LncRNA FOXP4-AS1 facilitates colorectal cancer invasion and migration by enhancing USP7 interaction with ZEB1 Running title: LncRNA FOXP4-AS1 raises CRC invasion and migration

LncRNA FOXP4-AS1 facilitates colorectal cancer invasion and migration by enhancing USP7 interaction with ZEB1 Running title: LncRNA FOXP4-AS1 raises CRC invasion and migration | 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 Article LncRNA FOXP4-AS1 facilitates colorectal cancer invasion and migration by enhancing USP7 interaction with ZEB1 Running title: LncRNA FOXP4-AS1 raises CRC invasion and migration Xiaoling Yang, Chenglong Shen, Yuchen Yuan, Jiazhe Shao, Haichen Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6691801/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Colorectal cancer (CRC) poses a threat to the health of people worldwide. Long noncoding RNAs (lncRNAs) have been reported to play a key role in regulating carcinogenesis, including CRC. In this study, the levels of lncRNA forkhead box P4 antisense RNA 1 (FOXP4-AS1) were analyzed in CRC cell lines and normal cell lines using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) method. The effects of FOXP4-AS1 on CRC cell metastasis were investigated. Then, the silver staining assay, western blot, RIP, Co-IP, and immunofluorescence were used to explore and validate the molecular mechanisms by which FOXP4-AS1 affects CRC progression. We discovered that FOXP4-AS1 expression was significantly elevated in CRC tissues and cell lines. Functionally, knockdown of FOXP4-AS1 expression inhibited CRC cell migration and invasion. In addition, silencing FOXP4-AS1 weakened CRC tumor growth in vivo . Mechanistically, we identified that FOXP4-AS1 enhanced the interaction of USP7 with ZEB1. Rescue experiments demonstrated that USP7 inhibitor P005091 rescued the promotion of cell migration, invasion and EMT by overexpression of FOXP4-AS1. Furthermore, ZEB1 overexpression reversed the impact of silencing FOXP4-AS1 on cell migration, invasion and EMT. LncRNA FOXP4-AS1 accelerates CRC malignant progression by strengthening the interaction between USP7 and ZEB1. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology Health sciences/Biomarkers Health sciences/Gastroenterology Health sciences/Oncology LncRNA FOXP4-AS1 USP7 ZEB1 colorectal cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer (CRC) is one of the common and serious malignant tumors threatening human life and health, with the third highest incidence and second highest mortality rate among various malignant tumors worldwide 1 . Multiple risk factors are associated with the development of the disease, such as, genetic inheritance and poor lifestyle habits 2 . Currently, the clinical treatment of CRC is mainly a comprehensive treatment based on surgery, and there is a significant improvement in the clinical outcome of CRC. However, the treatment of patients with advanced stage, post-surgical recurrence, and resistance to radiotherapy is still the difficulty of clinical treatment, and the 5-year survival rate has not been obviously improved 3 . Therefore, finding new early diagnostic markers for CRC, elucidating the mechanisms of CRC development, and identifying new therapeutic targets are the keys to inhibiting CRC tumor progression and reducing recurrence and mortality. The development of malignant tumors is an extremely complex biological phenomenon that is multifactorial, multigenic, and undergoes multiple stages before it finally develops. Both inactivation and activation of oncogenes cause genetic alterations thereby affecting various aspects of tumors, such as proliferation, invasion, metastasis and material metabolism 4 . Previous studies on tumor-related genes have been focused on coding protein genes, and with the continuous development and improvement of technologies such as high-throughput sequencing, a large number of long fragments of non-coding RNAs have been uncovered, and their significance in life activities has been gradually revealed 5 . Long noncoding RNAs (lncRNAs) are a class of molecules that do not encode proteins but have biological functions. LncRNAs are dysregulated in a large of human diseases, including CRC, and their dysregulation is intimately related to the progression of the disease, suggesting that they are potentially clinically useful 6 . LncRNAs act through kinds of mechanisms, mainly by interacting with DNA, miRNAs, mRNAs and proteins to interact and regulate gene transcription, mRNA stability and translation 7 . For example, lncRNA RMRP facilitates growth and metastasis of bladder cancer via miR-206 8 . Also, lncRNA BREA2 facilitates breast cancer metastasis via disrupting WWP2-mediated ubiquitination of Notch1 9 . LncRNA FOXP4-AS1, a new tumor-associated biomarker discovered in recent years, has been shown to be aberrantly expressed in malignant tumors, such as pancreatic, esophageal squamous cell carcinoma and nasopharyngeal carcinoma, and is capable of affecting a variety of biological functions in tumor cells. Liang et al. found FOXP4-AS1 is a poor prognostic biomarker for hepatocellular carcinoma and regulates the proliferation, invasion and angiogenesis of hepatocellular carcinoma cells 10 . Li et al. showed that FOXP4-AS1 is important in the progression of CRC and is linked with cell proliferation and apoptosis 11 . However, the exact mechanism by which LncRNA FOXP4-AS1 acts a role in CRC remains to be elucidated. Therefore, a more comprehensive understanding of the role of FOXP4-AS1 in CRC progression is expected to provide new perspectives for the diagnosis and treatment of this malignant tumor. With the above research background, this research aimed to investigate the mechanism of lncRNA FOXP4-AS1 in CRC. FOXP4-AS1 was enhanced in CRC tissues by TCGA database analysis. The high FOXP4-AS1 was further discovered to be linked to shorter overall survival (OS), disease-free survival (DFS) in CRC patients. FOXP4-AS1 was heightened in CRC tissues by analyzing specimens collected from CRC patients in the clinic. Cellular experiments revealed that down-regulation of FOXP4-AS1 suppressed cell metastasis and EMT. Mechanistically, our study displayed that FOXP4-AS1 enhanced the interaction of USP7 with ZEB1. The successful completion of this study will provide a new treatment option for CRC patients. Material and methods Acquisition of clinical specimens Blood samples were collected from our CRC patients and healthy volunteers. Clinical tissue specimens were obtained from patients who underwent surgery in our hospital. All patients at the hospital signed a written informed consent. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Changshu NO.2 People’s Hospital. Cell culture Human colon cancer cell lines HCT116, SW480 were obtained from the American Typical Culture Collection Center (ATCC, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco). All cells were cultured in 5% CO 2 humidified environment at 37°C. Cell transfection FOXP4-AS1 small interfering RNA (si-FOXP4-AS1#1/2) or overexpression of FOXP4-AS1 or ZEB1 vectors (pcDNA FOXP4-AS1 or ZEB1) and their corresponding controls were designed by GenePharma. FOXP4-AS1, ZEB1 were transfected through Lipofectamine 3000 (Thermo Fisher Scientific). To generate lentivirus, transfections were inoculated into 24-well plates for incubation, followed by addition of 0.5 mL of medium containing 8 µg/ml Polybrene (Sigma, USA) to each well and transfection of CRC cells. 48 h later, the complete medium was changed to 2 µg/mL puromycin, and screening was continued for 2 weeks to obtain stably transfected cell lines. Cell scratch healing assay Three parallel marking lines were scribed on the back of the 6-well plate with a marker pen in advance, and the cell suspension was prepared, the cell concentration was adjusted, and placed in the incubator at 37°C. After 24 h, the cells were scribed with the tip of the gun of 200 µL perpendicularly to the 6-well plate and perpendicularly to the marking line, and the point of intersection of the scribed and marking line was the observation point, and the cells were washed with PBS for 3 times. The cells were washed with PBS, and the serum-free RPMI 1640 or DMEM medium was used for normal fluid exchange, and the observation point was photographed by the microscope at 0, 24 h of incubation. Cell invasion assay Matrigel coated chambers with 8 µm pores were used. 5×10 4 cells/well were resuspended in 200 µL of serum-free DMEM medium added to the upper layer of the chambers, and medium containing 10% serum was added to the lower chamber and incubated for 48 h. After incubation, the cells were fixed with 600 µL of 4% paraformaldehyde for 30 min and stained with 600 µL of 0.1% crystal violet for 30 min. The uninvaded cells were gently wiped off with a cotton swab in the upper chamber, and the membrane of the transwell chambers was scraped off with a knife and fixed on slides, and the invaded cells were photographed with an inverted microscope (Olympus) and counted. qRT-PCR Total RNA of the cells and peripheral blood were extracted using Trizol reagent. 2 µg of total RNA extract was used to synthesize cDNA based on the cDNA reverse transcription kit ReverTra Ace qPCR RT. cDNA was used as a template for synthesizing cDNA based on the standard instructions of qRT-PCR fluorescence quantification kit (Toyobo, Japan) standard instructions for qPCR amplification reaction. Nucleocytoplasmic separation assay Nucleocytoplasmic separation assay was applied to measure the cellular localization of FOXP4-AS1. Cells were digested with 0.25% trypsin (Solarbio, China) and centrifuged at 1,000 g for 3 min. RNA was extracted from nuclei and cytoplasm and subjected to qRT-PCR based on the instructions of the Nucleocytoplasmic Isolation Kit (Norgen Biotek, Canada). U2 and S14 were used as internal reference genes. Western blot experiments Each group of CRC cell lines was taken separately and total protein was extracted from each group of cells by cell lysate. Protein samples were separated by electrophoresis and then transferred to PVDF membranes, which were blocked with 5% skimmed milk for 1 h. The membranes were incubated with a primary (1: 2000) antibody at 4°C overnight. On the following day, the membrane was incubated with HRP-coupled secondary antibody (1: 5000) for 2 h. Then, ECL luminescent chemistry was added to allow the bands to be developed and analyzed and processed by a Rio-Rad gel imaging system. RNA pull down assay In vitro transcripts of FOXP4-AS1 were labeled using biotin (Thermo Fisher). Then, biotin-labeled RNA was captured using streptavidin magnetic beads (Invitrogen, USA), and then the RNA beads were incubated with cell lysates. After washing out unbound proteins, the RNA-protein complexes were eluted from the magnetic beads by SDS-PAGE upsampling buffer. Proteins bound to FOXP4-AS1 were detected by western blot. SDS-PAGE silver stain assay Experiments were performed using a protein silver staining kit (Solarbio, China). The PAGE protein gel was fixed with fixative for 30 min, and the gel was transferred to sensitization solution for 30 min, after which it was rinsed with deionized water. The gel was transferred to the silver staining solution for 30 min, during which it was shaken continuously with a horizontal shaker, and finally the gel was transferred to the color development solution and displayed for 5 min. Subsequently, the color development solution was discarded and the termination solution was added to terminate the color development. The silver-stained gel was photographed with a protein imaging system. RNA immunoprecipitation (RIP) The RIP assay was performed according to the instructions of the EZ-Magna-RIP kit (Millipore, USA). Cells were lysed with lysis buffer. Cell lysates were then pre-washed with recombinant protein A/G agarose for 30 min at 4°C. Equal amounts of cell lysates were incubated with IgG or ZEB1 antibodies overnight. Capture RNA protein/antibody complexes were incubated with recombinant protein A/G agarose. RNA was eluted from the precipitated complexes and transcribed into cDNA. Binding of RNA to protein or antibody was detected by qRT-PCR. Co-immunoprecipitation (Co-IP) and IP assay Cells were lysed on ice for 30 min using RIPA buffer containing protease inhibitors. The supernatant was collected. USP7, ZEB1, or IgG antibodies were added to the supernatant and incubated overnight. Then, Protein A agarose beads (10 µL) were pre-treated by lysis buffer (Beyotime), and then mixed with the cell lysate and antibody complexes, and the antibodies were allowed to affix to the Protein A agarose beads by slow shaking for 2 h at 4°C. After the immunoprecipitation reaction, the complex was centrifuged at 3,000 rpm for 3 min. The supernatant was discarded. Finally, 15 µL of 2×SDS Sampling Buffer was added and boiled for 5 min. The precipitated proteins were then analyzed by western blot. Immunofluorescence 4×10 4 cells were cultured on coverslips in 24-well plates. Cells were then fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min. Then blocked with 5% BSA for 1 h, and incubated with primary antibody overnight. Cells were then rinsed with PBST and treated with the appropriate secondary antibody (1:1000). After incubation with DAPI (KGI, China), coverslips were mounted on slides and scanned serially with a microscope. Nude mouse xenografts Twelve BALB/c nude mice weighing 17–20 g and aged 4–6 weeks were purchased from the Shanghai Laboratory Animal Center. 2×10 7 lentivirus-infected cells in serum-free DMEM were injected subcutaneously into mice, which were divided into the Lv-sh-NC group and the Lv-sh-FOXP4-AS1 group. After 5 weeks, the tumors were removed, photographed, and measured with vernier calipers. Immunohistochemistry Tissue sections were deparaffinized, closed with 3% hydrogen peroxide for 15 min, washed with PBS, closed with 10% normal goat serum for 30 min, and incubated overnight with the addition of primary antibody Ki-67 (1: 5000, Abcam, USA). The sections were rinsed 3 times with PBS for 5 min each, and the secondary antibody was incubated for 1 h. The secondary antibody was visualized with diaminobenzidine and immersed in distilled water. Gradient alcohol dehydration, xylene transparency, and neutral gum closure. Ki-67 expression was measured via calculating the percentage of positive cells. Statistical analysis Overall survival (OS), disease-free survival (DFS) were calculated using Kaplan-Meier test. The diagnostic value of FOXP4-AS1 was determined using the receiver operating characteristic (ROC) curve. All analyses were performed using GraphPad Prism 8 software. p < 0.05 was considered a statistically significant difference. Results LncRNA FOXP4-AS1 is highly expressed in CRC To investigate the FOXP4-AS1 level in CRC, lncRNA FOXP4-AS1 level was analyzed by TCGA database, and it was discovered FOXP4-AS1 was highly expressed in CRC tissues (Fig. 1 A). The results from paired samples of CRC patients in the TCGA database also showed a high expression of FOXP4-AS1 (Fig. 1 B). ROC curve results disclosed that FOXP4-AS1 is a sensitive CRC diagnostic marker (Fig. 1 C). Survival analysis manifested that high FOXP4-AS1 was linked to shorter OS, DFS in CRC patients (Fig. 1 D, E). Subsequently, the FOXP4-AS1 levels in cancer tissues and their paracancerous normal tissues of 80 CRC patients in our hospital were measured through qRT-PCR. The high expression of FOXP4-AS1 in CRC was similarly found (Fig. 1 F). In addition, FOXP4-AS1 in the peripheral blood of CRC patients was significantly higher than that of FOXP4-AS1 in the peripheral blood of healthy volunteers (Fig. 1 G). Therefore, our study identified that FOXP4-AS1 is highly expressed in CRC and has a good diagnostic value. Knockdown of FOXP4-AS1 inhibits cell invasion, migration and EMT To evaluate the role of FOXP4-AS1 in CRC, siRNA was utilized to knockdown the FOXP4-AS1 expression in cells. After knockdown of FOXP4-AS1, FOXP4-AS1 was significantly reduced in cells (Fig. 2 A). The mRNA levels of E-cadherin and N-cadherin were measured by qRT-PCR, and it was found that knockdown of FOXP4-AS1 markedly increased the E-cadherin mRNA level in the cells, whereas the N-cadherin mRNA level was significantly decreased (Fig. 2 B, C). Western blot experiments verified this result (Fig. 2 D). The outcomes of scratch assay revealed that the migration ability of cells was obviously reduced after transfection of si-FOXP4-AS1 (Fig. 2 E, F). We also found that cell invasion ability was significantly suppressed after silencing FOXP4-AS1 (Fig. 2 G). The above findings confirmed that silencing FOXP4-AS1 weakened cell metastasis and EMT. FOXP4-AS1 interacts with ZEB1 Next, analysis by subcellular separation demonstrated that FOXP4-AS1 was primarily localized in the cytoplasm (Fig. 3 A). Thus, we hypothesized that FOXP4-AS1 may exert its function via interaction with specific proteins. To investigate the potential functions of FOXP4-AS1, positive-sense and antisense probes for FOXP4-AS1 were constructed, biotin-labeled, and subjected to RNA pull down and silver staining assay for isolation of proteins associated with FOXP4-AS1 (Fig. 3 B). Subsequently, the interaction of FOXP4-AS1 with ZEB1 was measured through western blot. The outcomes displayed that only the positive-sense probe of FOXP4-AS1 immunoprecipitated with the ZEB1 protein, whereas the negative control and antisense probe groups did not show this interaction (Fig. 3 B, C). This suggested that there is a direct target binding between FOXP4-AS1 and ZEB1 proteins. RNA pull down results revealed that more ZEB1 was enriched in FOXP4-AS1 compared to antisense (Fig. 3 D). RIP experiments disclosed that anti-ZEB1 enriched more FOXP4-AS1 compared to normal anti-IgG (Fig. 3 E). Subsequently, the FOXP4-AS1 level was overexpressed in cells and the transfection efficiency after overexpression of FOXP4-AS1 was determined through qRT-PCR. The results revealed that FOXP4-AS1 was clearly elevated in the cells after overexpression of FOXP4-AS1 (Fig. 3 F). Western blot assay revealed that ZEB1 protein level was significantly diminished or elevated in the cells after knockdown or overexpression of FOXP4-AS1 (Fig. 3 G). The above outcomes displayed that FOXP4-AS1 interacted with ZEB1. FOXP4-AS1 enhances the interaction of USP7 with ZEB1 Previously, it was shown that lncRNAs regulate protein expression through ubiquitination 12 . We wondered whether FOXP4-AS1 could inhibit the ubiquitination degradation of ZEB1, so we next searched for ubiquitination-regulating proteins that could bind to both FOXP4-AS1 and ZEB1, and identified USP7 in the USP family through literature research. The interaction of FOXP4-AS1 with USP7 was confirmed by RNA pull down and western blot experiments (Fig. 4 A). The results of RIP experiments showed that anti-USP7 enriched more FOXP4-AS1 compared to normal anti-IgG (Fig. 4 B). The interaction of USP7 with ZEB1 was subsequently verified by Co-IP (Fig. 4 C). Immunofluorescence experiments also confirmed this (Fig. 4 D). IP experiments verified that FOXP4-AS1 enhanced the interaction of USP7 with ZEB1 (Fig. 4 E). The above experiments confirmed that FOXP4-AS1 influence the strength of USP7 and ZEB1 binding. USP7 inhibitor P005091 rescues the promotion of cell invasion, migration and EMT by overexpression of FOXP4-AS1 To verify the mechanism by which FOXP4-AS1 affects CRC development via USP7, we performed rescue experiments. The results demonstrated that the USP7 inhibitor P005091 offset the influence of FOXP4-AS1 on cellular E-cadherin and N-cadherin mRNA expression (Fig. 5 A, B). Western blot results confirmed this (Fig. 5 C). Scratch and transwell assay findings proved that overexpression of FOXP4-AS1 promoted cell migration and invasion, which was rescued by the addition of USP7 inhibitor P005091 (Fig. 5 D-F). In conclusion, USP7 inhibitor P005091 returned the effects of FOXP4-AS1 overexpression on cell migration, invasion and EMT. Overexpression of ZEB1 reverses the effect of silencing FOXP4-AS1 on cell invasion, migration and EMT Next, ZEB1 was overexpressed in cells and qRT-PCR was applied to detect the transfection efficiency after ZEB1 overexpression in CRC cell lines. ZEB1 was obviously elevated in cells after overexpression of ZEB1 (Fig. 6 A). We found ZEB1 introduction abolished the repression effect of silencing FOXP4-AS1 on the mRNA and protein expression of E-cadherin and N-cadherin in cells (Fig. 6 B-D). In addition, the suppressive impact of FOXP4-AS1 silencing on cell metastasis were returned by ZEB1 overexpression (Fig. 6 E-G). Our results indicate that introduction of ZEB1 returned the effects of silencing FOXP4-AS1 on cell migration, invasion and EMT. Silencing of FOXP4-AS1 inhibits CRC tumor growth in vivo To better determine the role of FOXP4-AS1, we constructed a transplanted tumor model to evaluate the growth of CRC tumors in vivo. After transfecting Lv-shFOXP4-AS1 and Lv-shNC into nude mice for 5 weeks, tumor growth was observed. Compared with Lv-shNC, tumor growth was significantly slower in the Lv-shFOXP4-AS1 group (Fig. 7 A). The weight and volume of tumors were also significantly reduced after transfection with Lv-shFOXP4-AS1 (Fig. 7 B, C). In addition, immunohistochemical analysis results confirmed that the Ki67 level in the Lv-shFOXP4-AS1 group was clearly lower than that in the control group (Fig. 7 D). Furthermore, ZEB1 level showed the same trend(Fig. 7 E). Our results reveal that silencing FOXP4-AS1 inhibits CRC tumor growth in vivo. Discussion Currently, few therapeutic strategies are available to improve the survival of patients with advanced CRC. Therefore, elucidating their associated molecular mechanisms is critical to understanding CRC progression and clinical management. Interest in the use of lncRNAs in cancer patients has been increasing due to their high specificity and ease of testing in tissues, serum, plasma, urine, and saliva 13 . LncRNAs have biomarker potential in the diagnosis and prognosis of kinds of cancers, including cholangiocarcinomas, gastric cancers, hepatocellular carcinomas, pancreatic carcinomas, and breast cancers, and 1ncRNAs have been used to adjuvantly improve the specificity and sensitivity of existing biomarkers 14 . In this research, we confirmed that 1ncRNA FOXP4-AS1 was obviously enhanced in CRC. Subsequently, silencing of FOXP4-AS1 was found to inhibit cell migration, invasion, and EMT. These findings proved that FOXP4-AS1 plays a facilitating part in CRC development. Previous studies have shown that FOXP4-AS1 accelerates cervical cancer progression via regulating CBX4 through competitive binding to miR-136-5p 15 . Niu et al. discovered that the overexpression of FOXP4-AS1 in ESCC contributes to cancer progression through interaction with MLL2/H3K4me3 16 . Our results have similarities with previous studies. It is widely recognized that lncRNAs engage with binding factors to affect their target genes 17 . In this study, we continued to explore on this basis and found that FOXP4-AS1 interacted with ZEB1 through analysis, which was verified by a series of experiments. EMT plays a vital regulatory role in the proliferation and metastasis of cancer cells 18 . Previous literature reported that zinc finger E-box binding homeobox 1 (ZEB1) is a key transcription factor for the induction of EMT, inducing the promotion of EMT and thus cancer proliferation and metastasis 19 . ZEB1 is a member of the family of zinc finger transcription factors involved in embryonic development and formation, located on the on Chr10p 11.22, and its expression is linked to poor prognosis in a variety of malignant tumors, and modulation of its expression can regulate cancer progression 20 . For example, TRIM9 interacts with ZEB1 and promotes ZEB1 protein degradation through the ubiquitin proteasome pathway thereby inhibiting esophageal cancer progression 21 . The EMT-activating factor, ZEB1, affects cellular plasticity and mediates pancreatic cancer metastasis 22 . In addition, ZEB1 facilitates prostate cancer invasion and proliferation via the ERK1/2 23 . At this research, we disclosed that ZEB1 protein was clearly reduced or raised in cells after silencing or insertion of FOXP4-AS1. Overexpression of ZEB1 returned the suppressive effect of silencing FOXP4-AS1 on cellular E-cadherin and N-cadherin mRNA and protein expression. Furthermore, the suppressive impact of FOXP4-AS1 silencing on cell metastasis were returned by ZEB1 overexpression. Furthermore, we found an interaction between ZEB1 and USP7. Ubiquitin specific peptidase 7 (USP7) contains 1102 amino acids in its full length and is an approximately 135 kDa protein 24 . The USP7 gene is located on chromosome 16 and contains 35 exons. USP7 is a deubiquitinating enzyme that regulates important cellular functions by interacting with other proteins 25 . USP7 has been proven to act a major part in the malignant progression of several cancers, such as breast, ovarian, and prostate cancers 26 . Tang et al. demonstrated that USP7 enhanced the migration and invasion of breast cancer cells via antagonizing FBXW7-mediated degradation of ZMYND8 27 . Wang et al. found that USP7 promote the malignant progression of ovarian cancer by mediating TRAF4 deubiquitination through RSK4/PI3K/AKT, whereas knockdown of USP7 inhibited the metastasis and proliferation of ovarian cancer cells, and inhibited tumor growth in mice in vivo 28 . The USP7 inhibitor, the drug P005091, has been demonstrated to exert an anticancer effect by inhibiting the cyclic progression of cells and promoting apoptosis 29 . Therefore, scientists believe that the use of USP7 inhibitors may be a rational strategy for the treatment of CRC. In our investigation, USP7 interacted with FOXP4-AS1, and the use of USP7 inhibitor P005091 would rescue the promotion of cell migration, invasion and EMT via FOXP4-AS1 overexpression. However, there are some flaws in our study. For example, we should have recruited more volunteers to participate in the study, did not use a high-throughput screening method, and whether lncRNA FOXP4-AS1 also regulates the expression of ZEB1 through other mechanisms remains to be further investigated. In the future, we will explore further in depth. In conclusion, lncRNA FOXP4-AS1 was heightened in CRC and associated with shorter OS and DFS in CRC patients. In addition, lncRNA FOXP4-AS1 promotes CRC malignant progression by enhancing the interaction between USP7 and ZEB1, and targeting lncRNA FOXP4-AS1 has potential for the treatment of CRC patients. Declarations Competing interests The authors declare that they have no conflicts of interest to report regarding the present study. Ethics approval This study was approved by the Ethics Committee of Changshu NO.2 People’s Hospital. All participants were provided with written informed consent at the time of recruitment, and all experiments involving human tissue specimens comply with the Declaration of Helsinki. Animal studies were performed in compliance with the ARRIVE guidelines. Funding Guiding project of Jiangsu Provincial Health Commission (Z2021032), Suzhou Medical and Health science and technology Innovation Project (SKY2022022), Suzhou City clinical key disease diagnosis and treatment technology special project (LCZX202224), Changshu City Science and Technology Development Plan (CS202125), Key Laboratory of Digestive System Tumor Innovation and Diagnosis and Treatment (CS202313) Author Contribution Yichen li and Yuchen Yuan performed experiments, Chenlong Shen, Xiaoling Yang analyzed data and wrote the paper; Jiazhe Shao and Haichen Liu performed some experiments and analyzed data; Guoqiang Zhou guided the experiments and the analysis; Zhiliang Shi initiated the study, designed experiments. 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Lu, J. et al. Targeting Ubiquitin-Specific Protease 7 (USP7) in Cancer: A New Insight to Overcome Drug Resistance. Front. Pharmacol. 12 , 648491. https://doi.org/10.3389/fphar.2021.648491 (2021). Oliveira, R. I., Guedes, R. A. & Salvador, J. A. R. Highlights in USP7 inhibitors for cancer treatment. Front. Chem. 10 , 1005727. https://doi.org/10.3389/fchem.2022.1005727 (2022). Tang, K. et al. USP7 deubiquitinates epigenetic reader ZMYND8 to promote breast cancer cell migration and invasion. J. Biol. Chem. 300 (9), 107672. https://doi.org/10.1016/j.jbc.2024.107672 (2024). Wang, Y. et al. USP7 mediates TRAF4 deubiquitination to facilitate the malignant phenotype of ovarian cancer via the RSK4/PI3K/AKT axis. J. Cancer Res. Ther. 19 (1), 97–107. https://doi.org/10.4103/jcrt.jcrt_517_22 (2023). Lee, J. E., Park, C. M. & Kim, J. H. USP7 deubiquitinates and stabilizes EZH2 in prostate cancer cells. Genet. Mol. Biol. 43 (2). https://doi.org/10.1590/1678-4685-gmb-2019-0338 (2020). e20190338. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 22 Jul, 2025 Reviews received at journal 11 Jul, 2025 Reviewers agreed at journal 11 Jul, 2025 Reviews received at journal 16 Jun, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers invited by journal 16 Jun, 2025 Editor assigned by journal 16 Jun, 2025 Editor invited by journal 03 Jun, 2025 Submission checks completed at journal 02 Jun, 2025 First submitted to journal 18 May, 2025 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-6691801","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472521454,"identity":"6f35c90a-92fb-4003-baac-317e4e4b449d","order_by":0,"name":"Xiaoling Yang","email":"","orcid":"","institution":"Changshu Hospital affiliated to Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Yang","suffix":""},{"id":472521455,"identity":"61afab88-76a3-40de-97b9-2e562064ba8d","order_by":1,"name":"Chenglong Shen","email":"","orcid":"","institution":"Changshu Hospital affiliated to Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Chenglong","middleName":"","lastName":"Shen","suffix":""},{"id":472521456,"identity":"723bb1ce-a6a6-445a-be44-74fb66c4c55d","order_by":2,"name":"Yuchen Yuan","email":"","orcid":"","institution":"Meili People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Yuan","suffix":""},{"id":472521457,"identity":"4e489dc4-6b26-466c-a94e-5c3dfe0695f4","order_by":3,"name":"Jiazhe Shao","email":"","orcid":"","institution":"Changshu Hospital affiliated to Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Jiazhe","middleName":"","lastName":"Shao","suffix":""},{"id":472521459,"identity":"e5261289-bcd5-4f9b-b318-cbb6c4076af4","order_by":4,"name":"Haichen Liu","email":"","orcid":"","institution":"Changshu Hospital affiliated to Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Haichen","middleName":"","lastName":"Liu","suffix":""},{"id":472521460,"identity":"bdebdf56-4cf2-4b54-a73d-eb43c9d8269b","order_by":5,"name":"Yichen Li","email":"","orcid":"","institution":"Changshu Hospital affiliated to Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yichen","middleName":"","lastName":"Li","suffix":""},{"id":472521461,"identity":"3eb2f965-a9ed-4142-a380-ba2dbeb73ee0","order_by":6,"name":"Guoqiang Zhou","email":"","orcid":"","institution":"Changshu Hospital affiliated to Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Guoqiang","middleName":"","lastName":"Zhou","suffix":""},{"id":472521462,"identity":"dce8c3ba-4101-44ea-9f1b-11af15588e81","order_by":7,"name":"Zhiliang Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDCCA1Caj70BTDM2EKEFooiN5wCYQYoWiQQitfAdb37+4OOeOns2ybfHH/Mw2MhuOMD87AE+LZJnjhk2znh2mJlNOi+xmYchzXjDATZzA3xaDG7kMDbzHDjAxiadYwjUcjhxwwEeNgkitNTxsEmeAWn5T7QWZgk2CR6QlgOEtYD8MnPGgcMGbDw5hjPnGCQbzzzMZoZXCzDEHnz4cKDOnp/9jMGHNxV2sn3Hm5/h1YLuTiBmJkH9KBgFo2AUjALsAABNrUelb8JF7AAAAABJRU5ErkJggg==","orcid":"","institution":"Changshu Hospital affiliated to Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Zhiliang","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-05-18 12:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6691801/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6691801/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-34903-6","type":"published","date":"2026-01-04T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84881596,"identity":"b1d90daf-d9f3-4340-99d7-a468b66e364d","added_by":"auto","created_at":"2025-06-18 10:59:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9042765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLncRNA FOXP4-AS1 is highly expressed in CRC.\u003c/strong\u003e (A) Expression of lncRNA FOXP4-AS1 in TCGA colorectal cancer (CRC) tissues; (B) Expression of FOXP4-AS1 in paired samples of TCGA CRC patients; (C) ROC curves of FOXP4-AS1 in TCGA CRC patients; (D) GEPIA analysis of FOXP4-AS1 in CRC; (E) GEPIA analysis of disease-free survival of FOXP4-AS1 in CRC; (F) qRT-PCR was used to detect the level of lncRNA FOXP4-AS1 in the cancerous tissues and their paracancerous normal tissues of 80 CRC patients in our hospital; (G) qRT-PCR was used to detect the level of lncRNA FOXP4-AS1 in the peripheral blood of 80 CRC patients and 80 healthy volunteers in our hospital.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/9b17b12417b01c0459f38728.jpg"},{"id":84881603,"identity":"954798b7-79b8-410b-a5ea-7f22fa56c5de","added_by":"auto","created_at":"2025-06-18 10:59:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14519161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of FOXP4-AS1 inhibits cell invasion, migration and EMT. \u003c/strong\u003e(A) qRT-PCR was used to detect the transfection efficiency after knockdown of FOXP4-AS1 in CRC cell lines; (B+C) qRT-PCR was used to detect the mRNA levels of E-cadherin and N-cadherin; (D) Western blot was used to detect the protein levels; (E+F) Scratch assay was used to detect cell metastasis; (G) Transwell assay was used to detect cell invasion.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/cfd5cad6c9ad203a01fd83a3.jpg"},{"id":84881600,"identity":"380527ee-2b37-4cf2-bd5b-1875828415e8","added_by":"auto","created_at":"2025-06-18 10:59:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6151956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXP4-AS1 interacts with ZEB1.\u003c/strong\u003e (A) qRT-PCR was used to detect the localization of FOXP4-AS1 in cells after nucleoplasmic isolation; (B) SDS-PAGE was applied to isolate the pull down protein of FOXP4-AS1 after silver staining; (C) Western blot was used to detect the interaction between FOXP4-AS1 and ZEB1 (D) qRT-PCR was used to detect the FOXP4-AS1 interaction with ZEB1; (E) RIP and qRT-PCR was used to measure the interaction of FOXP4-AS1 with ZEB1; (F) qRT-PCR was used to detect the transfection efficiency after overexpression of FOXP4-AS1; (G) Western blot was used to detect the effect of knockdown or overexpression of FOXP4-AS1 on ZEB1 protein.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/1d65a1f49cb98cf357eca969.jpg"},{"id":84881597,"identity":"b5d3d708-ca1c-42a0-9530-8321a5fe9e64","added_by":"auto","created_at":"2025-06-18 10:59:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2084397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXP4-AS1 enhances the interaction of USP7 with ZEB1.\u003c/strong\u003e (A) RNA pull down and western blot was used to detect the interaction of FOXP4-AS1 with USP7; (B) RIP and qRT-PCR was used to detect the interaction of FOXP4-AS1 with USP7; (C) CO-IP was used to verify the interaction of USP7 with ZEB1; (D) Immunofluorescence was used to verify the interaction of USP7 interaction with ZEB1; (E) IP experiments was used to validate that FOXP4-AS1 enhanced USP7 interaction with ZEB1.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/d2861062d4c7c6349338e234.jpg"},{"id":84881612,"identity":"3843ae00-230e-4af7-92f4-3af02c228e10","added_by":"auto","created_at":"2025-06-18 10:59:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22926634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP7 inhibitor P005091 rescues the promotion of cell invasion, migration and EMT by overexpression of FOXP4-AS1.\u003c/strong\u003e (A+B) qRT-PCR was used to detect the effects of overexpression of FOXP4-AS1 in CRC cell lines and further addition of USP7 inhibitor P005091 on the mRNA levels of E-cadherin and N-cadherin; (C) Western blot was used to detect the E-cadherin and N-cadherin cadherin protein levels; (D+E) Scratch assay was used to detect cell metastatic ability; (F) Transwell assay was used to detect cell invasion ability.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/0ffb13d552ef4b0745073ce1.jpg"},{"id":84881604,"identity":"b5720a4e-f7c7-473f-8c18-31c8425127e2","added_by":"auto","created_at":"2025-06-18 10:59:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16991395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of ZEB1 reverses the effect of silencing FOXP4-AS1 on cell invasion, migration and EMT.\u003c/strong\u003e (A) qRT-PCR was used to detect the transfection efficiency after overexpression of ZEB1 in CRC cell lines; (B+C) qRT-PCR was applied to detect the mRNA levels of E-cadherin and N-cadherin; (D) Western blot was used to detect the protein levels of E-cadherin and N-cadherin; ( E+F) Scratch assay was used to detect cell metastatic ability; (G) Transwell assay was used to detect cell invasion ability.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/5efc326ee1ff05f9095df1a8.jpg"},{"id":84881605,"identity":"7eda0100-3284-469d-ba3c-14dc8294ccda","added_by":"auto","created_at":"2025-06-18 10:59:36","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5708954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing of FOXP4-AS1 inhibits CRC tumor growth in vivo.\u003c/strong\u003e (A) Constructing the transplanted tumor model and observing the growth of the tumor; (B) Weighing the tumor; (C) Measuring the tumor volume; (D) Immunohistochemistry was used to detect the expression of Ki67; (E) Immunohistochemistry was used to detect the expression of ZEB1.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/ad0f1eaa86642e0f10cbeaa5.jpg"},{"id":99545611,"identity":"c8a909da-ec5c-408e-b67d-c668f6bd0fa8","added_by":"auto","created_at":"2026-01-05 16:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":78427154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6691801/v1/8dfc2294-f2a3-4922-8789-e400fdd3ade0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LncRNA FOXP4-AS1 facilitates colorectal cancer invasion and migration by enhancing USP7 interaction with ZEB1 Running title: LncRNA FOXP4-AS1 raises CRC invasion and migration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is one of the common and serious malignant tumors threatening human life and health, with the third highest incidence and second highest mortality rate among various malignant tumors worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Multiple risk factors are associated with the development of the disease, such as, genetic inheritance and poor lifestyle habits\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Currently, the clinical treatment of CRC is mainly a comprehensive treatment based on surgery, and there is a significant improvement in the clinical outcome of CRC. However, the treatment of patients with advanced stage, post-surgical recurrence, and resistance to radiotherapy is still the difficulty of clinical treatment, and the 5-year survival rate has not been obviously improved\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, finding new early diagnostic markers for CRC, elucidating the mechanisms of CRC development, and identifying new therapeutic targets are the keys to inhibiting CRC tumor progression and reducing recurrence and mortality.\u003c/p\u003e \u003cp\u003eThe development of malignant tumors is an extremely complex biological phenomenon that is multifactorial, multigenic, and undergoes multiple stages before it finally develops. Both inactivation and activation of oncogenes cause genetic alterations thereby affecting various aspects of tumors, such as proliferation, invasion, metastasis and material metabolism\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Previous studies on tumor-related genes have been focused on coding protein genes, and with the continuous development and improvement of technologies such as high-throughput sequencing, a large number of long fragments of non-coding RNAs have been uncovered, and their significance in life activities has been gradually revealed\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Long noncoding RNAs (lncRNAs) are a class of molecules that do not encode proteins but have biological functions. LncRNAs are dysregulated in a large of human diseases, including CRC, and their dysregulation is intimately related to the progression of the disease, suggesting that they are potentially clinically useful\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. LncRNAs act through kinds of mechanisms, mainly by interacting with DNA, miRNAs, mRNAs and proteins to interact and regulate gene transcription, mRNA stability and translation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. For example, lncRNA RMRP facilitates growth and metastasis of bladder cancer via miR-206\u003csup\u003e8\u003c/sup\u003e. Also, lncRNA BREA2 facilitates breast cancer metastasis via disrupting WWP2-mediated ubiquitination of Notch1\u003csup\u003e9\u003c/sup\u003e. LncRNA FOXP4-AS1, a new tumor-associated biomarker discovered in recent years, has been shown to be aberrantly expressed in malignant tumors, such as pancreatic, esophageal squamous cell carcinoma and nasopharyngeal carcinoma, and is capable of affecting a variety of biological functions in tumor cells. Liang et al. found FOXP4-AS1 is a poor prognostic biomarker for hepatocellular carcinoma and regulates the proliferation, invasion and angiogenesis of hepatocellular carcinoma cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Li et al. showed that FOXP4-AS1 is important in the progression of CRC and is linked with cell proliferation and apoptosis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the exact mechanism by which LncRNA FOXP4-AS1 acts a role in CRC remains to be elucidated. Therefore, a more comprehensive understanding of the role of FOXP4-AS1 in CRC progression is expected to provide new perspectives for the diagnosis and treatment of this malignant tumor.\u003c/p\u003e \u003cp\u003eWith the above research background, this research aimed to investigate the mechanism of lncRNA FOXP4-AS1 in CRC. FOXP4-AS1 was enhanced in CRC tissues by TCGA database analysis. The high FOXP4-AS1 was further discovered to be linked to shorter overall survival (OS), disease-free survival (DFS) in CRC patients. FOXP4-AS1 was heightened in CRC tissues by analyzing specimens collected from CRC patients in the clinic. Cellular experiments revealed that down-regulation of FOXP4-AS1 suppressed cell metastasis and EMT. Mechanistically, our study displayed that FOXP4-AS1 enhanced the interaction of USP7 with ZEB1. The successful completion of this study will provide a new treatment option for CRC patients.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAcquisition of clinical specimens\u003c/h2\u003e \u003cp\u003eBlood samples were collected from our CRC patients and healthy volunteers. Clinical tissue specimens were obtained from patients who underwent surgery in our hospital. All patients at the hospital signed a written informed consent. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Changshu NO.2 People\u0026rsquo;s Hospital.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHuman colon cancer cell lines HCT116, SW480 were obtained from the American Typical Culture Collection Center (ATCC, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco). All cells were cultured in 5% CO\u003csub\u003e2\u003c/sub\u003e humidified environment at 37\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eCell transfection\u003c/h3\u003e\n\u003cp\u003eFOXP4-AS1 small interfering RNA (si-FOXP4-AS1#1/2) or overexpression of FOXP4-AS1 or ZEB1 vectors (pcDNA FOXP4-AS1 or ZEB1) and their corresponding controls were designed by GenePharma. FOXP4-AS1, ZEB1 were transfected through Lipofectamine 3000 (Thermo Fisher Scientific). To generate lentivirus, transfections were inoculated into 24-well plates for incubation, followed by addition of 0.5 mL of medium containing 8 \u0026micro;g/ml Polybrene (Sigma, USA) to each well and transfection of CRC cells. 48 h later, the complete medium was changed to 2 \u0026micro;g/mL puromycin, and screening was continued for 2 weeks to obtain stably transfected cell lines.\u003c/p\u003e\n\u003ch3\u003eCell scratch healing assay\u003c/h3\u003e\n\u003cp\u003eThree parallel marking lines were scribed on the back of the 6-well plate with a marker pen in advance, and the cell suspension was prepared, the cell concentration was adjusted, and placed in the incubator at 37\u0026deg;C. After 24 h, the cells were scribed with the tip of the gun of 200 \u0026micro;L perpendicularly to the 6-well plate and perpendicularly to the marking line, and the point of intersection of the scribed and marking line was the observation point, and the cells were washed with PBS for 3 times. The cells were washed with PBS, and the serum-free RPMI 1640 or DMEM medium was used for normal fluid exchange, and the observation point was photographed by the microscope at 0, 24 h of incubation.\u003c/p\u003e\n\u003ch3\u003eCell invasion assay\u003c/h3\u003e\n\u003cp\u003eMatrigel coated chambers with 8 \u0026micro;m pores were used. 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well were resuspended in 200 \u0026micro;L of serum-free DMEM medium added to the upper layer of the chambers, and medium containing 10% serum was added to the lower chamber and incubated for 48 h. After incubation, the cells were fixed with 600 \u0026micro;L of 4% paraformaldehyde for 30 min and stained with 600 \u0026micro;L of 0.1% crystal violet for 30 min. The uninvaded cells were gently wiped off with a cotton swab in the upper chamber, and the membrane of the transwell chambers was scraped off with a knife and fixed on slides, and the invaded cells were photographed with an inverted microscope (Olympus) and counted.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA of the cells and peripheral blood were extracted using Trizol reagent. 2 \u0026micro;g of total RNA extract was used to synthesize cDNA based on the cDNA reverse transcription kit ReverTra Ace qPCR RT. cDNA was used as a template for synthesizing cDNA based on the standard instructions of qRT-PCR fluorescence quantification kit (Toyobo, Japan) standard instructions for qPCR amplification reaction.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNucleocytoplasmic separation assay\u003c/h3\u003e\n\u003cp\u003eNucleocytoplasmic separation assay was applied to measure the cellular localization of FOXP4-AS1. Cells were digested with 0.25% trypsin (Solarbio, China) and centrifuged at 1,000 g for 3 min. RNA was extracted from nuclei and cytoplasm and subjected to qRT-PCR based on the instructions of the Nucleocytoplasmic Isolation Kit (Norgen Biotek, Canada). U2 and S14 were used as internal reference genes.\u003c/p\u003e\n\u003ch3\u003eWestern blot experiments\u003c/h3\u003e\n\u003cp\u003eEach group of CRC cell lines was taken separately and total protein was extracted from each group of cells by cell lysate. Protein samples were separated by electrophoresis and then transferred to PVDF membranes, which were blocked with 5% skimmed milk for 1 h. The membranes were incubated with a primary (1: 2000) antibody at 4\u0026deg;C overnight. On the following day, the membrane was incubated with HRP-coupled secondary antibody (1: 5000) for 2 h. Then, ECL luminescent chemistry was added to allow the bands to be developed and analyzed and processed by a Rio-Rad gel imaging system.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA pull down assay\u003c/h2\u003e \u003cp\u003eIn vitro transcripts of FOXP4-AS1 were labeled using biotin (Thermo Fisher). Then, biotin-labeled RNA was captured using streptavidin magnetic beads (Invitrogen, USA), and then the RNA beads were incubated with cell lysates. After washing out unbound proteins, the RNA-protein complexes were eluted from the magnetic beads by SDS-PAGE upsampling buffer. Proteins bound to FOXP4-AS1 were detected by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE silver stain assay\u003c/h2\u003e \u003cp\u003eExperiments were performed using a protein silver staining kit (Solarbio, China). The PAGE protein gel was fixed with fixative for 30 min, and the gel was transferred to sensitization solution for 30 min, after which it was rinsed with deionized water. The gel was transferred to the silver staining solution for 30 min, during which it was shaken continuously with a horizontal shaker, and finally the gel was transferred to the color development solution and displayed for 5 min. Subsequently, the color development solution was discarded and the termination solution was added to terminate the color development. The silver-stained gel was photographed with a protein imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP)\u003c/h2\u003e \u003cp\u003eThe RIP assay was performed according to the instructions of the EZ-Magna-RIP kit (Millipore, USA). Cells were lysed with lysis buffer. Cell lysates were then pre-washed with recombinant protein A/G agarose for 30 min at 4\u0026deg;C. Equal amounts of cell lysates were incubated with IgG or ZEB1 antibodies overnight. Capture RNA protein/antibody complexes were incubated with recombinant protein A/G agarose. RNA was eluted from the precipitated complexes and transcribed into cDNA. Binding of RNA to protein or antibody was detected by qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP) and IP assay\u003c/h2\u003e \u003cp\u003eCells were lysed on ice for 30 min using RIPA buffer containing protease inhibitors. The supernatant was collected. USP7, ZEB1, or IgG antibodies were added to the supernatant and incubated overnight. Then, Protein A agarose beads (10 \u0026micro;L) were pre-treated by lysis buffer (Beyotime), and then mixed with the cell lysate and antibody complexes, and the antibodies were allowed to affix to the Protein A agarose beads by slow shaking for 2 h at 4\u0026deg;C. After the immunoprecipitation reaction, the complex was centrifuged at 3,000 rpm for 3 min. The supernatant was discarded. Finally, 15 \u0026micro;L of 2\u0026times;SDS Sampling Buffer was added and boiled for 5 min. The precipitated proteins were then analyzed by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003e4\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were cultured on coverslips in 24-well plates. Cells were then fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min. Then blocked with 5% BSA for 1 h, and incubated with primary antibody overnight. Cells were then rinsed with PBST and treated with the appropriate secondary antibody (1:1000). After incubation with DAPI (KGI, China), coverslips were mounted on slides and scanned serially with a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNude mouse xenografts\u003c/h2\u003e \u003cp\u003eTwelve BALB/c nude mice weighing 17\u0026ndash;20 g and aged 4\u0026ndash;6 weeks were purchased from the Shanghai Laboratory Animal Center. 2\u0026times;10\u003csup\u003e7\u003c/sup\u003e lentivirus-infected cells in serum-free DMEM were injected subcutaneously into mice, which were divided into the Lv-sh-NC group and the Lv-sh-FOXP4-AS1 group. After 5 weeks, the tumors were removed, photographed, and measured with vernier calipers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue sections were deparaffinized, closed with 3% hydrogen peroxide for 15 min, washed with PBS, closed with 10% normal goat serum for 30 min, and incubated overnight with the addition of primary antibody Ki-67 (1: 5000, Abcam, USA). The sections were rinsed 3 times with PBS for 5 min each, and the secondary antibody was incubated for 1 h. The secondary antibody was visualized with diaminobenzidine and immersed in distilled water. Gradient alcohol dehydration, xylene transparency, and neutral gum closure. Ki-67 expression was measured via calculating the percentage of positive cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eOverall survival (OS), disease-free survival (DFS) were calculated using Kaplan-Meier test. The diagnostic value of FOXP4-AS1 was determined using the receiver operating characteristic (ROC) curve. All analyses were performed using GraphPad Prism 8 software. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLncRNA FOXP4-AS1 is highly expressed in CRC\u003c/h2\u003e \u003cp\u003eTo investigate the FOXP4-AS1 level in CRC, lncRNA FOXP4-AS1 level was analyzed by TCGA database, and it was discovered FOXP4-AS1 was highly expressed in CRC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results from paired samples of CRC patients in the TCGA database also showed a high expression of FOXP4-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). ROC curve results disclosed that FOXP4-AS1 is a sensitive CRC diagnostic marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Survival analysis manifested that high FOXP4-AS1 was linked to shorter OS, DFS in CRC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Subsequently, the FOXP4-AS1 levels in cancer tissues and their paracancerous normal tissues of 80 CRC patients in our hospital were measured through qRT-PCR. The high expression of FOXP4-AS1 in CRC was similarly found (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In addition, FOXP4-AS1 in the peripheral blood of CRC patients was significantly higher than that of FOXP4-AS1 in the peripheral blood of healthy volunteers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Therefore, our study identified that FOXP4-AS1 is highly expressed in CRC and has a good diagnostic value.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of FOXP4-AS1 inhibits cell invasion, migration and EMT\u003c/h2\u003e \u003cp\u003eTo evaluate the role of FOXP4-AS1 in CRC, siRNA was utilized to knockdown the FOXP4-AS1 expression in cells. After knockdown of FOXP4-AS1, FOXP4-AS1 was significantly reduced in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The mRNA levels of E-cadherin and N-cadherin were measured by qRT-PCR, and it was found that knockdown of FOXP4-AS1 markedly increased the E-cadherin mRNA level in the cells, whereas the N-cadherin mRNA level was significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Western blot experiments verified this result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The outcomes of scratch assay revealed that the migration ability of cells was obviously reduced after transfection of si-FOXP4-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). We also found that cell invasion ability was significantly suppressed after silencing FOXP4-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The above findings confirmed that silencing FOXP4-AS1 weakened cell metastasis and EMT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFOXP4-AS1 interacts with ZEB1\u003c/h2\u003e \u003cp\u003eNext, analysis by subcellular separation demonstrated that FOXP4-AS1 was primarily localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Thus, we hypothesized that FOXP4-AS1 may exert its function via interaction with specific proteins. To investigate the potential functions of FOXP4-AS1, positive-sense and antisense probes for FOXP4-AS1 were constructed, biotin-labeled, and subjected to RNA pull down and silver staining assay for isolation of proteins associated with FOXP4-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Subsequently, the interaction of FOXP4-AS1 with ZEB1 was measured through western blot. The outcomes displayed that only the positive-sense probe of FOXP4-AS1 immunoprecipitated with the ZEB1 protein, whereas the negative control and antisense probe groups did not show this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). This suggested that there is a direct target binding between FOXP4-AS1 and ZEB1 proteins. RNA pull down results revealed that more ZEB1 was enriched in FOXP4-AS1 compared to antisense (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). RIP experiments disclosed that anti-ZEB1 enriched more FOXP4-AS1 compared to normal anti-IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Subsequently, the FOXP4-AS1 level was overexpressed in cells and the transfection efficiency after overexpression of FOXP4-AS1 was determined through qRT-PCR. The results revealed that FOXP4-AS1 was clearly elevated in the cells after overexpression of FOXP4-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Western blot assay revealed that ZEB1 protein level was significantly diminished or elevated in the cells after knockdown or overexpression of FOXP4-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The above outcomes displayed that FOXP4-AS1 interacted with ZEB1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eFOXP4-AS1 enhances the interaction of USP7 with ZEB1\u003c/h2\u003e \u003cp\u003ePreviously, it was shown that lncRNAs regulate protein expression through ubiquitination \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We wondered whether FOXP4-AS1 could inhibit the ubiquitination degradation of ZEB1, so we next searched for ubiquitination-regulating proteins that could bind to both FOXP4-AS1 and ZEB1, and identified USP7 in the USP family through literature research. The interaction of FOXP4-AS1 with USP7 was confirmed by RNA pull down and western blot experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The results of RIP experiments showed that anti-USP7 enriched more FOXP4-AS1 compared to normal anti-IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The interaction of USP7 with ZEB1 was subsequently verified by Co-IP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Immunofluorescence experiments also confirmed this (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). IP experiments verified that FOXP4-AS1 enhanced the interaction of USP7 with ZEB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The above experiments confirmed that FOXP4-AS1 influence the strength of USP7 and ZEB1 binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eUSP7 inhibitor P005091 rescues the promotion of cell invasion, migration and EMT by overexpression of FOXP4-AS1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo verify the mechanism by which FOXP4-AS1 affects CRC development via USP7, we performed rescue experiments. The results demonstrated that the USP7 inhibitor P005091 offset the influence of FOXP4-AS1 on cellular E-cadherin and N-cadherin mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Western blot results confirmed this (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Scratch and transwell assay findings proved that overexpression of FOXP4-AS1 promoted cell migration and invasion, which was rescued by the addition of USP7 inhibitor P005091 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). In conclusion, USP7 inhibitor P005091 returned the effects of FOXP4-AS1 overexpression on cell migration, invasion and EMT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression of ZEB1 reverses the effect of silencing FOXP4-AS1 on cell invasion, migration and EMT\u003c/h2\u003e \u003cp\u003eNext, ZEB1 was overexpressed in cells and qRT-PCR was applied to detect the transfection efficiency after ZEB1 overexpression in CRC cell lines. ZEB1 was obviously elevated in cells after overexpression of ZEB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We found ZEB1 introduction abolished the repression effect of silencing FOXP4-AS1 on the mRNA and protein expression of E-cadherin and N-cadherin in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). In addition, the suppressive impact of FOXP4-AS1 silencing on cell metastasis were returned by ZEB1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G). Our results indicate that introduction of ZEB1 returned the effects of silencing FOXP4-AS1 on cell migration, invasion and EMT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eSilencing of FOXP4-AS1 inhibits CRC tumor growth in vivo\u003c/h2\u003e \u003cp\u003eTo better determine the role of FOXP4-AS1, we constructed a transplanted tumor model to evaluate the growth of CRC tumors in vivo. After transfecting Lv-shFOXP4-AS1 and Lv-shNC into nude mice for 5 weeks, tumor growth was observed. Compared with Lv-shNC, tumor growth was significantly slower in the Lv-shFOXP4-AS1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The weight and volume of tumors were also significantly reduced after transfection with Lv-shFOXP4-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). In addition, immunohistochemical analysis results confirmed that the Ki67 level in the Lv-shFOXP4-AS1 group was clearly lower than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Furthermore, ZEB1 level showed the same trend(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Our results reveal that silencing FOXP4-AS1 inhibits CRC tumor growth in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, few therapeutic strategies are available to improve the survival of patients with advanced CRC. Therefore, elucidating their associated molecular mechanisms is critical to understanding CRC progression and clinical management. Interest in the use of lncRNAs in cancer patients has been increasing due to their high specificity and ease of testing in tissues, serum, plasma, urine, and saliva\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. LncRNAs have biomarker potential in the diagnosis and prognosis of kinds of cancers, including cholangiocarcinomas, gastric cancers, hepatocellular carcinomas, pancreatic carcinomas, and breast cancers, and 1ncRNAs have been used to adjuvantly improve the specificity and sensitivity of existing biomarkers\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In this research, we confirmed that 1ncRNA FOXP4-AS1 was obviously enhanced in CRC. Subsequently, silencing of FOXP4-AS1 was found to inhibit cell migration, invasion, and EMT. These findings proved that FOXP4-AS1 plays a facilitating part in CRC development. Previous studies have shown that FOXP4-AS1 accelerates cervical cancer progression via regulating CBX4 through competitive binding to miR-136-5p\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Niu et al. discovered that the overexpression of FOXP4-AS1 in ESCC contributes to cancer progression through interaction with MLL2/H3K4me3\u003csup\u003e16\u003c/sup\u003e. Our results have similarities with previous studies.\u003c/p\u003e \u003cp\u003eIt is widely recognized that lncRNAs engage with binding factors to affect their target genes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In this study, we continued to explore on this basis and found that FOXP4-AS1 interacted with ZEB1 through analysis, which was verified by a series of experiments. EMT plays a vital regulatory role in the proliferation and metastasis of cancer cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Previous literature reported that zinc finger E-box binding homeobox 1 (ZEB1) is a key transcription factor for the induction of EMT, inducing the promotion of EMT and thus cancer proliferation and metastasis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. ZEB1 is a member of the family of zinc finger transcription factors involved in embryonic development and formation, located on the on Chr10p 11.22, and its expression is linked to poor prognosis in a variety of malignant tumors, and modulation of its expression can regulate cancer progression\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. For example, TRIM9 interacts with ZEB1 and promotes ZEB1 protein degradation through the ubiquitin proteasome pathway thereby inhibiting esophageal cancer progression\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The EMT-activating factor, ZEB1, affects cellular plasticity and mediates pancreatic cancer metastasis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In addition, ZEB1 facilitates prostate cancer invasion and proliferation via the ERK1/2\u003csup\u003e23\u003c/sup\u003e. At this research, we disclosed that ZEB1 protein was clearly reduced or raised in cells after silencing or insertion of FOXP4-AS1. Overexpression of ZEB1 returned the suppressive effect of silencing FOXP4-AS1 on cellular E-cadherin and N-cadherin mRNA and protein expression. Furthermore, the suppressive impact of FOXP4-AS1 silencing on cell metastasis were returned by ZEB1 overexpression. Furthermore, we found an interaction between ZEB1 and USP7.\u003c/p\u003e \u003cp\u003eUbiquitin specific peptidase 7 (USP7) contains 1102 amino acids in its full length and is an approximately 135 kDa protein\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The USP7 gene is located on chromosome 16 and contains 35 exons. USP7 is a deubiquitinating enzyme that regulates important cellular functions by interacting with other proteins\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. USP7 has been proven to act a major part in the malignant progression of several cancers, such as breast, ovarian, and prostate cancers\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Tang et al. demonstrated that USP7 enhanced the migration and invasion of breast cancer cells via antagonizing FBXW7-mediated degradation of ZMYND8\u003csup\u003e27\u003c/sup\u003e. Wang et al. found that USP7 promote the malignant progression of ovarian cancer by mediating TRAF4 deubiquitination through RSK4/PI3K/AKT, whereas knockdown of USP7 inhibited the metastasis and proliferation of ovarian cancer cells, and inhibited tumor growth in mice in vivo\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The USP7 inhibitor, the drug P005091, has been demonstrated to exert an anticancer effect by inhibiting the cyclic progression of cells and promoting apoptosis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, scientists believe that the use of USP7 inhibitors may be a rational strategy for the treatment of CRC. In our investigation, USP7 interacted with FOXP4-AS1, and the use of USP7 inhibitor P005091 would rescue the promotion of cell migration, invasion and EMT via FOXP4-AS1 overexpression.\u003c/p\u003e \u003cp\u003eHowever, there are some flaws in our study. For example, we should have recruited more volunteers to participate in the study, did not use a high-throughput screening method, and whether lncRNA FOXP4-AS1 also regulates the expression of ZEB1 through other mechanisms remains to be further investigated. In the future, we will explore further in depth.\u003c/p\u003e \u003cp\u003eIn conclusion, lncRNA FOXP4-AS1 was heightened in CRC and associated with shorter OS and DFS in CRC patients. In addition, lncRNA FOXP4-AS1 promotes CRC malignant progression by enhancing the interaction between USP7 and ZEB1, and targeting lncRNA FOXP4-AS1 has potential for the treatment of CRC patients.\u003c/p\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest to report regarding the present study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003eThis study was approved by the Ethics Committee of Changshu NO.2 People\u0026rsquo;s Hospital. All participants were provided with written informed consent at the time of recruitment, and all experiments involving human tissue specimens comply with the Declaration of Helsinki. Animal studies were performed in compliance with the ARRIVE guidelines.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eGuiding project of Jiangsu Provincial Health Commission (Z2021032), Suzhou Medical and Health science and technology Innovation Project (SKY2022022), Suzhou City clinical key disease diagnosis and treatment technology special project (LCZX202224), Changshu City Science and Technology Development Plan (CS202125), Key Laboratory of Digestive System Tumor Innovation and Diagnosis and Treatment (CS202313)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYichen li and Yuchen Yuan performed experiments, Chenlong Shen, Xiaoling Yang analyzed data and wrote the paper; Jiazhe Shao and Haichen Liu performed some experiments and analyzed data; Guoqiang Zhou guided the experiments and the analysis; Zhiliang Shi initiated the study, designed experiments. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e (3), 229\u0026ndash;263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3322/caac.21834\u003c/span\u003e\u003cspan address=\"10.3322/caac.21834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao, Q. et al. 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Biol.\u003c/em\u003e \u003cb\u003e43\u003c/b\u003e (2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/1678-4685-gmb-2019-0338\u003c/span\u003e\u003cspan address=\"10.1590/1678-4685-gmb-2019-0338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020). e20190338.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LncRNA FOXP4-AS1, USP7, ZEB1, colorectal cancer","lastPublishedDoi":"10.21203/rs.3.rs-6691801/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6691801/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) poses a threat to the health of people worldwide. Long noncoding RNAs (lncRNAs) have been reported to play a key role in regulating carcinogenesis, including CRC. In this study, the levels of lncRNA forkhead box P4 antisense RNA 1 (FOXP4-AS1) were analyzed in CRC cell lines and normal cell lines using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) method. The effects of FOXP4-AS1 on CRC cell metastasis were investigated. Then, the silver staining assay, western blot, RIP, Co-IP, and immunofluorescence were used to explore and validate the molecular mechanisms by which FOXP4-AS1 affects CRC progression. We discovered that FOXP4-AS1 expression was significantly elevated in CRC tissues and cell lines. Functionally, knockdown of FOXP4-AS1 expression inhibited CRC cell migration and invasion. In addition, silencing FOXP4-AS1 weakened CRC tumor growth \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, we identified that FOXP4-AS1 enhanced the interaction of USP7 with ZEB1. Rescue experiments demonstrated that USP7 inhibitor P005091 rescued the promotion of cell migration, invasion and EMT by overexpression of FOXP4-AS1. Furthermore, ZEB1 overexpression reversed the impact of silencing FOXP4-AS1 on cell migration, invasion and EMT. LncRNA FOXP4-AS1 accelerates CRC malignant progression by strengthening the interaction between USP7 and ZEB1.\u003c/p\u003e","manuscriptTitle":"LncRNA FOXP4-AS1 facilitates colorectal cancer invasion and migration by enhancing USP7 interaction with ZEB1 Running title: LncRNA FOXP4-AS1 raises CRC invasion and migration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 10:59:31","doi":"10.21203/rs.3.rs-6691801/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-22T14:26:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-11T20:08:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148066609198523217193322860025948475236","date":"2025-07-11T19:56:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T15:44:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63896544386004184773186629153635255732","date":"2025-06-16T14:54:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-16T13:49:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-16T13:10:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-03T11:38:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-02T07:39:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-18T12:49:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e21f5f07-02e4-41b3-86b9-1a4523498ff4","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50179933,"name":"Biological sciences/Cancer"},{"id":50179934,"name":"Biological sciences/Cell biology"},{"id":50179935,"name":"Biological sciences/Molecular biology"},{"id":50179936,"name":"Health sciences/Biomarkers"},{"id":50179937,"name":"Health sciences/Gastroenterology"},{"id":50179938,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-01-05T16:06:34+00:00","versionOfRecord":{"articleIdentity":"rs-6691801","link":"https://doi.org/10.1038/s41598-025-34903-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-04 15:57:27","publishedOnDateReadable":"January 4th, 2026"},"versionCreatedAt":"2025-06-18 10:59:31","video":"","vorDoi":"10.1038/s41598-025-34903-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34903-6","workflowStages":[]},"version":"v1","identity":"rs-6691801","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6691801","identity":"rs-6691801","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}