminobenzidine (DAB) was used for color development. Finally, tissues were counterstained with H&E, dehydrated with alcohol, and mounted for microscopic examination. Six tumor slices and three high-magnification fields of each slice were randomly selected for scanning. The ratio of positive area was analyzed by using ImageJ software. All of the above reagents, except the antibodies, were purchased from Wuhan Servicebio Technology CO., LTD (China). The lung tissues of 3 mice were randomly selected in each group for analysis using Image J, and the proportion of positive cells was used as an indicator with which to compare with the control group. For comparison between two groups, a t-test was used. 2.6. Actin Staining The 2 × 10 4 SKOV-3 cells in 24-well plates were fixed with 3.75% paraformaldehyde for 15 min, permeabilized with 0.5% Triton for 10 min, and finally stained with an appropriate amount of an FITC-labeled phalloidin stock solution (Solarbio, #CA1640) for 15 min. After dripping DAPI onto the slide, cells were observed under a fluorescence microscope (Leica). 2.7. F-actin Quantification F-actin was quantified using an enzyme-linked immunosorbent assay kit (CUSABIO Human F-actin ELISA Kit). Briefly, SKOV-3 cells in a 6-well plate with a density of 2 × 10 5 were transferred to a 1.5 mL centrifuge tube to generate a homogenous solution via an ultrasonic cell disruptor. The sample was then centrifuged at 5000 × g, 4 °C for 5 min, and supernatant was added to the embedded 96-well plate. The OD value of each well was measured on a plotted standard curve under the wavelength of 450 nm to calculate the actual F-actin concentration. 2.8. Downstream Gene Screening To explore the relevant targets for the mechanism of EPS8L1 in epithelial ovarian cancer, the comprehensive database GENE EXPRESSION OMNIBUS ( http://www.ncbi.nlm.nih.gov/geo ) and the public database String (https://cn.string-db.org/cgi/) were selected. To further confirm the relevant genes, RT-PCR assays with specific primers were performed. The mRNA expression levels of each gene in Sh-EPS8L1, OE-EPS8L1, and their corresponding vector control groups were obtained. After a comparison of two groups, genes displaying a large variance between cells were selected for the following verification and further verified by using a Western blot assay. 2.9. Quantitative RT-PCR Assay Real-time quantitative PCR was performed via the use of Trizol (Invitrogen) and reverse transcription via the use of a high-capacity cDNA (Sigma) kit. TNNI3, TNNC1, TNNT2, CD44, TIAM2, TIAM1, MTA1, NM23, CD147, CD82, EPS8L1, and GAPDH levels were measured using target primers (Invitrogen), as shown in Table S3. Relative expression levels were measured by comparing the ratio of the Ct value of the target with the Ct value of GAPDH. 2.10. Rac1 Activation Assay The PI3K-specific activator 740Y-P (20 μM) was added to cells in Sh-EPS8L1 and control groups for 24 h. The SKOV-3 cells were then scraped with a cell lysate that contained protease inhibitors. The lysates were centrifuged (10,000× g, 4 °C for 1 min) to remove cellular debris and then incubated with 10 μL of PAK-PBD beads from a Rac1 Activation Kit (cytoskeleton, CAT#BK035-S) for 60 min at 4°C on a shaker. Beads were washed in 20 μL of a 2 × Laemmli sample buffer and heated to boil for 2 min. The determination of Rac1-GTP activation was then performed with Western blot experiments. 2.11. Western Blot Analysis Cells were washed with phosphate-buffered saline (PBS) and harvested with a RIPA lysis collection buffer. Cell lysates were run on 10–15% SDS-polyacrylamide gels and then transferred to PVDF membranes, with primary antibodies incubated overnight at 4°C. After multiple washes, HRP-conjugated secondary antibodies were added for 2 h at room temperature and developed with an ECL kit. The detection of active Rac1 proteins requires pretreatment via a pull-down assay. EPS8L1 antibodies were purchased from Invitrogen (PA5-38746); TIAM2 antibodies were purchased from Abcam (ab199426); Rac1 antibodies was purchased from Abcam (ab155938); and GAPDH antibodies were purchased from Affinity (AF7021). 2.12. KEGG and GO Enrichment The gene annotation information of GO (Gene Ontology) was mainly obtained from Bioconductor, and the annotation information of KEGG (Kyoto Encyclopedia of Genes and Genomes) was obtained through the API of KEGG data. Briefly, we opened the website, entered EPS8L1, selected a species, and downloaded the corresponding annotation information. GO cluster and KEGG pathway enrichment was generated by the R program for analysis. The relevant information of EPS8L1 (gene ID: 54869) was down-loaded from the DAVID bioinformatics database (https://david.ncifcrf.gov/)and im-ported into the R program for analysis and to draw a plot. Partial enrichment codes for KEGG and GO in Table S4. 2.13. String Analysis In the String database (https://cn.string-db.org/cgi/), we entered the corresponding gene name and selected the retrieved database in order to generate results. Accordingly, we found a series of related proteins, such as TNNT2, TNNCI, and TNNI3. In addition, through the GENE EXPRESSION OMNIBUS database (http://www.ncbi.nlm.nih.gov/geo), the GSE18520 (microarray expression data of epithelial ovarian cancer) dataset was selected for the statistical analysis of Spearman’s correlation using the R program, and other related proteins were found, including TIAM2, CD44, CD82, and TIAM1. Subsequently, experimental verification was carried out on these proteins. 2.14. cfDNA Extraction and Purification 5 mL of plasma was collected from patients, 500 μL of proteinase K and 4 mL of buffer ACL (containing carrier RNA) were added and mixed well. After placing the mix at 60 °C for 30min to 1h, 9 mL of buffer ACB was added and mixed well, then was put on ice for 5min. The ACB lysis mixture was purified and eluted through the column to obtain cfDNA; cfDNA concentration was measured using the Qubit2000 Fluorometer. Statistical analysis: The experimental results were analyzed using the statis-tical software SPSS 20.0 with data presented as mean ± SD, and t-test was used for comparison between the two independent groups. 2.15. Statistical Analysis The data were expressed as the mean ± standard deviation (SD), and the results were analyzed with the statistical software SPSS 22.0. Each experiment was performed at least three times unless noted. If the data conformed to a normal distribution and homogeneity of variance, two independent-samples t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparisons between more than three groups. If the data did not conform to a normal distribution, a nonparametric rank sum test was used. The enumeration data were analyzed by using the chi-square test. A value of P < 0.05 indicated that the difference was statistically significant. Figures were plotted with GraphPad Prism 7. 3. Results 3.1. The Overexpression of EPS8L1 in Epithelial Ovarian Cancer was Correlated with Poor Clinical Outcomes and Positively Related to Tumor Migration In our previous work on the next-generation sequencing of ovarian tumors from 31 patients, the EPS8L1 gene was screened out with high expression [15] . To further interpret the previous RNA-Seq results, we validated the expression of the EPS8L1 gene in patients with ovarian cancer. Preoperative venous blood from 43 patients with serous ovarian cancer was collected. The concentrations of plasma cfDNA and EPS8L1-cfDNA from samples were then detected via qPCR. As shown in Table 1, the plasma cfDNA concentrations from peripheral blood of patients with different ages, unilateral or bilateral tumors, classifications, stages, and with or without lymph node metastasis showed no significant difference. In contrast, the EPS8L1-cfDNA concentration was significantly different ( P < 0.001 ) in terms of the FIGO stage (stages I and II as the early-stage groups; stages III and IV as the advanced-stage groups). The level of EPS8L1-cfDNA in patients with lymph node metastasis was significantly higher than that in patients without metastasis ( P < 0.001 ). Table 1. Relationship of plasma cfDNA concentration and EPS8L1-cfDNA level in 43 patients with ovarian serous cystadenocarcinoma. Characteristic Cases cf-DNA Concentration（ng/μL） P Relative Level of EPS8L1-cfDNA P Age < 55 25 0.203 ± 0.051 0.917 2.70 ± 0.42 0.065 ≥ 55 18 0.201 ± 0.737 2.95 ± 0.44 Primary Tumor Site Unilateral 15 0.186 ± 0.051 0.218 2.83 ± 0.51 0.797 Bilateral 28 0.210 ± 0.064 2.79 ± 0.41 Organization Type High-grade serous ovarian cancer 30 0.201 ± 0.064 0.954 2.78 ± 0.45 0.663 Low-grade serous ovarian cancer 13 0.203 ± 0.053 2.85 ± 0.43 FIGO Staging Ⅰ + Ⅱ 19 0.190 ± 0.040 0.220 2.49 ± 0.33 < 0.001 Ⅲ + Ⅳ 24 0.211 ± 0.072 3.05 ± 0.35 Lymph node metastasis Yes 22 0.214 ± 0.067 0.173 3.13 ± 0.23 < 0.001 No 21 0.189 ± 0.051 2.46 ± 0.34 Therefore, the relationship between mRNA expression and the clinicopathological indicators of patients was analyzed by detecting the expression of the EPS8L1 gene in the tumor tissues of 43 patients with qRT-PCR. Consistently, the results showed that the expression of the EPS8L1 gene was not related to a patient's age, primary site, and other factors; however, it was positively related to FIGO stages ( P = 0.031) and lymph node metastasis ( P < 0.001 ) (Table 2). The relationship between the expression of EPS8L1-mRNA in tissues and the clinicopathological indicators of patients was consistent with the results of cfDNA concentration, both suggesting that the EPS8L1 gene might be related to the progression and metastasis of epithelial ovarian cancer, and probably serve as a new biomarker to predict the clinical outcomes of ovarian cancer patients. Table 2. The correlation between the expression of EPS8L1-mRNA and clinicopathological indicators of patients. Characteristic No. of Cases EPS8L1-mRNA P Age < 55 25 2.579 ± 0.506 0.987 ≥ 55 18 2.576 ± 0.735 Primary Tumor Site Unilateral 15 2.426 ± 0.641 0.232 Bilateral 28 2.659 ± 0.579 Organization Type High-grade serous ovarian cancer 30 2.627 ± 0.614 0.478 Low-grade serous ovarian cancer 13 2.487 ± 0.154 FIGO Staging Ⅰ + Ⅱ 18 2.357 ± 0.462 0.031 Ⅲ + Ⅳ 25 2.737 ± 0.652 Lymph Node Metastasis Yes 22 3.196 ± 0.136 < 0.001 No 21 1.994 ± 0.516 We further performed a GO clustering analysis of EPS8L1 to identify relevant genes in different functions. The results showed that, for biological processes, cell–substrate adhesion was the key function, with most genes clustered. Regarding the cellular components, the collagen-containing extracellular matrix was mainly clustered. For molecular function, most genes were concentrated in the active part of the endopeptidase. The clustered genes mostly contributed to cellular movement, suggesting that EPS8L1 might regulate tumor invasion and metastasis through cell–extracellular matrix adhesion and other associated biological functions (Figure 1a). The KEGG pathway enrichment showed that EPS8L1 was mainly overexpressed in signaling pathways such as vascular smooth muscle contraction, actin cytoskeleton regulation, and axonal pathways that positively related to the migration and motility of cells (Figure 1b). Using the TCGA database, we further analyzed the relationship between clinicopathological indicators and EPS8L1-mRNA expression in 379 patients with epithelial ovarian cancer. Consistently, the high expression of EPS8L1 was significantly related to the FIGO stages of patients (P = 0.042 < 0.05), shown in Table S5. The above results further demonstrated that EPS8L1 was positively related to the cellular invasion and migration in epithelial ovarian cancer. The expression of EPS8L1 protein in human normal ovarian epithelial cells (IOSE80) and human epithelial ovarian cancer cells (CAOV3, SKOV-3, A2780, ES-2, OVCAR3) were measured. As shown in Figure 1c, among the five ovarian cancer cell lines, SKOV-3 cells exhibited the highest level of EPS8L1 protein expression, suggesting the necessities of EPS8L1 and probably important role in SKOV-3 cells. We hypothesized alterations in EPS8L1 expression levels might induce a more significant effect on SKOV-3 cells which was feasible for measurement. Therefore, we selected the SKOV-3 cell line for subsequent knockdown or overexpression experiments. After constructing SKOV-3 cells with EPS8L1-knockdown or overexpression via lentivirus transfection, the level of EPS8L1 protein in transfected SKOV-3 cells were detected as shown in Figure 1d. The results demonstrated that the expression of EPS8L1 decreased by 43.61% (0.409 ± 0.16 vs 0.725 ± 0.20, P < 0.001) in the knockdown group, and increased by 35.24% (1.07 ± 0.03 vs 0.69 ± 0.02, P < 0.001) in the overexpression group. Therefore, the cell model with EPS8L1-knockdown or overexpression was successfully constructed. 3.2. The Knockdown of EPS8L1 Inhibits the Migration of Ovarian Cancer Cells by Suppressing Cellular Actin Formation and Cytoskeleton Remodeling To study the effect of EPS8L1, the migration of SKOV-3 cells after EPS8L1 knock-down was detected via a scratch healing assay. The quantification was represented by the migration ratio, in which a smaller value referred to a slower migration rate. The results showed that the migration ratio of the Sh-EPS8L1 group (16.7%) was significantly lower than that of the control group (35.5%), with P < 0.05 (Figure 2a). Thus, the migration speed of the Sh-EPS8L1 group was significantly slower than that of the control, indicating the regulation of cell migration by EPS8L1. This result suggested that EPS8L1 may affect the migration of ovarian cancer cells. Multiple G-actin are linked together to form actin chains, and two strings of actin chains are twisted into fibrillar actin (F-actin) to constitute the cytoskeleton of eukaryotes, which is closely related to the chemotactic ability of cells(Nürnberg et al., 2011) . To illustrate the status of cytoskeleton remodeling during the EPS8L1 regulation of cellular migration and metastasis, F-actin in Sh-EPS8L1 and EPS8L1 (as control) cells was stained with FITC-labeled phalloidin. As observed, the brightness of cells in the control group demonstrated the formation of more filopodia that enable cells to migrate in one direction(Cappellini et al., 2015) . In contrast, the amount of filopodia in Sh-EPS8L1 cells was significantly reduced, and no filopodium with an invasive structure was detected, indicating the inhibition of filopodia formation by suppressing EPS8L1. Then, EGF was given to stimulate the cells to further evaluate the effect of EPS8L1 knockdown. As shown in Figure 2b, the polymerization of cortical ac-tin was observed, and the number of central actin bundles decreased in the control group. This indicated that EGF stimulation induced the reorganization of cortical actin to promote cytoskeleton remodeling and cell migration. On the contrary, cells in the Sh-EPS8L1 group exhibited no obvious response to EGF stimulation. In the quantification of F-actin via an ELISA kit, the amount of F-actin in Sh-EPS8L1 cells was 3.67 mg/mL, while in the control cells it was 6.39 mg/mL. The difference between the two groups was statistically significant (Figure 2c). Consistently, the corresponding study in EPS8L1 overexpression cells demonstrated the same results, as shown in Fig. S1. These results suggested that the regulation of cytoskeleton remodeling and cell migration by EGF stimulation was dependent on the presence of EPS8L1. Thus, EPS8L1 might regulate the migration of ovarian cancer by regulating the formation of filopodia and cytoskeleton remodeling. 3.3. EPS8L1 Promotes the Colonization of Ovarian Tumor Cells in vivo by Regulating the Expressions of MMP-2 and MMP-9 To further verify the participation of EPS8L1 in the migration and metastasis of ovarian cancer, in vivo colonization experiments were performed. After 8 weeks of tumor growth via a tail vein injection of GFP-Sh-EPS8L1 and GFP-EPS8L1 (control), mice were imaged by Bruker in vivo Fx to evaluate the colonization and distribution of ovarian tumors. As shown in Figure 3a, local fluorescence enhancement was observed dramatically in the lungs of mice in the control group, suggesting the development of lung colonization. In contrast, the low expression of EPS8L1 decreased the colonization of ovarian tumors in mice. Furthermore, tumors in the control group were shown to be widely distributed throughout the body compared with that of the Sh-EPS8L1 group, especially in the cervical lymph node group (including submaxillary lymph nodes, superficial cervical lymph nodes, and deep cervical lymph nodes) on dual sides of mice(Bobek et al., 2005) . The dissected lungs were imaged to evaluate the tumor growth. A higher number of tumor cells in lungs of the control than that of the Sh-EPS8L1 groups was shown (Figure 3b). In H&E staining (Figure 3c), mice injected with normal EPS8L1-expressed SKOV-3 cells displayed a significantly higher intensity of tumors per lung slice com-pared to mice injected with Sh-EPS8L1 cells (median of 22 ± 1.78 vs. 4.6 ± 1.01). More photographs for different mice and the ink-staining experiment for lungs with colonization to demonstrate the location of small tumor lesions are shown in Fig. S2. The matrix metalloproteinase (MMP) family regulated extracellular matrix degradation and cell adhesion remodeling to promote tumor cells’ diffusion and angiogenesis, which played an important role in tumor invasion and metastasis(Kapoor et al., 2016) . In the immuno-histochemistry of lung tissues of the Sh-EPS8L1 group, the ratios of MMP-2 and MMP-9 protein expression were 0.719 ± 0.09 and 0.111 ± 0.02, while those of the MMP-2 and MMP-9 proteins in the control group were 0.992 ± 0.00 and 0.990 ± 0.00, respectively. As compared in Figure 3d, the difference between two groups was statistically significant with MMP-2 (P < 0.05) and MMP-9 (P < 0.01) expression, indicating that EPS8L1 promoted the colonization of ovarian cancer cells in vivo by regulating the expression of MMP-2 and MMP-9. Consistently, the corresponding study in EPS8L1 overexpression demonstrated the same results, as shown in Fig. S3. The parallel study of the effect of EPS8L1 overexpression on colonization and metastasis of SKOV-3 ovarian cancer cells was shown in Fig. S4 indicating that the overexpressed EPS8L1 induced the colonization and metastasis. 3.4. EPS8L1 Regulates the Expression of Downstream TIAM2 To further explore the relevant targets of EPS8L1, the microarray expression dataset (GSE18520) of epithelial ovarian cancer from GENE EXPRESSION OMNIBUS (http://www.ncbi.nlm.nih.gov/geo) was selected and a Spearman’s correlation analysis was generated (Figure 4a). Moreover, a public database, String (https://cn.string-db.org/cgi/), and an online analysis (http://gepia.cancer-pku.cn/) were recruited to analyze genes related to EPS8L1 (Figure 4b and Fig. S5). It was found that the genes TIAM2 (r = 0.470, P < 0.001), TNNT2, TNNC1, and TNNI3 (all r = 0.67, P < 0.05) are possibly related to EPS8L1 (Fig. S6). The RT-PCR results verified that the EPS8L1 gene was highly expressed in OE-EPS8L1 and lowly expressed in Sh-EPS8L1 cells supporting the successful establishment of our experimental models. The expressions of TNNT2, CD82, TIAM1, and TIAM2 were significantly different between OE-EPS8L1 and Sh-EPS8L1, being 46.64-fold (P < 0.01), 13.37-fold (P < 0.01), 9.71-fold (P < 0.01), and 4.61-fold (P < 0.01), respectively (Figure 4c). Subsequently, four groups of SKOV-3 cells with OE-EPS8L1 or Sh-EPS8L1 or their corresponding vectors as control groups were used to evaluate the related genes at the protein level. The results showed that, among these differentially expressed genes, only TIAM2 was obviously related to EPS8L1 at the protein level (Figure 4d). The decreased EPS8L1 expression resulted in decreased TIAM2 expression, while increased EPS8L1 expression also in-creased TIAM2 expression. The TNNT2 gene with the greatest difference at the mRNA level demonstrated no significant difference at the protein level, indicating that the TNNT2 gene might be a transcription factor in post-transcriptional regulation or that the half-life of the TNNT2 protein is too short to be captured. We also performed a validation of EPS8L1-overexpressing ES-2 cells (Fig. S7), and the results were similar to those in SKOV-3 cells demonstrating the TIAM2 expression varied with EPS8L1 level. These results indicated that TIAM2 was positively related to EPS8L1 and may be regulated by the upstream molecule EPS8L1. To further clarify the regulatory relationship be-tween EPS8L1 and TIAM2, small interfering RNA (siRNA) was utilized to knockdown EPS8L1 and TIAM2. As shown in Figure 4e, the protein expression of TIAM2 decreased when EPS8L1 was knocked down. Contrarily, no significant change in EPS8L1 expression was observed with TIAM2 knockdown. These results indicated that EPS8L1 was an upstream regulatory molecule of TIAM2 and that TIAM2 was a downstream molecule of EPS8L1 (Figure 4f). 3.5. EPS8L1 is Stimulated by EGF with the Presence of TIAM2 and is Positively Related to the Conversion of Rac1-GDP into Rac1-GTP As has been reported, an Eps8-Abi1-Sos1 complex regulates the depolymerization and assembly of microtubules by actin and activates Rac1-GTPase to regulate the cytoskeleton(Scita et al., 2001) . To investigate whether EPS8L1 (a member of Eps8 family) promotes the activation of Rac1, the Rac1-GTP concentration was measured. After stimulating Sh-EPS8L1 with EGF, the expression of the EPS8L1 protein increased with the prolongation of the stimulation time (Figure 5a). Simultaneously, the expressions of the TIAM2 protein and Rac1-GTP increased as well. The total Rac1 amount was un-changed, indicating that the proportion of Rac1-GTP increased. However, as shown in Figure 5b, EGF stimulation did not induce Rac1 activation in the TIAM2-knockdown SKOV-3 cells (TIAM2-Si). Meanwhile, scratch healing (Figure 5c) and transwell migration (Figure 5d) results with EGF stimulation after EPS8L1 and TIAM2 knockdown were consistent with those of protein expression. After TIAM2 knockdown, the migration of the cells to all directions was reduced. The EPS8L1 expression could be recovered to some extent by EGF stimulation, indicating that EPS8L1 is an effector molecule of EGF stimulation; however, the expression of TIAM2 did not change significantly, suggesting the non-response of TIAM2 to the stimulation of EGF. Previous research pointed out(Van Leeuwen et al., 2003) that the activation of Rac1 is dependent on the GEF (guanine nucleotide exchange factor) activity of TIAM1 in the case of lysophosphatidic acid (LPA) or platelet growth factor (PDGF) induction. As an analog of TIAM1, a similar mechanism may exist for TIAM2. Therefore, TIAM2 and the activation of Rac1 cannot respond to the stimulation of EGF. These results demonstrated that the Rac1-GTP conversion activated by EGF stimulation was dependent on the presence of EPS8L1. 3.6. The Regulatory Role of EPS8L1 and TIAM2 in Rac1-MAPK Activation As reported, TIAM2 was essential for the Rac1-dependent cell motility mediated by PI3K (Cooke et al., 2021). However, the role of upstream EPS8L1 was unknown. In this study, 740Y-P was used as a membrane-permeable agonist to activate PI3K(Williams & Doherty, 1999) . As demonstrated in Figure 6a, the expression of TIAM2 was restored after 740Y-P treatment, while the levels of EPS8L1 and Rac1-GTP activation did not recover compared to the control group. Thus, a PI3K agonist could stimulate the recovery of TIAM2 independently with the presence of EPS8L1; however, TIAM2 could not mediate Rac1 activation by itself, indicating the decisive role of EPS8L1 and essential role of TIAM2 in the EPS8L1-TIAM2-driven Rac1 activation. With regard to the participation of the MAPK signaling pathway in the extracellular matrix, adhesion, motility, and angiogenesis of tumor cells(Chen et al., 2015) , we evaluated whether it might be a downstream regulatory pathway of EPS8L1 for tumor metastasis. Consistent with the previous section, the EPS8L1 protein level would not be increased further with EGF stimulation in the normal EPS8L1 expression group (negative vs. positive control in Figure 6b); therefore, TIAM2 in the normal EPS8L1 expression group would not be increased with EGF stimulation. However, the Rac1-GTP and phosphorylation levels of key proteins in the MAPK pathway, including P38, Erk, and Jnk, were increased significantly, indicating that EGF stimulation could also activate Rac1-MAPK in the normal presence of EPS8L1 and TIAM2. In the Sh-EPS8L1 group, EGF stimulation recovered EPS8L1 and TIAM2 accordingly compared with non-EGF treatment; however, Rac1-GTP was not increased to the level in the normal EPS8L1 expression group with EGF stimulation. Similarly, the phosphorylation levels of P38, Erk, and Jnk in the Sh-EPS8L1 group were upregulated with EGF stimulation to some extent but still lower than those in the control group. These results demonstrated that the suppression of EPS8L1 led to the inactivation of the MAPK pathway, indicating that the MAPK pathway might contribute to the EPS8L1-mediated metastasis and migration of ovarian cancer cells. 4. Discussion Ovarian cancer is usually diagnosed at an advanced stage (stages III–IV), with a high mortality rate and developed metastases at multiple tissues, leading to no re-markable improvement in the 5-year survival rate(Penny, 2020) . This being the case, the investigation of metastasis in ovarian cancer is key to developing effective treatments and prognoses of patients. Studies have shown that the Eps8 gene is related to tumor proliferation(Yang & Huang, 2015) and metastasis, in addition to probably participating in the integrin-dependent Rac1 pathway(Yap et al., 2009) , cytoskeleton alteration (Fregnan et al., 2011), and regulation of focal adhesion kinase(Shahoumi et al., 2020) . The high Eps8 expression in head and neck squamous cell carcinoma also promoted invasion and metastasis(Wang et al., 2009) in pancreatic cancer cells(Welsch et al., 2007). As demonstrated, Eps8-Abi1-Sos1 regulated Rac activity in the epithelial-mesenchymal transition (EMT) of ovarian cancer cells(Scita et al., 1999)(Innocenti et al., 2003) . Eps8 knockdown greatly reduces the ability of glioblastomas to invade and metastasize(Cattaneo et al., 2012) . As the most important homologous aspect of the Eps8 gene, EPS8L1 might have major functionality in regulating the migration and metastasis of tumor cells; however, no study on the mechanism of EPS8L1 in tumors, including ovarian cancer, has been reported. In our previous work, EPS8L1 was found to be upregulated in clinical ovarian cancer samples(Zhang et al., 2019) . In this study, we further illustrated the mechanism of overexpressed EPS8L1 in ovarian cancer, especially in tumor migration and metastasis. The analysis of clinical cases demonstrated that EPS8L1 was related to the FIGO staging and lymph node metastasis of patients with ovarian cancer. Additionally, EPS8L1 knockdown decreased the migration speed of ovarian cancer cells in the scratch-healing and transwell experiments. To exclude the possibility that scratch closure was caused by cell proliferation, cells were pretreated with a selected concentration of mitomycin C (Fig. S8) to inhibit cell proliferation but not cell migration(Liang et al., 2007) . Thus, the scratch healing in our study was ensured to be induced by migration. Regarding the establishment of an in vivo model with the metastasis of ovarian cancer cells, ortho-topic transplantation(Yi et al., 2005) , footpad injection(Fan et al., 2014) , intraperitoneal injection(Rose et al., 1996) , and tail vein injection(Peng et al., 2009) have been reported. Originally, we injected SKOV-3 cells into mice intra-peritoneally to simulate the progress of intraperitoneal dissemination and metastasis to mimic the development of ovarian cancer in the clinic. After 4–5 weeks post-injection, malignant ascites was found in the abdominal cavity of the mice, but the overall condition of the mice was poor. Only the abdomen colonization of tumors was observed, with no metastases in the lungs, liver, or other organs. We then selected the vein injection with the superior effect to establish a colonization model. To evaluate tumor growth and distribution, the injected SKOV-3 cells were labeled with GFP-fluorescence to provide images under a Bruker in vivo Fx imager. No fluorescence signal was detected in the lungs of the EPS8L1 knockdown group, with only scattered tumor cells being able to be observed by directly scanning dissected lungs, which may be due to the difficulty of low fluorescent intensity from a few tumor cells in penetrating tissues. As has been reported, Eps8 is required for the formation of dynamically actin-based cell protrusions and cell-cell junctions(Scita et al., 2001) ; therefore, we carried out an actin-staining experiment on whether EPS8L1 also induces actin‑rich membrane ruffle and filopodia formation. As is observed in Figure 2b, the formation of F-actin-composed cortical actin is an important indicator of cells with good contraction and movement ability(Small et al., 2002) . Moreover, the capping activity of EPS8L1 is crucially required for the formation of cortical actin. As has been demonstrated, EPS8L1 knock-down resulted in a remarkable change in cell shape and actin structures that morpho-logically resembled a less-motile cell phenotype. We hypothesized that the downregulation of Eps8L1 leads to impaired cell protrusion and filopodia formation as a result of inefficient F-actin. Surprisingly, when EPS8L1 is knocked down, cells can still recover migration ability to some extent after being stimulated by EGF. We speculated that other molecular mechanisms for migration might compensate in the case of Eps8 loss. However, EGF stimulation cannot fully reverse the reduction in actin capping and Rac-GEF activity induced by EPS8L1 downregulation, demonstrating the key regulatory role of EPS8L1. A moderately strong correlation between EPS8L1 and TIAM2 (r = 0.48, p < 0.001) was found. Its analog, TIAM1, catalyzes the conversion of Rac1 to promote the formation of lamellipodia(Raftopoulou & Hall, 2004)(Nobes & Hall, 1995)(Hall & Nobes, 2000) and cytoskeleton remodeling(Chiu et al., 1999)(Mertens et al., 2003) , which are important for cell migration and metastasis. Similarly, TIAM2 was associated with tumor progression and unfavorable prognoses (Jiang et al., 2021)(Woroniuk et al., 2018). In our study, we first identified EPS8L1 and further illustrated its relationship with downstream TIAM2. We then demonstrated their indispensable role in regulating Rac1 activation to participate in actin remodeling and the MAPK pathway, which finally induced migration. These conclusions would assist the diagnosis and staging of malignant ovarian cancer with EPS8L1 overexpression. As a substrate, EGF stimulation increases the phosphorylation of EPS8L1 by epi-dermal growth factor receptor (EGFR), and then enhances the function of downstream TIAM2 to activate Rac1. In our study, the expression of TIAM2, restored by the PI3K agonist 740Y-P, did not compensate for the reduced activation of Rac1 in EPS8L1 knockdown cells. In addition, the levels of p-P38, p-Erk, and p-Jnk were lower in ovarian cancer cells with EPS8L1 knockdown, regardless of stimulation with EGF or not, suggesting that the MAPK signaling pathway is downstream regulated by EPS8L1. Therefore, EPS8L1 could be a promising target for the intervention of Rac activation and the MAPK signaling pathway that participated in tumor migration. 5. Conclusion In this study, the regulation of ovarian cancer metastasis is depicted in Scheme 1. EPS8L1 promotes the migration and metastasis of ovarian cancer cells, which was mainly stimulated by extracellular signals such as EGF. The overexpression of EPS8L1 activates the downstream TIAM2 to induce its GEF activity, which facilitates the con-version of Rac1-GDP into Rac1-GTP and regulates downstream MAPK cascade signaling to affect cytoskeleton remodeling and ultimately promote the migration and metastasis of ovarian cancer cells. In addition, the mechanism of EPS8L1 in the pro-motion metastasis may also be related to its regulation of the expression of MMP-2 and MMP-9. These results provide an important scientific basis for understanding the mechanism of the EPS8L1 gene in the development of ovarian cancer, especially in migration and metastasis. In the future, EPS8L1 will be studied intensively as a novel biomarker for prognoses and a new therapeutic target for ovarian cancer patients. Declarations Data availability statement: For all data requests, please contact the corresponding author. Conflicts of Interest disclosure: All authors declare no conflicts of interest. Ethics approval statement: The study was approved by the Institutional Animal Care and Use Committee (IACUC) of Kunming Medical University according to the laboratory guidelines for the ethical review of animal welfare (kmmu2020060). Patient consent statement: All patients were informed, with consent obtained prior to the study. Funding: This work was supported by the National Natural Science Foundation of China (grant no. 81860463, 82160344, and 82160524); Scientific Research Fund of Yunnan Provincial Department of Education（No.2021Y326）；Yunnan Provincial Department of S&T-KMU Joint Foundation (no. 2018FE001-003 and 202101AY070001-068); Research Project on Undergraduate Educational and Teaching Reforms in Yunnan Province (no. JG2023001) and the Yunnan Provincial Department of Education Project (no. 2022J0213). Contribution to authorship: Y.W. performed all of the experiments and composed the manuscript. L.Z. provided the human specimens, clinical information, and data analysis. X.L., H.Z.,M.W., L.M.Z., Y.S., J. Z., M.L., and H.Z. provided support with data collection, experimental materials, and techniques. Y.J. and C.Q. designed the research and revised the manuscript. All authors read and approved the final manuscript. 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Additional Declarations No competing interests reported. Supplementary Files SI.docx scheme1.png Scheme 1. The mechanism of EPS8L1-TIAM2 regulation in F-actin remodeling by EGF in ovarian cancer cells. EGF stimulation enhances the phosphorylation of EPS8L1, activates the downstream function of TIAM2, and induces its specific GEF activity, which in turn activate the conversion of Rac1-GDP into Rac1-GTP, regulates cytoskeleton remodeling by activating the MAPK pathway, and ultimately promotes the migration of ovarian cancer. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4263533","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291184622,"identity":"f6f5420d-b0ed-45fb-84f5-27b5b507af2e","order_by":0,"name":"Yuting Wang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Wang","suffix":""},{"id":291184623,"identity":"d75e922a-e8cb-4566-aa82-1d233a1a49fc","order_by":1,"name":"Lei Zhang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":291184626,"identity":"e9aa644b-f498-451b-8098-381fc175e72a","order_by":2,"name":"Xianqiang Luo","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianqiang","middleName":"","lastName":"Luo","suffix":""},{"id":291184630,"identity":"9f6043b7-0ac0-4d2a-9d29-3d2f887614dc","order_by":3,"name":"Hongmei Zhu","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongmei","middleName":"","lastName":"Zhu","suffix":""},{"id":291184633,"identity":"31d43df3-3cff-4bed-bce9-7a3119fb8a8f","order_by":4,"name":"Meichen Wang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Meichen","middleName":"","lastName":"Wang","suffix":""},{"id":291184634,"identity":"cfd1decb-30e3-4d51-82e7-2fc38e3bdf6c","order_by":5,"name":"Min Luo","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Luo","suffix":""},{"id":291184637,"identity":"4079458c-277d-4acc-8177-6481e511a912","order_by":6,"name":"Hongyu Zhou","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Zhou","suffix":""},{"id":291184639,"identity":"27c549e1-4646-46fd-af3c-c11d44ca22ff","order_by":7,"name":"Limei Zhang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Limei","middleName":"","lastName":"Zhang","suffix":""},{"id":291184640,"identity":"264526ab-2a76-4a77-a930-03dad92ab49e","order_by":8,"name":"Yan Song","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Song","suffix":""},{"id":291184641,"identity":"821d5481-e919-4d19-a2e6-79eeeebe88c4","order_by":9,"name":"Junfei Zhang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Junfei","middleName":"","lastName":"Zhang","suffix":""},{"id":291184642,"identity":"602a0c71-b3e2-43d1-872b-c9429ec8c2e7","order_by":10,"name":"Yinnong Jia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACxmYgkQBmMh+GiiUQrYUtmTgtSIDHmDgtzO3MzyQe7rgjZ86/5rPRzZzDDPzsOQYMP3fgcxibmUTimWfGljPebk7O3XaYQbLnjQFj7xl8WhiAWtoOJ264cXbzYZAWgxs5BsyMbfi0sH+DajnzGKzFnrAWHqgt53uYwQ4zkCCspdgCqMXY4AabsXHutnQeiTPPCg724tFi2H98482fbYflDM4ffiydu81ajr89eeODn/i0NDCwSIBZEglgigdEHMCtgYFBHhg1H8AsfrzqRsEoGAWjYCQDAO45VO0ju5ubAAAAAElFTkSuQmCC","orcid":"","institution":"Kunming Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yinnong","middleName":"","lastName":"Jia","suffix":""},{"id":291184643,"identity":"5fa35512-5061-4be5-b44f-c2f865fb4320","order_by":11,"name":"Chen Qing","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Qing","suffix":""}],"badges":[],"createdAt":"2024-04-14 03:59:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4263533/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4263533/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54859270,"identity":"412c2f35-c35e-4f78-a52e-543fb43c6e7a","added_by":"auto","created_at":"2024-04-17 19:02:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13695989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe potential role of EPS8L1 in cell migration and the overexpression of EPS8L1 protein in the ovarian cancer cells.\u003c/strong\u003e(a) GO clustering analysis for EPS8L1. (b) Scattering plot of enriched KEGG pathways for EPS8L1. (c) The expression of EPS8L1 protein in normal ovarian epithelial cells and different ovarian cancer cells was detected by western blot and analyzed by grayscale (compared with IOSE80 group: ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). (d) The expression of EPS8L1 protein in knockdown/overexpression SKOV-3 cells (Sh-EPS8L1 or OE- EPS8L1) and the corresponding empty vectors (OE-Ctrl group or Sh-Ctrl group) were detected by western blot and compared (***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/b2cd8fbced6de01d73ea424f.png"},{"id":54859274,"identity":"9601af46-f465-48e4-b4ee-1769dd132799","added_by":"auto","created_at":"2024-04-17 19:02:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30565242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe inhibition of ovarian cancer cell migration and cytoskeleton reorganization with EPS8L1 knockdown.\u003c/strong\u003e (a) The scratch healing assay of control and Sh-EPS8L1 SKOV-3 cells at 0 h and 24 h. Images were obtained via microscopy and analyzed with Image J. Data are shown as the mean ± SE, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01. (b) Fluorescent micrographs of cell staining with phalloidin (for F-actin, green), DAPI (for nuclei, blue), and the merged images of control and Sh-EPS8L1 SKOV-3 cells (alone or with 40 ng/mL EGF). Images were acquired under identical parameters with a 10 μm scale bar. (c) Quantification of actin in Sh-EPS8L1 and control groups were performed with an F-actin ELISA kit. Data are shown as the mean ± SEM; n ≥ 3. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/30bd9ceeb775900ea4d42c46.png"},{"id":54859276,"identity":"b13cc7df-de74-4029-a24f-7f67f9b0351f","added_by":"auto","created_at":"2024-04-17 19:02:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49364483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEPS8L1 knockdown inhibits the cell colonization of ovarian tumor in vivo. \u003c/strong\u003e(a) Fluorescent images of control and Sh-EPS8L1 SKOV-3 cells carrying GFP fluorescence in nude mice were taken by a Bruker in vivo FX imager to observe the fluorescence distribution. (b) The fluorescent intensity of lung tissue in xenograft mice with GFP-labeled control and Sh-EPS8L1 cells. (c) Tumor colonization on lungs (indicated by arrows) and H\u0026amp;E staining of lung nodules (× 100 and × 400 magnification), with three slices per mouse. The total number of lung colonizations per slice was determined. Data are shown as the median ± SE; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 (n = four to seven mice). (d) Immunohistochemistry analysis of MMP-2 and MMP-9 in lung tissues (× 200 and × 400 magnification). The lung tissues of three mice were randomly selected in each group for analysis using Image J, and the proportion of positive cells was used as an indicator to compare with the control group. Data are shown as the mean ± SE, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/948f8660b85a1dc71cd198da.png"},{"id":54859272,"identity":"44b2330a-907d-4e83-be76-92d4ec2d7e43","added_by":"auto","created_at":"2024-04-17 19:02:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15530016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEPS8L1 regulated the expression of TIAM2. \u003c/strong\u003e(a) A Spearman’s analysis was performed on the dataset (GSE18520) in the GEO database. A correlation coefficient r = 0.4 to 0.6 was considered as a moderate correlation, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (b) String network analysis of genes related to EPS8L1. TNNT2, TNNI3, and TNNC1 were strongly associated, with a correlation coefficient r = 0.71 (Fig. S6). (c) The mRNA expression detected via the qRT-PCR of related genes in the OE-EPS8L1 group and Sh-EPS8L1 group; the fold change is equal to the ratio of mRNA expression (OE-EPS8L1) and mRNA expression (Sh-EPS8L1). Data are shown as the mean ± SE, with *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (d) Western blot analysis of protein ex-pression in OE-EPS8L1 and Sh-EPS8L1 cells. The quantification of EPS8L1 and TIAM2 proteins are expressed as percentages of GAPDH. (e) The expression levels of EPS8L1 and TIAM2 proteins after knocking down EPS8L1 with siRNA. (f) The expression levels of EPS8L1 and TIAM2 proteins after knocking down TIAM2 with siRNA. Data are shown as the mean ± SD (n = three); data of multiple groups were statistically analyzed by using a one-way ANOVA; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/dd412254391f50856c7c427e.png"},{"id":54859273,"identity":"30fc2f84-223a-4f63-8bc2-1a75e0d2a470","added_by":"auto","created_at":"2024-04-17 19:02:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32972312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEGF activated the conversion of Rac1-GDP into Rac1-GTP by stimulating EPS8L1 in the presence of TIAM2. \u003c/strong\u003e(a–b) Activation of Rac1 in EPS8L1 and TIAM2 knockdown cells. Cells were lysed and equal amount of proteins were incubated with the GST-PAK1 protein to extract GTP-Rac1. The expression of GTP-Rac1 was evaluated with Western blot via the use of an anti-Rac1 antibody. Data are shown as the mean ± SD (n = three). Data of multiple groups were compared by using a one-way ANOVA; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ImageJ software analyzed the grayscale of protein expression. (c) Migration of EPS8L1-Si cells and TIAM2-Si cells with EGF stimulation. Data are shown as the mean ± SD (n = three). Data of multiple groups were compared by using the Student's t-test; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001. (d) Transwell assay of EPS8L1-Si cells and TIAM2-Si cells with EGF stimulation. Data are shown as the mean ± SD (n = three). Data of multiple groups were compared by using the Student's t-test; *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/e3bb61a5783395f6aefee6a4.png"},{"id":54859275,"identity":"3f7b889a-ccff-438a-bc8c-7b483af0a6f2","added_by":"auto","created_at":"2024-04-17 19:02:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14903795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe regulatory role of EPS8L1 and TIAM2 in Rac1-MAPK activation.\u003c/strong\u003e (a) Activation of Rac1 with PI3K agonist (740Y-P) for 24 h in EPS8L1-knockdown cells. Cells were lysed and equal amounts of proteins were incubated with the GST-PAK1 protein to extract GTP-Rac1. The expression of GTP-Rac1 was evaluated via Western blot with the use of an anti-Rac1 antibody. (b) Activation of MAPK family with EGF (40 ng/ml) stimulation in EPS8L1-knockdown cells. All data are shown as the mean ± SD (n = three). Data of multiple groups were compared with a one-way ANOVA; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/a5de038b9f954a00fbd1d574.png"},{"id":55506717,"identity":"cc5a59ed-2cb7-4d79-a3d9-3ca117b95ce2","added_by":"auto","created_at":"2024-04-29 11:54:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3715776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/2231eb5c-ecef-4ed6-bec7-f21d68e3bbfd.pdf"},{"id":54859277,"identity":"630fc326-b5d9-4087-a91f-e8ee2dcaecdd","added_by":"auto","created_at":"2024-04-17 19:02:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":58766321,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/d465b11ca1e6c3c78c6cde8c.docx"},{"id":54859269,"identity":"3e8cbd9c-0f5d-4917-97e0-2fbb78099fe2","added_by":"auto","created_at":"2024-04-17 19:02:46","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":76770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. The mechanism of EPS8L1-TIAM2 regulation in F-actin remodeling by EGF in ovarian cancer cells.\u003c/strong\u003e EGF stimulation enhances the phosphorylation of EPS8L1, activates the downstream function of TIAM2, and induces its specific GEF activity, which in turn activate the conversion of Rac1-GDP into Rac1-GTP, regulates cytoskeleton remodeling by activating the MAPK pathway, and ultimately promotes the migration of ovarian cancer.\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4263533/v1/bb1884990417e4756d5299a7.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"The overexpression of EPS8L1 upregulates TIAM2 to promote cytoskeleton remodeling by activating the Rac1/MAPK signaling pathway in the migration of ovarian cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe incidence rate of ovarian cancer ranks third among gynecological malignant tumors in the female reproductive system; however, its mortality rate ranks at the top, being as high as 66%\u0026nbsp;(Sung et al., 2021)(Cao et al., 2021). In the clinic, 85%\u0026ndash;90% of ovarian cancer is classified as epithelial ovarian cancer, among which serous cystadenocarcinoma is the most common type, accounting for 40%\u0026nbsp;(Cho, 2009). During treatment, most patients experience relapse due to the systemic metastasis featured in ovarian cancer, resulting in a less than 30% 5-year survival rate(Penny, 2020)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e. Therefore, the investigation of an effective target with which to inhibit the migration and metastasis of ovarian cancer is significant for reducing the mortality rate.\u003c/p\u003e\n\u003cp\u003eIn 1993, Fazioli et al. identified epidermal growth factor receptor kinase substrate 8 (Eps8) for the first time in fibroblasts(Fazioli et al., 1993). Eps8 consisted of an N-terminal phosphotyrosine binding (PTB) region, an intermediate Src-homology 3 (SH\u003csub\u003e3\u003c/sub\u003e) domain, and a C-terminal effector region capping on the barbed end of filamentous actin to promote elongation(Tocchetti et al., 2003). Three analogs are in this family, namely epidermal growth factor receptor kinase substrate 8-like protein 1, 2, and 3 (EPS8L1, EPS8L2, and EPS8L3)(Matoskova et al., 1995)\u0026nbsp;. Previous publications have reported that the overexpression of Eps8 promotes the proliferation of esophageal cancer\u0026nbsp;(Bashir et al., 2010), non-small-cell lung cancer(Wen et al., 2019)\u0026nbsp;, and pancreatic cancer(Tod et al., 2017)\u0026nbsp;, as well as the migration and metastasis of oral squamous cell carcinoma(Yap et al., 2009)\u0026nbsp;, colon cancer(Maa et al., 2007)\u0026nbsp;,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand breast cancer(Chen et al., 2015)\u0026nbsp;. At a later point in time, Offenhauser et al. demonstrated that EPS8 participated in cell migration and metastasis via activating the Rac-GEF of Sos-1 and inducing tyrosine kinase receptor-mediated cell remodeling by binding to actin(Helfand et al., 2003)\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003eIn our previous work(Zhang et al., 2019)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e, we collected 31 epithelial ovarian cancer samples and 10 adjacent normal tissue samples from patients with ovarian cancer, after which we performed next-generation sequencing. The \u003cem\u003eEps8l1\u003c/em\u003e gene was screened out as a differentially expressed gene with a high possibility of participating in the occurrence and development of epithelial ovarian cancer. Consistently, the bioinformatics analysis demonstrated that the clustered genes of \u003cem\u003eEps8l1\u003c/em\u003e concentrated on the function of cell movement and migration; however, the mechanism of \u003cem\u003eEps8l1\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eits protein, EPS8L1, in ovarian cancer, especially in metastasis, was not elucidated. To illustrate the role of EPS8L1 in the regulatory pathway, we analyzed the clinical information and outcomes of patients with ovarian cancer. Additionally, in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003ein vivo experiments were performed to investigate the regulation of EPS8L1 in cell migration.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e\u003cem\u003e2.1. Cell Culture and Transfection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCells from the human ovarian cancer cell lines SKOV-3 and ES-2 were purchased from the cell bank of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. Cells were cultured in McCoy\u0026apos;s 5A medium, containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin, at 37\u0026deg;C in a humidified environment with 5% CO2. SKOV-3 cells are typically serous ovarian cancer cells, being the most common type in epithelial ovarian cancer; therefore, the SKOV-3 cell line was selected for the following experiments, including the establish-ment of EPS8L1 knockdown (Sh-EPS8L1) and overexpression (OE-EPS8L1) cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the lentiviral transfection, the Sh-RNA with the designed sequence (shown in Tables S1 and S2) was applied. The designing and packaging of the above lentiviruses were completed by the Jikai Gene Company (Shanghai, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCells in the logarithmic growth was trypsinized, and complete medium were added to adjust the cell density of 2x10\u003csup\u003e4\u003c/sup\u003e/mL. Then, cells were seeded in a 24-well plate in a 500 \u0026mu;L/well and cultured overnight. In the preliminary experiment, the optimal MOI value for lentiviral infection of SKOV3 was determined as 10, as a result, the cor-responding virus volume was calculated as V= (MOI X number of cells)/virus titer. Then, an appropriate amount of virus solution and 5 \u0026mu;g/mL of Polybrene (enhancement solution) were added to 500 \u0026mu;L medium. Similarly, a control was constructed. After incubating for 24 hours, the virus-containing medium was replaced with complete medium for continuous culture. After 48 hours, the infection efficiency was observed under fluorescence microscopy. At the same time, SKOV3 cells were seeded in a 96-well plate with cell density of 4x10\u003csup\u003e4\u003c/sup\u003e/mL, and cultured overnight. Medium containing different concentrations of puromycin (1, 2, 4, 8, 10 \u0026mu;g/mL) was added to each well. The proportion of viable cells was observed daily, and the puromycin-containing medium was changed every other day. We screened that 2\u0026mu;g/mL was the lowest concentration (kill all cells within 2 days). After transfection, cells were cultured in a 24-well plate, and when the density reached 80%, culture medium containing 2 \u0026mu;g/mL puromycin was added. The puromycin-containing medium was changed daily or every other day according to the color and cultured until 14 days. The transfected cell line was considered to be stably established by the result more than 95% of the cells with green fluorescence were obtained under the fluorescence microscope.\u003c/p\u003e\n\u003cp\u003eFor the transfection of siRNA, cells were seeded in 6-well plates at a density of 105 cells per well, and transfected with relevant siRNA (80 pmol), followed by transfection with the LIPO3000 (Sigma) transfection reagent according to the manufacturer\u0026apos;s instructions. Then, the expression ability of siRNA molecules was analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2. Scratch Healing Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter cells were seeded into 6-well plates at 3 \u0026times; 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecells per well with 90%-100% confluency, the complete medium was discarded and treated with 4 \u0026mu;g/mL mitomycin C (GLPbio) for 1 h. A sterilized micropipette tip was applied to scratch the cell layer, and the floating cells were washed off. Multiple spots on the scratch were imaged using a Zeiss microscope. Images were taken at 100x magnification. Cell migration measurements were performed using ImageJ software, and the mobility ratio was calculated by the migrated area divided by the original area.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3. Transwell Migration Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOn a transwell migration plate (Corning, 8.0 \u0026mu;m pore size), 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e transfected SKOV-3 cells were resuspended in the upper chamber with 400 \u0026mu;L of serum-free McCoy\u0026rsquo;s 5A medium and the lower chamber was filled with 500 \u0026mu;L of McCoy\u0026apos;s 5A medium containing 40 ng/mL EGF. After 24 h of incubation, cells on the surface of the cavity that had been invaded were stained with 0.5% crystal violet and imaged under a fluorescence microscope. The number of migrated cells was counted using ImageJ software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4. In Vivo Colonization Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 2- to 3-week immunodeficient female Balb/c-nu mice (ASM Pharmaceutical Research Institute Co., Ltd., Hunan, China) were injected with 5 \u0026times; 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eGFP-labeled SKOV-3 cells via a tail vein injection. After 8 weeks, the distribution of fluorescent signal was observed under a small-animal in vivo imager (Bruker in vivo FX, Germany) for whole-body imaging. After this, the mice were sacrificed and dissected organs were imaged. For the evaluation of pulmonary colonization, lungs were cut into 5 \u0026mu;m pieces and stained with H\u0026amp;E. Three sections throughout the lung per mice were screened, and the number of metastases was counted. In addition, the lungs were stained with MMP-2 and MMP-9 to quantify the expression levels of the corresponding proteins.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5. Immunohistochemistry (IHC) Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter being embedded with paraffin, the tumor tissues were stored in a citrate antigen retrieval buffer (pH = 6.0). Next, the tissue was blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and covered with 3% BSA at room temperature for 30 min. Then, primary antibody MMP-9 (1:200, Cell Signaling Technology, 13667) and MMP-2 (1:200, Cell Signaling Technology, 40994S) were added and incubated overnight at 4\u0026deg;C. After washing tissues with PBS, the corresponding secondary antibodies (HRP-labeled) were added and incubated at room temperature for 50 min. Then, freshly prepared diaminobenzidine (DAB) was used for color development. Finally, tissues were counterstained with H\u0026amp;E, dehydrated with alcohol, and mounted for microscopic examination. Six tumor slices and three high-magnification fields of each slice were randomly selected for scanning. The ratio of positive area was analyzed by using ImageJ software. All of the above reagents, except the antibodies, were purchased from Wuhan Servicebio Technology CO., LTD (China). The lung tissues of 3 mice were randomly selected in each group for analysis using Image J, and the proportion of positive cells was used as an indicator with which to compare with the control group. For comparison between two groups, a t-test was used.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.6. Actin Staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 2 \u0026times; 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eSKOV-3 cells in 24-well plates were fixed with 3.75% paraformaldehyde for 15 min, permeabilized with 0.5% Triton for 10 min, and finally stained with an appropriate amount of an FITC-labeled phalloidin stock solution (Solarbio, #CA1640) for 15 min. After dripping DAPI onto the slide, cells were observed under a fluorescence microscope (Leica).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.7. F-actin Quantification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eF-actin was quantified using an enzyme-linked immunosorbent assay kit (CUSABIO Human F-actin ELISA Kit). Briefly, SKOV-3 cells in a 6-well plate with a density of 2 \u0026times; 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ewere transferred to a 1.5 mL centrifuge tube to generate a homogenous solution via an ultrasonic cell disruptor. The sample was then centrifuged at 5000 \u0026times; g, 4 \u0026deg;C for 5 min, and supernatant was added to the embedded 96-well plate. The OD value of each well was measured on a plotted standard curve under the wavelength of 450 nm to calculate the actual F-actin concentration.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.8. Downstream Gene Screening\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the relevant targets for the mechanism of EPS8L1 in epithelial ovarian cancer, the comprehensive database GENE EXPRESSION OMNIBUS (\u003ca href=\"http://www.ncbi.nlm.nih.gov/geo\"\u003ehttp://www.ncbi.nlm.nih.gov/geo\u003c/a\u003e) and the public database String (https://cn.string-db.org/cgi/) were selected. To further confirm the relevant genes, RT-PCR assays with specific primers were performed. The mRNA expression levels of each gene in Sh-EPS8L1, OE-EPS8L1, and their corresponding vector control groups were obtained. After a comparison of two groups, genes displaying a large variance between cells were selected for the following verification and further verified by using a Western blot assay.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.9. Quantitative RT-PCR Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eReal-time quantitative PCR was performed via the use of Trizol (Invitrogen) and reverse transcription via the use of a high-capacity cDNA (Sigma) kit. TNNI3, TNNC1, TNNT2, CD44, TIAM2, TIAM1, MTA1, NM23, CD147, CD82, EPS8L1, and GAPDH levels were measured using target primers (Invitrogen), as shown in Table S3. Relative expression levels were measured by comparing the ratio of the Ct value of the target with the Ct value of GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.10. Rac1 Activation Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe PI3K-specific activator 740Y-P (20 \u0026mu;M) was added to cells in Sh-EPS8L1 and control groups for 24 h. The SKOV-3 cells were then scraped with a cell lysate that contained protease inhibitors. The lysates were centrifuged (10,000\u0026times; g, 4 \u0026deg;C for 1 min) to remove cellular debris and then incubated with 10 \u0026mu;L of PAK-PBD beads from a Rac1 Activation Kit (cytoskeleton, CAT#BK035-S) for 60 min at 4\u0026deg;C on a shaker. Beads were washed in 20 \u0026mu;L of a 2 \u0026times; Laemmli sample buffer and heated to boil for 2 min. The determination of Rac1-GTP activation was then performed with Western blot experiments.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.11. Western Blot Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCells were washed with phosphate-buffered saline (PBS) and harvested with a RIPA lysis collection buffer. Cell lysates were run on 10\u0026ndash;15% SDS-polyacrylamide gels and then transferred to PVDF membranes, with primary antibodies incubated overnight at 4\u0026deg;C. After multiple washes, HRP-conjugated secondary antibodies were added for 2 h at room temperature and developed with an ECL kit. The detection of active Rac1 proteins requires pretreatment via a pull-down assay. EPS8L1 antibodies were purchased from Invitrogen (PA5-38746); TIAM2 antibodies were purchased from Abcam (ab199426); Rac1 antibodies was purchased from Abcam (ab155938); and GAPDH antibodies were purchased from Affinity (AF7021). \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.12. KEGG and GO Enrichment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe gene annotation information of GO (Gene Ontology) was mainly obtained from Bioconductor, and the annotation information of KEGG (Kyoto Encyclopedia of Genes and Genomes) was obtained through the API of KEGG data. Briefly, we opened the website, entered EPS8L1, selected a species, and downloaded the corresponding annotation information. GO cluster and KEGG pathway enrichment was generated by the R program for analysis. The relevant information of EPS8L1 (gene ID: 54869) was down-loaded from the DAVID bioinformatics database (https://david.ncifcrf.gov/)and im-ported into the R program for analysis and to draw a plot. Partial enrichment codes for KEGG and GO in Table S4.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.13. String Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the String database (https://cn.string-db.org/cgi/), we entered the corresponding gene name and selected the retrieved database in order to generate results. Accordingly, we found a series of related proteins, such as TNNT2, TNNCI, and TNNI3. In addition, through the GENE EXPRESSION OMNIBUS database (http://www.ncbi.nlm.nih.gov/geo), the GSE18520 (microarray expression data of epithelial ovarian cancer) dataset was selected for the statistical analysis of Spearman\u0026rsquo;s correlation using the R program, and other related proteins were found, including TIAM2, CD44, CD82, and TIAM1. Subsequently, experimental verification was carried out on these proteins. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.14. cfDNA\u0026nbsp;Extraction and Purification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e5 mL of plasma was collected from patients, 500 \u0026mu;L of proteinase K and 4 mL of buffer ACL (containing carrier RNA) were added and mixed well. After placing the mix at 60 \u0026deg;C for 30min to 1h, 9 mL of buffer ACB was added and mixed well, then was put on ice for 5min. The ACB lysis mixture was purified and eluted through the column to obtain cfDNA; cfDNA concentration was measured using the Qubit2000 Fluorometer. Statistical analysis: The experimental results were analyzed using the statis-tical software SPSS 20.0 with data presented as mean \u0026plusmn; SD, and t-test was used for comparison between the two independent groups.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.15. Statistical Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data were expressed as the mean \u0026plusmn; standard deviation (SD), and the results were analyzed with the statistical software SPSS 22.0. Each experiment was performed at least three times unless noted. If the data conformed to a normal distribution and homogeneity of variance, two independent-samples t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparisons between more than three groups. If the data did not conform to a normal distribution, a nonparametric rank sum test was used. The enumeration data were analyzed by using the chi-square test. A value of P \u0026lt; 0.05 indicated that the difference was statistically significant. Figures were plotted with GraphPad Prism 7.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e3.1. The Overexpression of EPS8L1 in Epithelial Ovarian Cancer was Correlated with Poor Clinical Outcomes and Positively Related to Tumor Migration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn our previous work on the next-generation sequencing of ovarian tumors from 31 patients, the EPS8L1 gene was screened out with high expression\u003csup\u003e\u0026nbsp;[15]\u003c/sup\u003e. To further interpret the previous RNA-Seq results, we validated the expression of the EPS8L1 gene in patients with ovarian cancer. Preoperative venous blood from 43 patients with serous ovarian cancer was collected. The concentrations of plasma cfDNA and EPS8L1-cfDNA from samples were then detected via qPCR. As shown in Table 1, the plasma cfDNA concentrations from peripheral blood of patients with different ages, unilateral or bilateral tumors, classifications, stages, and with or without lymph node metastasis showed no significant difference. In contrast, the EPS8L1-cfDNA concentration was significantly different (\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e) in terms of the FIGO stage (stages I and II as the early-stage groups; stages III and IV as the advanced-stage groups). The level of EPS8L1-cfDNA in patients with lymph node metastasis was significantly higher than that in patients without metastasis (\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eRelationship of plasma cfDNA concentration and EPS8L1-cfDNA level in 43 patients with ovarian serous cystadenocarcinoma.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003eCases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003ecf-DNA Concentration（ng/\u0026mu;L）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003eRelative Level of EPS8L1-cfDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003e\u0026lt; 55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.203 \u0026plusmn; 0.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e0.917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.70 \u0026plusmn; 0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003e\u0026ge; 55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.201 \u0026plusmn; 0.737\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.95 \u0026plusmn; 0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003ePrimary Tumor Site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eUnilateral\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.186 \u0026plusmn; 0.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e0.218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.83 \u0026plusmn; 0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e0.797\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eBilateral\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.210 \u0026plusmn; 0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.79 \u0026plusmn; 0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eOrganization Type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eHigh-grade serous ovarian cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.201 \u0026plusmn; 0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e0.954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.78 \u0026plusmn; 0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e0.663\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eLow-grade serous ovarian cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.203 \u0026plusmn; 0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.85 \u0026plusmn; 0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eFIGO Staging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eⅠ + Ⅱ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.190 \u0026plusmn; 0.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e0.220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.49 \u0026plusmn; 0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eⅢ + Ⅳ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.211 \u0026plusmn; 0.072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e3.05 \u0026plusmn; 0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eLymph node metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.214 \u0026plusmn; 0.067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e0.173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e3.13 \u0026plusmn; 0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.72519083969466%\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.595419847328245%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.84732824427481%\"\u003e\n \u003cp\u003e0.189 \u0026plusmn; 0.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.114503816793894%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.51145038167939%\"\u003e\n \u003cp\u003e2.46 \u0026plusmn; 0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.206106870229007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTherefore, the relationship between mRNA expression and the clinicopathological indicators of patients was analyzed by detecting the expression of the EPS8L1 gene in the tumor tissues of 43 patients with qRT-PCR. Consistently, the results showed that the expression of the EPS8L1 gene was not related to a patient\u0026apos;s age, primary site, and other factors; however, it was positively related to FIGO stages (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.031) and lymph node metastasis (\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e) (Table 2). The relationship between the expression of EPS8L1-mRNA in tissues and the clinicopathological indicators of patients was consistent with the results of cfDNA concentration, both suggesting that the EPS8L1 gene might be related to the progression and metastasis of epithelial ovarian cancer, and probably serve as a new biomarker to predict the clinical outcomes of ovarian cancer patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eThe correlation between the expression of EPS8L1-mRNA and clinicopathological indicators of patients.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003eNo. of Cases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003eEPS8L1-mRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003e\u0026lt; 55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.579 \u0026plusmn; 0.506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e0.987\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003e\u0026ge; 55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.576 \u0026plusmn; 0.735\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003ePrimary Tumor Site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eUnilateral\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.426 \u0026plusmn; 0.641\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e0.232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eBilateral\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.659 \u0026plusmn; 0.579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eOrganization Type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eHigh-grade serous ovarian cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.627 \u0026plusmn; 0.614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e0.478\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eLow-grade serous ovarian cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.487 \u0026plusmn; 0.154\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eFIGO Staging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eⅠ + Ⅱ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.357 \u0026plusmn; 0.462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eⅢ + Ⅳ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e2.737 \u0026plusmn; 0.652\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eLymph Node Metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e3.196 \u0026plusmn; 0.136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.091603053435115%\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.366412213740457%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.091603053435115%\"\u003e\n \u003cp\u003e1.994 \u0026plusmn; 0.516\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.450381679389313%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eWe further performed a GO clustering analysis of EPS8L1 to identify relevant genes in different functions. The results showed that, for biological processes, cell\u0026ndash;substrate adhesion was the key function, with most genes clustered. Regarding the cellular components, the collagen-containing extracellular matrix was mainly clustered. For molecular function, most genes were concentrated in the active part of the endopeptidase. The clustered genes mostly contributed to cellular movement, suggesting that EPS8L1 might regulate tumor invasion and metastasis through cell\u0026ndash;extracellular matrix adhesion and other associated biological functions (Figure 1a). The KEGG pathway enrichment showed that EPS8L1 was mainly overexpressed in signaling pathways such as vascular smooth muscle contraction, actin cytoskeleton regulation, and axonal pathways that positively related to the migration and motility of cells (Figure 1b). Using the TCGA database, we further analyzed the relationship between clinicopathological indicators and EPS8L1-mRNA expression in 379 patients with epithelial ovarian cancer. Consistently, the high expression of EPS8L1 was significantly related to the FIGO stages of patients (P = 0.042 \u0026lt; 0.05), shown in Table S5. The above results further demonstrated that EPS8L1 was positively related to the cellular invasion and migration in epithelial ovarian cancer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression of EPS8L1 protein in human normal ovarian epithelial cells (IOSE80) and human epithelial ovarian cancer cells (CAOV3, SKOV-3, A2780, ES-2, OVCAR3) were measured. As shown in Figure 1c, among the five ovarian cancer cell lines, SKOV-3 cells exhibited the highest level of EPS8L1 protein expression, suggesting the necessities of EPS8L1 and probably important role in SKOV-3 cells. We hypothesized alterations in EPS8L1 expression levels might induce a more significant effect on SKOV-3 cells which was feasible for measurement. Therefore, we selected the SKOV-3 cell line for subsequent knockdown or overexpression experiments. After constructing SKOV-3 cells with EPS8L1-knockdown or overexpression via lentivirus transfection, the level of EPS8L1 protein in transfected SKOV-3 cells were detected as shown in Figure 1d. The results demonstrated that the expression of EPS8L1 decreased by 43.61% (0.409 \u0026plusmn; 0.16 vs 0.725 \u0026plusmn; 0.20,\u003cem\u003e\u0026nbsp;P\u003c/em\u003e \u0026lt; 0.001) in the knockdown group, and increased by 35.24% (1.07 \u0026plusmn; 0.03 vs 0.69 \u0026plusmn; 0.02, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) in the overexpression group. Therefore, the cell model with EPS8L1-knockdown or overexpression was successfully constructed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2. The Knockdown of EPS8L1 Inhibits the Migration of Ovarian Cancer Cells by Suppressing Cellular Actin Formation and Cytoskeleton Remodeling\u003c/p\u003e\n\u003cp\u003eTo study the effect of EPS8L1, the migration of SKOV-3 cells after EPS8L1 knock-down was detected via a scratch healing assay. The quantification was represented by the migration ratio, in which a smaller value referred to a slower migration rate. The results showed that the migration ratio of the Sh-EPS8L1 group (16.7%) was significantly lower than that of the control group (35.5%), with P \u0026lt; 0.05 (Figure 2a). Thus, the migration speed of the Sh-EPS8L1 group was significantly slower than that of the control, indicating the regulation of cell migration by EPS8L1. This result suggested that EPS8L1 may affect the migration of ovarian cancer cells. Multiple G-actin are linked together to form actin chains, and two strings of actin chains are twisted into fibrillar actin (F-actin) to constitute the cytoskeleton of eukaryotes, which is closely related to the chemotactic ability of cells(N\u0026uuml;rnberg et al., 2011)\u0026nbsp;. To illustrate the status of cytoskeleton remodeling during the EPS8L1 regulation of cellular migration and metastasis, F-actin in Sh-EPS8L1 and EPS8L1 (as control) cells was stained with FITC-labeled phalloidin. As observed, the brightness of cells in the control group demonstrated the formation of more filopodia that enable cells to migrate in one direction(Cappellini et al., 2015)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e. In contrast, the amount of filopodia in Sh-EPS8L1 cells was significantly reduced, and no filopodium with an invasive structure was detected, indicating the inhibition of filopodia formation by suppressing EPS8L1. Then, EGF was given to stimulate the cells to further evaluate the effect of EPS8L1 knockdown. As shown in Figure 2b, the polymerization of cortical ac-tin was observed, and the number of central actin bundles decreased in the control group. This indicated that EGF stimulation induced the reorganization of cortical actin to promote cytoskeleton remodeling and cell migration. On the contrary, cells in the Sh-EPS8L1 group exhibited no obvious response to EGF stimulation. In the quantification of F-actin via an ELISA kit, the amount of F-actin in Sh-EPS8L1 cells was 3.67 mg/mL, while in the control cells it was 6.39 mg/mL. The difference between the two groups was statistically significant (Figure 2c). Consistently, the corresponding study in EPS8L1 overexpression cells demonstrated the same results, as shown in Fig. S1. These results suggested that the regulation of cytoskeleton remodeling and cell migration by EGF stimulation was dependent on the presence of EPS8L1. Thus, EPS8L1 might regulate the migration of ovarian cancer by regulating the formation of filopodia and cytoskeleton remodeling.\u003c/p\u003e\n\u003cp\u003e3.3. EPS8L1 Promotes the Colonization of Ovarian Tumor Cells in vivo by Regulating the Expressions of MMP-2 and MMP-9\u003c/p\u003e\n\u003cp\u003eTo further verify the participation of EPS8L1 in the migration and metastasis of ovarian cancer, in vivo colonization experiments were performed. After 8 weeks of tumor growth via a tail vein injection of GFP-Sh-EPS8L1 and GFP-EPS8L1 (control), mice were imaged by Bruker in vivo Fx to evaluate the colonization and distribution of ovarian tumors. As shown in Figure 3a, local fluorescence enhancement was observed dramatically in the lungs of mice in the control group, suggesting the development of lung colonization. In contrast, the low expression of EPS8L1 decreased the colonization of ovarian tumors in mice. Furthermore, tumors in the control group were shown to be widely distributed throughout the body compared with that of the Sh-EPS8L1 group, especially in the cervical lymph node group (including submaxillary lymph nodes, superficial cervical lymph nodes, and deep cervical lymph nodes) on dual sides of mice(Bobek et al., 2005)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e. The dissected lungs were imaged to evaluate the tumor growth. A higher number of tumor cells in lungs of the control than that of the Sh-EPS8L1 groups was shown (Figure 3b). In H\u0026amp;E staining (Figure 3c), mice injected with normal EPS8L1-expressed SKOV-3 cells displayed a significantly higher intensity of tumors per lung slice com-pared to mice injected with Sh-EPS8L1 cells (median of 22 \u0026plusmn; 1.78 vs. 4.6 \u0026plusmn; 1.01). More photographs for different mice and the ink-staining experiment for lungs with colonization to demonstrate the location of small tumor lesions are shown in Fig. S2. The matrix metalloproteinase (MMP) family regulated extracellular matrix degradation and cell adhesion remodeling to promote tumor cells\u0026rsquo; diffusion and angiogenesis, which played an important role in tumor invasion and metastasis(Kapoor et al., 2016)\u0026nbsp;. In the immuno-histochemistry of lung tissues of the Sh-EPS8L1 group, the ratios of MMP-2 and MMP-9 protein expression were 0.719 \u0026plusmn; 0.09 and 0.111 \u0026plusmn; 0.02, while those of the MMP-2 and MMP-9 proteins in the control group were 0.992 \u0026plusmn; 0.00 and 0.990 \u0026plusmn; 0.00, respectively. As compared in Figure 3d, the difference between two groups was statistically significant with MMP-2 (P \u0026lt; 0.05) and MMP-9 (P \u0026lt; 0.01) expression, indicating that EPS8L1 promoted the colonization of ovarian cancer cells in vivo by regulating the expression of MMP-2 and MMP-9. Consistently, the corresponding study in EPS8L1 overexpression demonstrated the same results, as shown in Fig. S3. The parallel study of the effect of EPS8L1 overexpression on colonization and metastasis of SKOV-3 ovarian cancer cells was shown in Fig. S4 indicating that the overexpressed EPS8L1 induced the colonization and metastasis.\u003c/p\u003e\n\u003cp\u003e3.4. EPS8L1 Regulates the Expression of Downstream TIAM2\u003c/p\u003e\n\u003cp\u003eTo further explore the relevant targets of EPS8L1, the microarray expression dataset (GSE18520) of epithelial ovarian cancer from GENE EXPRESSION OMNIBUS (http://www.ncbi.nlm.nih.gov/geo) was selected and a Spearman\u0026rsquo;s correlation analysis was generated (Figure 4a). Moreover, a public database, String (https://cn.string-db.org/cgi/), and an online analysis (http://gepia.cancer-pku.cn/) were recruited to analyze genes related to EPS8L1 (Figure 4b and Fig. S5). It was found that the genes TIAM2 (r = 0.470, P \u0026lt; 0.001), TNNT2, TNNC1, and TNNI3 (all r = 0.67, P \u0026lt; 0.05) are possibly related to EPS8L1 (Fig. S6). The RT-PCR results verified that the EPS8L1 gene was highly expressed in OE-EPS8L1 and lowly expressed in Sh-EPS8L1 cells supporting the successful establishment of our experimental models. The expressions of TNNT2, CD82, TIAM1, and TIAM2 were significantly different between OE-EPS8L1 and Sh-EPS8L1, being 46.64-fold (P \u0026lt; 0.01), 13.37-fold (P \u0026lt; 0.01), 9.71-fold (P \u0026lt; 0.01), and 4.61-fold (P \u0026lt; 0.01), respectively (Figure 4c). Subsequently, four groups of SKOV-3 cells with OE-EPS8L1 or Sh-EPS8L1 or their corresponding vectors as control groups were used to evaluate the related genes at the protein level. The results showed that, among these differentially expressed genes, only TIAM2 was obviously related to EPS8L1 at the protein level (Figure 4d). The decreased EPS8L1 expression resulted in decreased TIAM2 expression, while increased EPS8L1 expression also in-creased TIAM2 expression. The TNNT2 gene with the greatest difference at the mRNA level demonstrated no significant difference at the protein level, indicating that the TNNT2 gene might be a transcription factor in post-transcriptional regulation or that the half-life of the TNNT2 protein is too short to be captured. We also performed a validation of EPS8L1-overexpressing ES-2 cells (Fig. S7), and the results were similar to those in SKOV-3 cells demonstrating the TIAM2 expression varied with EPS8L1 level. These results indicated that TIAM2 was positively related to EPS8L1 and may be regulated by the upstream molecule EPS8L1. To further clarify the regulatory relationship be-tween EPS8L1 and TIAM2, small interfering RNA (siRNA) was utilized to knockdown EPS8L1 and TIAM2. As shown in Figure 4e, the protein expression of TIAM2 decreased when EPS8L1 was knocked down. Contrarily, no significant change in EPS8L1 expression was observed with TIAM2 knockdown. These results indicated that EPS8L1 was an upstream regulatory molecule of TIAM2 and that TIAM2 was a downstream molecule of EPS8L1 (Figure 4f).\u003c/p\u003e\n\u003cp\u003e3.5. EPS8L1 is Stimulated by EGF with the Presence of TIAM2 and is Positively Related to the Conversion of Rac1-GDP into Rac1-GTP\u003c/p\u003e\n\u003cp\u003eAs has been reported, an Eps8-Abi1-Sos1 complex regulates the depolymerization and assembly of microtubules by actin and activates Rac1-GTPase to regulate the cytoskeleton(Scita et al., 2001)\u0026nbsp;. To investigate whether EPS8L1 (a member of Eps8 family) promotes the activation of Rac1, the Rac1-GTP concentration was measured. After stimulating Sh-EPS8L1 with EGF, the expression of the EPS8L1 protein increased with the prolongation of the stimulation time (Figure 5a). Simultaneously, the expressions of the TIAM2 protein and Rac1-GTP increased as well. The total Rac1 amount was un-changed, indicating that the proportion of Rac1-GTP increased. However, as shown in Figure 5b, EGF stimulation did not induce Rac1 activation in the TIAM2-knockdown SKOV-3 cells (TIAM2-Si). Meanwhile, scratch healing (Figure 5c) and transwell migration (Figure 5d) results with EGF stimulation after EPS8L1 and TIAM2 knockdown were consistent with those of protein expression. After TIAM2 knockdown, the migration of the cells to all directions was reduced. The EPS8L1 expression could be recovered to some extent by EGF stimulation, indicating that EPS8L1 is an effector molecule of EGF stimulation; however, the expression of TIAM2 did not change significantly, suggesting the non-response of TIAM2 to the stimulation of EGF. Previous research pointed out(Van Leeuwen et al., 2003)\u0026nbsp;that the activation of Rac1 is dependent on the GEF (guanine nucleotide exchange factor) activity of TIAM1 in the case of lysophosphatidic acid (LPA) or platelet growth factor (PDGF) induction. As an analog of TIAM1, a similar mechanism may exist for TIAM2. Therefore, TIAM2 and the activation of Rac1 cannot respond to the stimulation of EGF. These results demonstrated that the Rac1-GTP conversion activated by EGF stimulation was dependent on the presence of EPS8L1.\u003c/p\u003e\n\u003cp\u003e3.6. The Regulatory Role of EPS8L1 and TIAM2 in Rac1-MAPK Activation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs reported, TIAM2 was essential for the Rac1-dependent cell motility mediated by PI3K\u0026nbsp;(Cooke et al., 2021). However, the role of upstream EPS8L1 was unknown. In this study, 740Y-P was used as a membrane-permeable agonist to activate PI3K(Williams \u0026amp; Doherty, 1999)\u0026nbsp;. As demonstrated in Figure 6a, the expression of TIAM2 was restored after 740Y-P treatment, while the levels of EPS8L1 and Rac1-GTP activation did not recover compared to the control group. Thus, a PI3K agonist could stimulate the recovery of TIAM2 independently with the presence of EPS8L1; however, TIAM2 could not mediate Rac1 activation by itself, indicating the decisive role of EPS8L1 and essential role of TIAM2 in the EPS8L1-TIAM2-driven Rac1 activation.\u003c/p\u003e\n\u003cp\u003eWith regard to the participation of the MAPK signaling pathway in the extracellular matrix, adhesion, motility, and angiogenesis of tumor cells(Chen et al., 2015) , we evaluated whether it might be a downstream regulatory pathway of EPS8L1 for tumor metastasis. Consistent with the previous section, the EPS8L1 protein level would not be increased further with EGF stimulation in the normal EPS8L1 expression group (negative vs. positive control in Figure 6b); therefore, TIAM2 in the normal EPS8L1 expression group would not be increased with EGF stimulation. However, the Rac1-GTP and phosphorylation levels of key proteins in the MAPK pathway, including P38, Erk, and Jnk, were increased significantly, indicating that EGF stimulation could also activate Rac1-MAPK in the normal presence of EPS8L1 and TIAM2. In the Sh-EPS8L1 group, EGF stimulation recovered EPS8L1 and TIAM2 accordingly compared with non-EGF treatment; however, Rac1-GTP was not increased to the level in the normal EPS8L1 expression group with EGF stimulation. Similarly, the phosphorylation levels of P38, Erk, and Jnk in the Sh-EPS8L1 group were upregulated with EGF stimulation to some extent but still lower than those in the control group. These results demonstrated that the suppression of EPS8L1 led to the inactivation of the MAPK pathway, indicating that the MAPK pathway might contribute to the EPS8L1-mediated metastasis and migration of ovarian cancer cells.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOvarian cancer is usually diagnosed at an advanced stage (stages III\u0026ndash;IV), with a high mortality rate and developed metastases at multiple tissues, leading to no re-markable improvement in the 5-year survival rate(Penny, 2020)\u0026nbsp;. This being the case, the investigation of metastasis in ovarian cancer is key to developing effective treatments and prognoses of patients. Studies have shown that the Eps8 gene is related to tumor proliferation(Yang \u0026amp; Huang, 2015)\u0026nbsp; \u0026nbsp;and metastasis, in addition to probably participating in the integrin-dependent Rac1 pathway(Yap et al., 2009)\u0026nbsp;, cytoskeleton alteration\u0026nbsp;(Fregnan et al., 2011), and regulation of focal adhesion kinase(Shahoumi et al., 2020)\u0026nbsp;. The high Eps8 expression in head and neck squamous cell carcinoma also promoted invasion and metastasis(Wang et al., 2009)\u0026nbsp;in pancreatic cancer cells(Welsch et al., 2007). As demonstrated, Eps8-Abi1-Sos1 regulated Rac activity in the epithelial-mesenchymal transition (EMT) of ovarian cancer cells(Scita et al., 1999)(Innocenti et al., 2003)\u0026nbsp;. Eps8 knockdown greatly reduces the ability of glioblastomas to invade and metastasize(Cattaneo et al., 2012)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e. As the most important homologous aspect of the Eps8 gene, EPS8L1 might have major functionality in regulating the migration and metastasis of tumor cells; however, no study on the mechanism of EPS8L1 in tumors, including ovarian cancer, has been reported. In our previous work, EPS8L1 was found to be upregulated in clinical ovarian cancer samples(Zhang et al., 2019)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e. In this study, we further illustrated the mechanism of overexpressed EPS8L1 in ovarian cancer, especially in tumor migration and metastasis.\u003c/p\u003e\n\u003cp\u003eThe analysis of clinical cases demonstrated that EPS8L1 was related to the FIGO staging and lymph node metastasis of patients with ovarian cancer. Additionally, EPS8L1 knockdown decreased the migration speed of ovarian cancer cells in the scratch-healing and transwell experiments. To exclude the possibility that scratch closure was caused by cell proliferation, cells were pretreated with a selected concentration of mitomycin C (Fig. S8) to inhibit cell proliferation but not cell migration(Liang et al., 2007)\u0026nbsp;. Thus, the scratch healing in our study was ensured to be induced by migration. Regarding the establishment of an in vivo model with the metastasis of ovarian cancer cells, ortho-topic transplantation(Yi et al., 2005)\u0026nbsp;, footpad injection(Fan et al., 2014)\u0026nbsp;, intraperitoneal injection(Rose et al., 1996)\u0026nbsp;, and tail vein injection(Peng et al., 2009)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e have been reported. Originally, we injected SKOV-3 cells into mice intra-peritoneally to simulate the progress of intraperitoneal dissemination and metastasis to mimic the development of ovarian cancer in the clinic. After 4\u0026ndash;5 weeks post-injection, malignant ascites was found in the abdominal cavity of the mice, but the overall condition of the mice was poor. Only the abdomen colonization of tumors was observed, with no metastases in the lungs, liver, or other organs. We then selected the vein injection with the superior effect to establish a colonization model. To evaluate tumor growth and distribution, the injected SKOV-3 cells were labeled with GFP-fluorescence to provide images under a Bruker in vivo Fx imager. No fluorescence signal was detected in the lungs of the EPS8L1 knockdown group, with only scattered tumor cells being able to be observed by directly scanning dissected lungs, which may be due to the difficulty of low fluorescent intensity from a few tumor cells in penetrating tissues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs has been reported, Eps8 is required for the formation of dynamically actin-based cell protrusions and cell-cell junctions(Scita et al., 2001)\u0026nbsp;; therefore, we carried out an actin-staining experiment on whether EPS8L1 also induces actin‑rich membrane ruffle and filopodia formation. As is observed in Figure 2b, the formation of F-actin-composed cortical actin is an important indicator of cells with good contraction and movement ability(Small et al., 2002)\u0026nbsp;. Moreover, the capping activity of EPS8L1 is crucially required for the formation of cortical actin. As has been demonstrated, EPS8L1 knock-down resulted in a remarkable change in cell shape and actin structures that morpho-logically resembled a less-motile cell phenotype. We hypothesized that the downregulation of Eps8L1 leads to impaired cell protrusion and filopodia formation as a result of inefficient F-actin. Surprisingly, when EPS8L1 is knocked down, cells can still recover migration ability to some extent after being stimulated by EGF. We speculated that other molecular mechanisms for migration might compensate in the case of Eps8 loss. However, EGF stimulation cannot fully reverse the reduction in actin capping and Rac-GEF activity induced by EPS8L1 downregulation, demonstrating the key regulatory role of EPS8L1.\u003c/p\u003e\n\u003cp\u003eA moderately strong correlation between EPS8L1 and TIAM2 (r = 0.48, p \u0026lt; 0.001) was found. Its analog, TIAM1, catalyzes the conversion of Rac1 to promote the formation of lamellipodia(Raftopoulou \u0026amp; Hall, 2004)(Nobes \u0026amp; Hall, 1995)(Hall \u0026amp; Nobes, 2000)\u0026nbsp;and cytoskeleton remodeling(Chiu et al., 1999)(Mertens et al., 2003)\u0026nbsp;, which are important for cell migration and metastasis. Similarly, TIAM2 was associated with tumor progression and unfavorable prognoses\u0026nbsp;(Jiang et al., 2021)(Woroniuk et al., 2018). In our study, we first identified EPS8L1 and further illustrated its relationship with downstream TIAM2. We then demonstrated their indispensable role in regulating Rac1 activation to participate in actin remodeling and the MAPK pathway, which finally induced migration. These conclusions would assist the diagnosis and staging of malignant ovarian cancer with EPS8L1 overexpression.\u003c/p\u003e\n\u003cp\u003eAs a substrate, EGF stimulation increases the phosphorylation of EPS8L1 by epi-dermal growth factor receptor (EGFR), and then enhances the function of downstream TIAM2 to activate Rac1. In our study, the expression of TIAM2, restored by the PI3K agonist 740Y-P, did not compensate for the reduced activation of Rac1 in EPS8L1 knockdown cells. In addition, the levels of p-P38, p-Erk, and p-Jnk were lower in ovarian cancer cells with EPS8L1 knockdown, regardless of stimulation with EGF or not, suggesting that the MAPK signaling pathway is downstream regulated by EPS8L1. Therefore, EPS8L1 could be a promising target for the intervention of Rac activation and the MAPK signaling pathway that participated in tumor migration.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, the regulation of ovarian cancer metastasis is depicted in Scheme 1. EPS8L1 promotes the migration and metastasis of ovarian cancer cells, which was mainly stimulated by extracellular signals such as EGF. The overexpression of EPS8L1 activates the downstream TIAM2 to induce its GEF activity, which facilitates the con-version of Rac1-GDP into Rac1-GTP and regulates downstream MAPK cascade signaling to affect cytoskeleton remodeling and ultimately promote the migration and metastasis of ovarian cancer cells. In addition, the mechanism of EPS8L1 in the pro-motion metastasis may also be related to its regulation of the expression of MMP-2 and MMP-9. These results provide an important scientific basis for understanding the mechanism of the EPS8L1 gene in the development of ovarian cancer, especially in migration and metastasis. In the future, EPS8L1 will be studied intensively as a novel biomarker for prognoses and a new therapeutic target for ovarian cancer patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e For all data requests, please contact the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest disclosure:\u0026nbsp;\u003c/strong\u003eAll authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement:\u003c/strong\u003e The study was approved by the Institutional Animal Care and Use Committee (IACUC) of Kunming Medical University according to the laboratory guidelines for the ethical review of animal welfare (kmmu2020060).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent statement:\u003c/strong\u003e All patients were informed, with consent obtained prior to the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (grant no. 81860463, 82160344, and 82160524); Scientific Research Fund of Yunnan Provincial Department of Education（No.2021Y326）；Yunnan Provincial Department of S\u0026amp;T-KMU Joint Foundation (no. 2018FE001-003 and 202101AY070001-068); Research Project on Undergraduate Educational and Teaching Reforms in Yunnan Province (no. JG2023001) and the Yunnan Provincial Department of Education Project (no. 2022J0213).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution to authorship:\u0026nbsp;\u003c/strong\u003eY.W. performed all of the experiments and composed the manuscript. L.Z. provided the human specimens, clinical information, and data analysis. X.L., H.Z.,M.W., L.M.Z., Y.S., J. Z., M.L., and H.Z. provided support with data collection, experimental materials, and techniques. Y.J. and C.Q. designed the research and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe authors affirm that human research participants provided informed consent for publication of all the images.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBashir, M., Kirmani, D., Bhat, H. F., Baba, R. A., Hamza, R., Naqash, S., Wani, N. A., Andrabi, K. 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R20 Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation. Oncogene, 28(27), 2524\u0026ndash;2534. https://doi.org/10.1038/onc.2009.105\u003c/li\u003e\n \u003cli\u003eYi, X. F., Yuan, S. T., Lu, L. J., Ding, J., \u0026amp; Feng, Y. J. (2005). A clinically relevant orthotopic implantation nude mouse model of human epithelial ovarian cancer - Based on consecutive observation. International Journal of Gynecological Cancer, 15(5), 850\u0026ndash;855. https://doi.org/10.1111/j.1525-1438.2005.00147.x\u003c/li\u003e\n \u003cli\u003eZhang, L., Luo, M., Yang, H., Zhu, S., Cheng, X., \u0026amp; Qing, C. (2019). Next-generation sequencing-based genomic profiling analysis reveals novel mutations for clinical diagnosis in Chinese primary epithelial ovarian cancer patients. Journal of Ovarian Research, 12(1), 4\u0026ndash;9. https://doi.org/10.1186/s13048-019-0494-4\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ovarian cancer, EPS8L1, cell migration and metastasis, TIAM2, Rac1/MAPK, cytoskeleton remodeling","lastPublishedDoi":"10.21203/rs.3.rs-4263533/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4263533/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: EPS8L1, an analog of epidermal growth factor receptor pathway substrate 8 (Eps8), was screened out in our previous work from clinical samples of patients with ovarian cancer. We also found that EPS8L1 was involved in various biological activities. In this study, the participation and mechanisms of EPS8L1 ion the migration and metastasis of ovarian cancer were investigated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In vitro scratch-healing, transwell assay, and actin-staining studies were performed in SKOV-3 ovarian cancer cells with EPS8L1 overexpression or knockdown. An ovarian cancer mouse model with lung colonization was established to evaluate the in vivo colonization and migration. To identify correlated proteins, a bioinformatics assay was conducted and verified via qRT-PCR and Western blot.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: EPS8L1 knockdown inhibited cellular migration in vitro and reduced tumor colonization in vivo. The actin-staining and ELISA experiments suggested that EPS8L1 regulated actin formation as well as cytoskeleton remodeling. Furthermore, mRNA and protein expression confirmed that EPS8L1 regulated the downstream T-cell lymphoma invasion and metastasis 2 (TIAM2) molecule and stimulated the activation of Rac1. Additionally, the phosphorylation levels of P38, Erk, and Jnk in the MAPK pathway decreased after EPS8L1 knockdown.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: The upregulation of EPS8L1 could promote the migration and metastasis of ovarian cancer cells by regulating cytoskeleton remodeling. The mechanism underlying this might be that EPS8L1 regulates TIAM2 to induce the transformation of Rac-GDP into Rac-GTP and then activates the downstream MAPK pathway. As a regulatory factor in cell migration and metastasis, EPS8L1 could be a new prognostic biomarker and a promising therapeutic target for ovarian cancer patients.\u003c/p\u003e","manuscriptTitle":"The overexpression of EPS8L1 upregulates TIAM2 to promote cytoskeleton remodeling by activating the Rac1/MAPK signaling pathway in the migration of ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 19:02:41","doi":"10.21203/rs.3.rs-4263533/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c167f0d-35d4-4082-aa1e-02d385b69c1b","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-29T10:46:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-17 19:02:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4263533","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4263533","identity":"rs-4263533","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}