Downregulation of HOPX promotes the invasion and migration abilities of hepatocellular carcinoma cells through EMT-related transcription factor SNAIL

Downregulation of HOPX promotes the invasion and migration abilities of hepatocellular carcinoma cells through EMT-related transcription factor SNAIL | 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 Downregulation of HOPX promotes the invasion and migration abilities of hepatocellular carcinoma cells through EMT-related transcription factor SNAIL Rong Wang, Haizhou Xu, Changhuan Hu, Kun Chen, Shaowen Tang, Xiaoli Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6140393/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract HOPX acts as a tumor suppressor in certain cancers, but the function of HOPX, as well as its mechanism of action in hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, using in vitro and in vivo animal models, the effect of HOPX on the development of HCC was explored. In our study, the HOPX expression at both protein and mRNA level were found to be downregulated in HCC cells and tumor tissues. Restoration of HOPX expression was found to inhibit HCC cell invasion and migration capabilities, but produced no effect on growth. Moreover, HOPX prevented metastasis in an HCC cell metastatic mouse model. Further investigations showed that HOPX could suppress HCC cell epithelial-to-mesenchymal transition (EMT) by inhibiting SNAIL, an EMT transcription factor that is required for the metastasis-inhibition activity of HOPX to proceed. Our study identified HOPX as a suppressor of the development of HCC, which implies that HOPX suppresses HCC cells invasion and migration by inhibiting SNAIL. Biological sciences/Cancer Biological sciences/Cell biology HOPX EMT Metastasis Hepatocellular carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Worldwide, hepatocellular carcinoma (HCC) was reported as the sixth most frequent cancer and the fourth most frequent cause of cancer-related death(Bray et al., 2018 ). Despite intensive search for new treatment strategies, the prognosis of HCC is still poor(de Jesus and Dettino, 2018 ). HCC cell metastatic and invasive behavior is the main cause of unsuccessful therapy and death of HCC patients(Tung-Ping Poon et al., 2000 ). Therefore, it is crucial that the mechanisms that underlie invasion and metastasis in HCC are explored and that more effective approaches are identified. HOPX, known also as HOP, NECC1, LAGY and OB1, was first identified as a gene related with the development of the heart(Chen et al., 2002 ; Shin et al., 2002 ). HOPX belongs to the family of protein that contain the well conserved homeodomain of 60 amino acids and is its smallest constituent. HOPX is commonly expressed in various tissues, and is vital for the regulation of cell differentiation, proliferation and migration(Mariotto et al., 2016 ). During recent times, the expression of HOPX has been reported to be silenced or downregulated in a number of types of human carcinoma, including breast cancer and lung cancer(Kikuchi et al., 2017 ), colorectal cancer(Katoh et al., 2012 ), gastric cancer(Ooki et al., 2010 ), uterine endometrial cancer(Yamaguchi et al., 2009 ) and pancreatic cancer(Waraya et al., 2012 ). Studies show that increased expression of HOPX inhibits the progression of tumors, while endogenous HOPX knockdown restores the antagonistic effect of the tumor. Although most studies indicate the tumor suppressor function of HOPX, it was found that HOPX is upregulated in invasive pancreatic cancer(Walsh et al., 2011 ), thyroid cancer(Pauws et al., 2004 ) and sarcoma(Kovarova et al., 2013 ), which increases tumor cell invasion and migration. Thus, HOPX may also function an oncogene or tumor suppressor in different types of cancer. Epithelial-to-mesenchymal transition (EMT), a complicated reprogramming process and, is a crucial early step for tumor metastasis. EMT is often characterized by a decreased epithelial cadherin (ECADHERIN) expression and increased in mesenchymal marker, including VIMENTIN and FIBRONECTIN, expression(Kalluri and Weinberg, 2009 ; Lamouille et al., 2014 ). The improper induction of EMT can increase the migration and invasion capabilities of tumor cells, initiating the tumor metastasis cascade(Valastyan and Weinberg, 2011 ). The tumor suppressor function of HOPX has been recognized in HCC, while it was found that HOPX inhibits HCC cell migration and invasion capabilities. Furthermore, SNAIL was identified as the main EMT-inducing transcriptional factor inhibited by HOPX. Materials and methods Cell culture Human HCC cell lines, HepG2, SK-HEP1, Hep3B, Huh7 and PLC cells, as well as normal human LO2 liver cells, were purchased from the Chinese Academy of Sciences Cell Bank (China). DMEM medium (GIBCO BRL, USA) supplemented with 10% FBS (fetal bovine serum; GIBCO BRL, USA), 100 U/ml streptomycin and 100 U/ml penicillin was used to grow the cells. These cells were stored in a CO 2 incubator at 37°C in a controlled 5% CO 2 atmosphere. Reagents The following reagents were used in this study: anti-HOPX antibody (Santa Cruz Biotechnology, USA); primary antibodies against ECADHERIN, α-CATENIN, VIMENTIN, FIBRONECTIN, SNAIL and horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA); Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies Inc., Japan); pLEGFP-N1-SNAIL and pLEGFP-N1 and pSin-EF2-puro-HOPX, pSin-EF2-puro-Vector plasmids (Vigene Bioscience, China). DNA sequencing was used to confirm the accuracy of all plasmids. siRNA oligonucleotides that have been confirmed to target HOPX and SNAIL were obtained from GenePharma (China). Supplementary Table 1 shows the siRNA sequences used for HOPX and SNAIL. A GAPDH antibody from Kangchen Biotech, China, was used. Plasmid transfection and RNA interference In order to generate cell lines transfected in a stable manner, 293FT cells were co-transfected with lentivirus packing expression plasmids. Thereafter, for 48 h the viral supernatants were cultured with HCC cells. Once infection had taken place, selection of permanent clones was made using 0.5 mg/ml puromycin (Sigma-Aldrich). Infection efficiency was confirmed using western blotting and Real-time PCR. For RNA interference, the cells were kept in in 6-well plates and allowed to grow and reach 80% confluency. Lipofectamine 2000 (LF2000; Invitrogen, USA) was used to transfect them on the next day. Following transfection for 48 h, HOPX expression was confirmed using western blotting . Real-time PCR Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA), following the manufacturer’s instructions. Standard PrimeScript RT reagent kit (Perfect Real Time, Japan) protocol was followed to conduct first-strand cDNA synthesis and amplification. Previously described protocol(Xiang et al., 2015 ) was used to conduct Real-time PCR analysis. Supplementary Table 1 shows the primer sequences used. Cell proliferation assay CCK-8 assay was used as previously detailed[19] to explore cell proliferation. In each well in a 96-well plate, 1 × 10 3 cells in 180 µl of medium were added. After 24 h, twenty microliters of CCK-8 reagent (100 µl/ml) were added into each well, after incubation for a given time period (1, 2, 3, 4 and 5 days). Absorbance at 450 nm was measured with a microplate reader (Thermo Fisher Scientific, USA). Colony-formation assay The 6-well plates were filled with 4×10 2 cells per well. Two weeks later, 4% paraformaldehyde was applied for 15 minutes for the fixing of cells and the cells were visualized through 0.5% crystal violet staining for 30 min. Wound healing assay A cellular monolayer was grown in 6-well plates until it reached confluency and was then scratched to wound the surface using the tip of a pipette and washed with PBS. DMEM containing 1% FBS in the presence of 50 ng/mL HGFwas used to wound the cells. The wounds were photographed after 24 h (SK-HEP1 and Huh7 cells) or 48 h (LO2 cells). Cell invasion and migration assays An 8 µm pore filter chamber (6.5 mm in diameter, 8 µm pore size, Corning, USA) was used for the migration assay. To the upper chamber, 5 × 10 4 cells in low serum (1% FBS) DMEM were added. Afterwards, to the lower chamber, 50 ng/ml of HGF with 0.6 ml of low serum (1% FBS) DMEM was added, and incubation was conducted at 37°C under 5% CO 2 for 24 h. Following incubation, 4% formaldehyde was used for fixing of the filters and hematoxylin was used for 10 min for staining. The upper surface of the filters were cleaned using a cotton swab to remove excess cells. An inverted microscope was used to observe the stained cells. A thin layer of 0.25 mg/ml Matrigel Basement Membrane Matrix (BD Biosciences, Bedford, MA) was used to coat the inserts to prepare them for the invasion assay. Then, to the upper chamber, 5 × 10 4 cells in low serum (1% FBS) DMEM were added, and to the lower chamber, 50 ng/ml of HGF with 0.6 ml of low serum (1% FBS) DMEM was added. After incubation at 37°C with 5% CO 2 and the cells were permitted for 48 hours to invade the Matrigel layer. Thereafter, the same steps that are described for the cell migration assays were followed. Experimental metastasis Medical Experimental Animal Center, Guangdong Province, China provided 5–6 weeks old athymic female BALB/c nude mice. All animals were fed a standard diet ad libitum and housed in a temperature-controlled animal facility with a 12/12 hours light/dark cycle. All procedures were performed according to the Sun Yat-sen University Guide for Care and Use of Laboratory Animals and were approved by the Bioethics Committee of Sun Yat-sen University. The study is reported in adherence to ARRIVE guidelines. In order to determine whether HOPX could decrease metastasis, vector or HOPX overexpressing HCC cells in a stable manner were created and administered into the female nude mice through tail vein injection. After eight weeks the mice were euthanized and their lungs were sampled for tissue sectioning. Euthanasia was performed via intraperitoneal injection of sodium pentobarbital (200 mg/kg). Statistical analysis Comparison of the date was done using the Student’s t -test. Statistically significance was regarded to be shown by data with a p value of < 0.05. HOPX is downregulated in HCC cells and tumor tissues We used RT PCR and western blotting analysis to identify HOPX expression in primary HCC tumors and HCC cells. The results show that compared with HOPX expression in normal liver cells, in all HCC cell lines expression is markedly decreased (Fig. 1 A). Similar results were observed in primary HCC tumor tissues (Fig. 1 B). Compared with samples of non-malignant liver tissues, protein and mRNA level HOPX expression was decreased in primary HCC tumor tissues. HOPX does not affect HCC cells proliferation Huh7 and SK-HEP1 cells with the control vector or HOPX expression plasmid transfected were used to determine the function of HOPX in HCC. HOPX expression in Huh7 and SK-HEP1 cells increased significantly at both mRNA and protein level after stable transfection with the HOPX plasmid (Fig. 2 A). The influence of HOPX on HCC cell proliferation was investigated using colony-formation and CCK-8 assays. The data reveals that HOPX overexpression and silencing in HCC cells and normal liver cells, respectively, has a minimal effect on cell viability and colonization (Fig. 2 B and C). HOPX inhibits HCC cells invasion and migration The monolayer wound healing assay found that ectopic expression of HOPX in HCC cells spread at wound edges much slower, compared with the control (Fig. 3 A). A Transwell migration assay was conducted to confirm this result. The lower chamber had a significantly lower number of HOPX overexpressing cells that had migrated into it, compared with the number of control cells (Fig. 3 B). Furthermore, a Transwell assay with Matrigel was carried out to explore the effect of HOPX on the invasive potential of the cells. As illustrated in Fig. 3 C, overexpression of HOPX significantly suppressed HCC cell invasion. Additionally, the effect of HOPX downregulation on cell invasion and migration was determined. In contrast, HOPX silencing in normal liver cells increased its invasion and migration potential (Fig. 3 D, E and F). HOPX inhibits EMT in HCC cells` EMT is crucial for tumor progression and plays an important role in tumor migratory and invasive behaviors(Valastyan and Weinberg, 2011 ). In order to explore the role of EMT in suppression effect of HOPX on HCC cells invasion and migration, the impact of HOPX on mesenchymal (VIMENTIN and FIBRONECTIN) and epithelial (ECADHERIN and α-CATENIN) marker expression were determined. Western blotting analysis reveals that overexpression of HOPX in Huh7and SK-HEP1 cells leads to a decreased expression of mesenchymal markers and elevated expression of epithelial markers (Fig. 4 ). In contrast, the epithelial markers are upregulated, while mesenchymal markers are downregulated in HOPX knockdown LO2 cells (Fig. 4 ). SNAIL is essential for the invasion and migration inhibition of HOPX EMT is regulated by several transcription factor families(Ansieau et al., 2014 ; Diaz et al., 2014 ). The expression levels of SNAIL, SLUG, ZEB1, ZEB2, TWIST1 and FOXC2 were analyzed using real-time PCR to identify HOPX target genes. As shown in Fig. 5 A, only SNAIL was induced by HOPX in all cell lines investigated. SNAIL was found to be downregulated in HOPX overexpressing HCC cells and upregulated in HOPX silenced normal liver cells. Western blotting analysis also confirmed the regulation of SNAIL by HOPX (Fig. 5 B). In order to explore whether SNAIL expression is necessary for the inhibition effect on invasion and migration induced by HOPX, we re-established SNAIL expression of HOPX overexpressing HCC cells. Co-transfection with the SNAIL expression vector suppressed the expression of E-CADHERIN induced by HOPX and increased the expression of VIMENTIN (Fig. 5 C). Moreover, significant elimination of the inhibitory effect produced by HOPX on HCC cell invasion (Fig. 6 C) and migration (Fig. 6 A and B) was achieved by SNAIL expression vector co-transfection. These results indicate that the invasion and migration inhibition effect induced by HOPX definitely requires SNAIL. HOPX prevents metastasis of HCC cells in vivo For the determination whether HOPX can decrease metastasis, a lung colonization model was employed. Eight weeks after injection of Huh7 or SK-HEP1 cells that overexpress HOPX in a stable manner, the mice were sacrificed and the lungs were removed. The results reveal that the formation of lung metastases decreased by 41.98% and 46.61% in Huh7 and SK-HEP1 cell metastatic mouse models, respectively, compared with the control group (Fig. 7 ). Discussion Although in a variety of normal tissues, HOPX expression is ubiquitous, similar expression is not seen in malignant tissues of the same type(Ooki et al., 2010 ). HOPX has been found to have an effect on cancer prognosis. For example, HOPX downregulation in breast cancer is linked to a poor clinical outcome(Kikuchi et al., 2017 ), and stable transfection with a HOPX expressing vector was found to suppress lung cancer cell growth(Katoh et al., 2012 ). HOPX has been found to be downregulated in nasopharyngeal carcinoma and head and neck cancer(Yap et al., 2016 ), and it can inhibit cancer cells growth, invasion, and tumorigenesis. Conversely, HOPX is upregulated in invasive pancreatic cancer(Walsh et al., 2011 ) and sarcoma(Kovarova et al., 2013 ), while it has also been shown to function as a prometastatic gene, suggesting that HOPX may play contrary roles in cancer development. Additionally, certain genes that are similar to HOPX have been shown to play both tumor-suppressing and tumor-promoting roles, depending on the type of cancer(Bosch-Presegue and Vaquero, 2011 ; Robbs et al., 2008 ). In the present study, we reveal the role of HOPX as a tumor suppressor in HCC. We first evaluated the expression of HOPX in HCC cells and normal liver LO2 cells. Western blotting and real-time PCR analysis reveal decreased expression of HOPX in HCC cells at both mRNA and protein levels. HOPX is downregulated at protein and mRNA level in primary HCC tumor tissues, compared with corresponding non-cancerous tissues. We then investigated whether HOPX is a tumor suppressor in HCC progression. Stable overexpression of HOPX was found to inhibit HCC cell migration and invasion using functional assays. Notably, the stable overexpression of HOPX in HCC cells has a minimal effect on growth but significantly decreases invasion and migration potential, indicating that HOPX impacts cell motility and invasion without affecting proliferation. We further investigated whether HOPX could reduce metastasis in vivo. Not surprisingly, lungs from mice in the HOPX overexpression group display an apparent decrease in metastatic foci, compared with the control group. Several studies have shown that HOPX inhibits cancer cell invasion and migration, but the molecular mechanisms remain elusive(Kovarova et al., 2013 ; Yap et al., 2016 ). The results of many studies show that EMT is a crucial process involving epithelial tumor cell dissociation, as well as dissemination to distant sites(Kalluri and Weinberg, 2009 ; Lamouille et al., 2014 ). Further to this fact, we observed a decrease in mesenchymal markers and enhancement of epithelial marker expression in HOPX overexpressing HCC cells, suggesting that HOPX can inhibit HCC cell invasion and migration through EMT inhibition. EMT, which is regulated by multiple transcription factors, including ZEB1, ZEB2, SNAIL, TWIST and SLUG, is a complex process(Chaffer and Weinberg, 2011 ; Ell and Kang, 2013 ; Fischer et al., 2015 ). Among them, SNAIL is the most important transcription factor and is associated with a poor clinical outcome in HCC, melanoma, squamous cell carcinoma, as well as lung, colorectal, ovary and breast cancers (Miyoshi et al., 2004 ; Peinado et al., 2007 ; Smith et al., 2014 ). In order to identify the mechanism of HOPX inhibition of EMT, we investigated if the expression of EMT transcription factors are suppressed by HOPX and identified SNAIL to be a target molecule of HOPX. The overexpression of SNAIL abolishes the HCC cell migration and invasion inhibition effect of HOPX, indicating that the inhibition effect of tumor metastasis induced by HOPX definitely requires SNAIL. In conclusion, our results demonstrate that HOPX inhibits HCC cell invasion and migration through SNAIL induced EMT modulation. Our findings indicate that HOPX is a potential therapy target for HCC. Declarations Acknowledgements Not applicable Author contributions Qingfeng Xiang and Qing Zhang developed the concept and designed the study. Qingfeng Xiang wrote the manuscript. Rong Wang, Haizhou Xu Changhuan Hu and Shaowen Tang performed the experiments and assisted in creating figures. Kun Chen and Xiaoli Wu reviewed and corrected the manuscript. The final manuscript was read and approved by all authors. Funding The Natural Science Foundation of Xiaogan City (XGKJ2024010007) supported this study. Data availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. References Ansieau, S., Collin, G., Hill, L., 2014. EMT or EMT-Promoting Transcription Factors, Where to Focus the Light? Front Oncol 4, 353. Bosch-Presegue, L., Vaquero, A., 2011. The dual role of sirtuins in cancer. Genes Cancer 2(6), 648-662. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R.L., Torre, L.A., Jemal, A., 2018. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. Chaffer, C.L., Weinberg, R.A., 2011. A perspective on cancer cell metastasis. Science 331(6024), 1559-1564. 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Supplementary Files SupplementaryTable1.docx Cite Share Download PDF Status: Published Journal Publication published 13 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Apr, 2025 Reviews received at journal 08 Apr, 2025 Reviews received at journal 23 Mar, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers invited by journal 23 Mar, 2025 Editor assigned by journal 23 Mar, 2025 Editor invited by journal 16 Mar, 2025 Submission checks completed at journal 13 Mar, 2025 First submitted to journal 02 Mar, 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-6140393","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":433614085,"identity":"db8acd93-1672-4eac-9d11-b67b40c7794d","order_by":0,"name":"Rong Wang","email":"","orcid":"","institution":"The affiliated Qingyuan Hospital (Qingyuan people's hospital), Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Wang","suffix":""},{"id":433614089,"identity":"fae0cc7e-a3c7-4a72-9311-d8a0f9043980","order_by":1,"name":"Haizhou Xu","email":"","orcid":"","institution":"Xiaogan First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haizhou","middleName":"","lastName":"Xu","suffix":""},{"id":433614091,"identity":"62f2c194-2fa9-4617-81a6-915fec020bd2","order_by":2,"name":"Changhuan Hu","email":"","orcid":"","institution":"Xiaogan First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Changhuan","middleName":"","lastName":"Hu","suffix":""},{"id":433614095,"identity":"8b88431b-70d8-4f1d-a62d-5986a381b759","order_by":3,"name":"Kun Chen","email":"","orcid":"","institution":"Xiaogan First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Chen","suffix":""},{"id":433614096,"identity":"a784f8e5-2867-4812-b35b-5f0f12bb2221","order_by":4,"name":"Shaowen Tang","email":"","orcid":"","institution":"Xiaogan First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shaowen","middleName":"","lastName":"Tang","suffix":""},{"id":433614097,"identity":"baa7699f-49e0-444a-8cac-a4d7d390448c","order_by":5,"name":"Xiaoli Wu","email":"","orcid":"","institution":"Xiaogan First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Wu","suffix":""},{"id":433614101,"identity":"b8fcfcec-f9e4-403c-b9a7-647fd92a67c8","order_by":6,"name":"Qing Zhang","email":"","orcid":"","institution":"Xiaogan First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Zhang","suffix":""},{"id":433614102,"identity":"8333f757-4634-40a6-b5bc-e6f5ba3b0b7b","order_by":7,"name":"Qingfeng Xiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIie3RMQrCMBSA4ScPosOTbNLQYq/wpFAdBK+iN3DqBQpOHsDiJZycKxm62QO4KIJzBZEODrYubm1GwXxDpvyElwdgWb8IP+eQZhrTtCjNk8CDTCwOydr8rWAKOQW6Jwzucoa3yyNi6sRUaCDw5SBtTlQsxoF3ZELs7/RyAqNkO29OJELoqtWLRJ1sCOZ8akkEdp9VwkRIZ03CIJFIobpXiYMEZomKKXKhmoVRcPXJTvssnGd7VUY8Y6mvRVFOfem2JDWk7zqc9uu1TmmyQcuyrP/1BuwlOl2gL7cJAAAAAElFTkSuQmCC","orcid":"","institution":"Xiaogan First People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Qingfeng","middleName":"","lastName":"Xiang","suffix":""}],"badges":[],"createdAt":"2025-03-02 16:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6140393/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6140393/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-15581-w","type":"published","date":"2025-08-13T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79668527,"identity":"68565baf-7c6c-4458-bc2d-96343c742432","added_by":"auto","created_at":"2025-04-01 10:45:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":113879,"visible":true,"origin":"","legend":"\u003cp\u003eHOPX expression in HCC tumors and cell lines. (A) Real-time PCR was used to analyze HOPX mRNA expression in HCC cell lines and normal liver cell LO2. HOPX protein expression in HCC cell lines and a normal LO2 liver cells was analysed using western blotting. (B) Expression of HOPX at protein level in HCC tumors was evaluated using western blotting, while the same at mRNA level was evaluated using real-time PCR. Results are expressed as mean ± SD of three separate trials. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. the control group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/60d85dcf69b8b521750ac251.png"},{"id":79670933,"identity":"b8e725dc-0d1a-467c-97bb-25641750ae6c","added_by":"auto","created_at":"2025-04-01 11:09:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262751,"visible":true,"origin":"","legend":"\u003cp\u003eHOPX does not affect HCC cell proliferation. (A) Protein and mRNA expression of HOPX in Huh7 and SK-HEP1 cells overexpressing the vector or HOPX in a stable manner, or LO2 cells with HOPX siRNA (si-2 and si-1) or control NC transfection.\u003cstrong\u003e \u003c/strong\u003e(B) CCK-8 assay was performed to examine the effect of HOPX on cell viability of the transfected cells. (C) The impact of HOPX on colonization of transfected cells was explored using a colony formation assay. Results are expressed as mean ± SD of three separate trials. *, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 vs. the control group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/cd55d59470225070c6c47d80.png"},{"id":79668534,"identity":"203ac000-4f9c-4054-8547-4f705538d1ef","added_by":"auto","created_at":"2025-04-01 10:45:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":457046,"visible":true,"origin":"","legend":"\u003cp\u003eHCC cell migration and invasion is suppressed \u003cem\u003ein vitro\u003c/em\u003e by HOPX. (A and B) Transwell assay without Matrigel and wound healing assay were used to determine the migration abilities of Huh7 and SK-HEP1 cells that overexpress the vector or HOPX in a stable manner. (C) A Transwell assay with Matrigel was used to determine the invasion abilities of Huh7 and SK-HEP1 cells that overexpress the vector or HOPX in a stable manner. (D and E) Transwell assay without Matrigel and wound healing assay were used to measure migration abilities of LO2 cells that express HOPX siRNAs (si-2 and si-1) or control NC in an impermanent manner. (F) Transwell assay with Matrigel was used to measure the invasion abilities of LO2 cells that express HOPX siRNAs (si-2 and si-1) or control NC in an impermanent manner. Results are expressed as mean ± SD of three separate trials. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. the control group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/3088c6b5c098b04bcef77d31.png"},{"id":79668528,"identity":"74806af0-4c75-4d17-8ddb-ce16881d9e08","added_by":"auto","created_at":"2025-04-01 10:45:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":114121,"visible":true,"origin":"","legend":"\u003cp\u003eHOPX inhibits EMT in HCC cells. VIMENTIN, FIBRONECTIN ECADHERIN and α-CADHERIN, expression levels in Huh7 and SK-HEP1 cells overexpressing the vector or HOPX in a stable manner, and LO2 cells transfected with HOPX siRNAs (si-2 and si-1) or control NC, and the subsequent analysis was conducted through western blotting.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/e5e37aa9b42de161ab822f07.png"},{"id":79669191,"identity":"8adbdbf9-f64a-43f0-add6-51ea6b867eda","added_by":"auto","created_at":"2025-04-01 10:53:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163784,"visible":true,"origin":"","legend":"\u003cp\u003eHOPX suppresses SNAIL expression in HCC cells. (A) Real-time PCR was used on Huh7 and SK-HEP1 cells overexpressing the vector or HOPX in a stable manner, or LO2 cells transfected with HOPX siRNAs (si-2 and si-1) or control NC to determine the mRNA expression levels of EMT-inducing transcription factors. (B) Western blotting and Real-time PCR analysis of SNAIL expression in Huh7, as well as SK-HEP1 cells overexpressing the vector or HOPX in a stable manner, and LO2 cells transfected with HOPX siRNAs (si-2 and si-1) or control NC. (C) Vector #2 or SNAIL was transfected into Huh7 and SK-HEP1 cells that overexpress the Vector #1 or HOPX in a stable manner. The expression of ECADHERIN, α-CADHERIN, VIMENTIN and FIBRONECTIN were measured using western blotting. Results are expressed as mean ± SD of three separate trials. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. the control group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/96be4ca9a235583dd053f83e.png"},{"id":79670126,"identity":"d5090d4d-4bd4-4878-b333-7789512c82a0","added_by":"auto","created_at":"2025-04-01 11:01:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":506636,"visible":true,"origin":"","legend":"\u003cp\u003eSNAIL mediates the metastasis-inhibition effect on HCC cells by acting as a functional target of HOPX. Vector #2 or SNAIL was transfected into Huh7 and SK-HEP1 cells that overexpress Vector #1 or HOPX in a stable manner. The migration and invasion abilities of the cells were determined using wound healing assays (A) and Transwell assays with or without Matrigel, respectively (B and C). Results are expressed as mean ± SD of three separate trials. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. the control group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/53da2c515a302c52fbef52af.png"},{"id":79668537,"identity":"d6e4bd1d-64d8-46ae-87fc-59230cbdef53","added_by":"auto","created_at":"2025-04-01 10:45:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":304441,"visible":true,"origin":"","legend":"\u003cp\u003eHOPX inhibits HCC cell metastasis \u003cem\u003ein vivo\u003c/em\u003e. \u0026nbsp;The nude mice were administered with Huh7 and SK-HEP1 cells transfected in a stable manner with the vector or HOPX through tail vein injection, and were assigned to different groups, with eight mice in each group. The mice were sacrificed eight weeks after injection to evaluate lung metastases. Results are expressed as mean ± SD of three separate trials. *, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 vs. the control group.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/df5e786e5db9bfc8943e01cc.png"},{"id":89310584,"identity":"b4103dcb-c97c-4b0b-8dd4-4c468b6aa187","added_by":"auto","created_at":"2025-08-18 16:08:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2506363,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/9e97cf15-d17b-4ccf-9b38-90c09b85b36e.pdf"},{"id":79670124,"identity":"21e0f564-1889-4f42-8701-ee9a27b9dbe9","added_by":"auto","created_at":"2025-04-01 11:01:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12321,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6140393/v1/05a32d7e097ed52f6ebc5b98.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Downregulation of HOPX promotes the invasion and migration abilities of hepatocellular carcinoma cells through EMT-related transcription factor SNAIL","fulltext":[{"header":"Background","content":"\u003cp\u003eWorldwide, hepatocellular carcinoma (HCC) was reported as the sixth most frequent cancer and the fourth most frequent cause of cancer-related death(Bray et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Despite intensive search for new treatment strategies, the prognosis of HCC is still poor(de Jesus and Dettino, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). HCC cell metastatic and invasive behavior is the main cause of unsuccessful therapy and death of HCC patients(Tung-Ping Poon et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Therefore, it is crucial that the mechanisms that underlie invasion and metastasis in HCC are explored and that more effective approaches are identified.\u003c/p\u003e \u003cp\u003eHOPX, known also as HOP, NECC1, LAGY and OB1, was first identified as a gene related with the development of the heart(Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Shin et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). HOPX belongs to the family of protein that contain the well conserved homeodomain of 60 amino acids and is its smallest constituent. HOPX is commonly expressed in various tissues, and is vital for the regulation of cell differentiation, proliferation and migration(Mariotto et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). During recent times, the expression of HOPX has been reported to be silenced or downregulated in a number of types of human carcinoma, including breast cancer and lung cancer(Kikuchi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), colorectal cancer(Katoh et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), gastric cancer(Ooki et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), uterine endometrial cancer(Yamaguchi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and pancreatic cancer(Waraya et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Studies show that increased expression of HOPX inhibits the progression of tumors, while endogenous HOPX knockdown restores the antagonistic effect of the tumor. Although most studies indicate the tumor suppressor function of HOPX, it was found that HOPX is upregulated in invasive pancreatic cancer(Walsh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), thyroid cancer(Pauws et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and sarcoma(Kovarova et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which increases tumor cell invasion and migration. Thus, HOPX may also function an oncogene or tumor suppressor in different types of cancer.\u003c/p\u003e \u003cp\u003eEpithelial-to-mesenchymal transition (EMT), a complicated reprogramming process and, is a crucial early step for tumor metastasis. EMT is often characterized by a decreased epithelial cadherin (ECADHERIN) expression and increased in mesenchymal marker, including VIMENTIN and FIBRONECTIN, expression(Kalluri and Weinberg, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lamouille et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The improper induction of EMT can increase the migration and invasion capabilities of tumor cells, initiating the tumor metastasis cascade(Valastyan and Weinberg, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe tumor suppressor function of HOPX has been recognized in HCC, while it was found that HOPX inhibits HCC cell migration and invasion capabilities. Furthermore, SNAIL was identified as the main EMT-inducing transcriptional factor inhibited by HOPX.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman HCC cell lines, HepG2, SK-HEP1, Hep3B, Huh7 and PLC cells, as well as normal human LO2 liver cells, were purchased from the Chinese Academy of Sciences Cell Bank (China). DMEM medium (GIBCO BRL, USA) supplemented with 10% FBS (fetal bovine serum; GIBCO BRL, USA), 100 U/ml streptomycin and 100 U/ml penicillin was used to grow the cells. These cells were stored in a CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C in a controlled 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eThe following reagents were used in this study: anti-HOPX antibody (Santa Cruz Biotechnology, USA); primary antibodies against ECADHERIN, α-CATENIN, VIMENTIN, FIBRONECTIN, SNAIL and horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA); Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies Inc., Japan); pLEGFP-N1-SNAIL and pLEGFP-N1 and pSin-EF2-puro-HOPX, pSin-EF2-puro-Vector plasmids (Vigene Bioscience, China).\u003c/p\u003e \u003cp\u003eDNA sequencing was used to confirm the accuracy of all plasmids. siRNA oligonucleotides that have been confirmed to target HOPX and SNAIL were obtained from GenePharma (China). Supplementary Table\u0026nbsp;1 shows the siRNA sequences used for HOPX and SNAIL. A GAPDH antibody from Kangchen Biotech, China, was used.\u003c/p\u003e\n\u003ch3\u003ePlasmid transfection and RNA interference\u003c/h3\u003e\n\u003cp\u003eIn order to generate cell lines transfected in a stable manner, 293FT cells were co-transfected with lentivirus packing expression plasmids. Thereafter, for 48 h the viral supernatants were cultured with HCC cells. Once infection had taken place, selection of permanent clones was made using 0.5 mg/ml puromycin (Sigma-Aldrich). Infection efficiency was confirmed using western blotting and Real-time PCR. For RNA interference, the cells were kept in in 6-well plates and allowed to grow and reach 80% confluency. Lipofectamine 2000 (LF2000; Invitrogen, USA) was used to transfect them on the next day. Following transfection for 48 h, HOPX expression was confirmed using western blotting .\u003c/p\u003e\n\u003ch3\u003eReal-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from cells using TRIzol reagent (Invitrogen, USA), following the manufacturer\u0026rsquo;s instructions. Standard PrimeScript RT reagent kit (Perfect Real Time, Japan) protocol was followed to conduct first-strand cDNA synthesis and amplification. Previously described protocol(Xiang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was used to conduct Real-time PCR analysis. Supplementary Table\u0026nbsp;1 shows the primer sequences used.\u003c/p\u003e\n\u003ch3\u003eCell proliferation assay\u003c/h3\u003e\n\u003cp\u003eCCK-8 assay was used as previously detailed[19] to explore cell proliferation. In each well in a 96-well plate, 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells in 180 \u0026micro;l of medium were added. After 24 h, twenty microliters of CCK-8 reagent (100 \u0026micro;l/ml) were added into each well, after incubation for a given time period (1, 2, 3, 4 and 5 days). Absorbance at 450 nm was measured with a microplate reader (Thermo Fisher Scientific, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony-formation assay\u003c/h2\u003e \u003cp\u003eThe 6-well plates were filled with 4\u0026times;10\u003csup\u003e2\u003c/sup\u003e cells per well. Two weeks later, 4% paraformaldehyde was applied for 15 minutes for the fixing of cells and the cells were visualized through 0.5% crystal violet staining for 30 min.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003eA cellular monolayer was grown in 6-well plates until it reached confluency and was then scratched to wound the surface using the tip of a pipette and washed with PBS. DMEM containing 1% FBS in the presence of 50 ng/mL HGFwas used to wound the cells. The wounds were photographed after 24 h (SK-HEP1 and Huh7 cells) or 48 h (LO2 cells).\u003c/p\u003e\n\u003ch3\u003eCell invasion and migration assays\u003c/h3\u003e\n\u003cp\u003eAn 8 \u0026micro;m pore filter chamber (6.5 mm in diameter, 8 \u0026micro;m pore size, Corning, USA) was used for the migration assay. To the upper chamber, 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells in low serum (1% FBS) DMEM were added. Afterwards, to the lower chamber, 50 ng/ml of HGF with 0.6 ml of low serum (1% FBS) DMEM was added, and incubation was conducted at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. Following incubation, 4% formaldehyde was used for fixing of the filters and hematoxylin was used for 10 min for staining. The upper surface of the filters were cleaned using a cotton swab to remove excess cells. An inverted microscope was used to observe the stained cells. A thin layer of 0.25 mg/ml Matrigel Basement Membrane Matrix (BD Biosciences, Bedford, MA) was used to coat the inserts to prepare them for the invasion assay. Then, to the upper chamber, 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells in low serum (1% FBS) DMEM were added, and to the lower chamber, 50 ng/ml of HGF with 0.6 ml of low serum (1% FBS) DMEM was added. After incubation at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e and the cells were permitted for 48 hours to invade the Matrigel layer. Thereafter, the same steps that are described for the cell migration assays were followed.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperimental metastasis\u003c/h2\u003e \u003cp\u003eMedical Experimental Animal Center, Guangdong Province, China provided 5\u0026ndash;6 weeks old athymic female BALB/c nude mice. All animals were fed a standard diet ad libitum and housed in a temperature-controlled animal facility with a 12/12 hours light/dark cycle. All procedures were performed according to the Sun Yat-sen University Guide for Care and Use of Laboratory Animals and were approved by the Bioethics Committee of Sun Yat-sen University. The study is reported in adherence to ARRIVE guidelines. In order to determine whether HOPX could decrease metastasis, vector or HOPX overexpressing HCC cells in a stable manner were created and administered into the female nude mice through tail vein injection. After eight weeks the mice were euthanized and their lungs were sampled for tissue sectioning. Euthanasia was performed via intraperitoneal injection of sodium pentobarbital (200 mg/kg).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eComparison of the date was done using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Statistically significance was regarded to be shown by data with a \u003cem\u003ep\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHOPX is downregulated in HCC cells and tumor tissues\u003c/h2\u003e \u003cp\u003eWe used RT PCR and western blotting analysis to identify HOPX expression in primary HCC tumors and HCC cells. The results show that compared with HOPX expression in normal liver cells, in all HCC cell lines expression is markedly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similar results were observed in primary HCC tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Compared with samples of non-malignant liver tissues, protein and mRNA level HOPX expression was decreased in primary HCC tumor tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHOPX does not affect HCC cells proliferation\u003c/h2\u003e \u003cp\u003eHuh7 and SK-HEP1 cells with the control vector or HOPX expression plasmid transfected were used to determine the function of HOPX in HCC. HOPX expression in Huh7 and SK-HEP1 cells increased significantly at both mRNA and protein level after stable transfection with the HOPX plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The influence of HOPX on HCC cell proliferation was investigated using colony-formation and CCK-8 assays. The data reveals that HOPX overexpression and silencing in HCC cells and normal liver cells, respectively, has a minimal effect on cell viability and colonization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHOPX inhibits HCC cells invasion and migration\u003c/h2\u003e \u003cp\u003eThe monolayer wound healing assay found that ectopic expression of HOPX in HCC cells spread at wound edges much slower, compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A Transwell migration assay was conducted to confirm this result. The lower chamber had a significantly lower number of HOPX overexpressing cells that had migrated into it, compared with the number of control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, a Transwell assay with Matrigel was carried out to explore the effect of HOPX on the invasive potential of the cells. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, overexpression of HOPX significantly suppressed HCC cell invasion. Additionally, the effect of HOPX downregulation on cell invasion and migration was determined. In contrast, HOPX silencing in normal liver cells increased its invasion and migration potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E and F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHOPX inhibits EMT in HCC cells`\u003c/h2\u003e \u003cp\u003eEMT is crucial for tumor progression and plays an important role in tumor migratory and invasive behaviors(Valastyan and Weinberg, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In order to explore the role of EMT in suppression effect of HOPX on HCC cells invasion and migration, the impact of HOPX on mesenchymal (VIMENTIN and FIBRONECTIN) and epithelial (ECADHERIN and α-CATENIN) marker expression were determined. Western blotting analysis reveals that overexpression of HOPX in Huh7and SK-HEP1 cells leads to a decreased expression of mesenchymal markers and elevated expression of epithelial markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, the epithelial markers are upregulated, while mesenchymal markers are downregulated in HOPX knockdown LO2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSNAIL is essential for the invasion and migration inhibition of HOPX\u003c/h2\u003e \u003cp\u003eEMT is regulated by several transcription factor families(Ansieau et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Diaz et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The expression levels of SNAIL, SLUG, ZEB1, ZEB2, TWIST1 and FOXC2 were analyzed using real-time PCR to identify HOPX target genes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, only SNAIL was induced by HOPX in all cell lines investigated. SNAIL was found to be downregulated in HOPX overexpressing HCC cells and upregulated in HOPX silenced normal liver cells. Western blotting analysis also confirmed the regulation of SNAIL by HOPX (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to explore whether SNAIL expression is necessary for the inhibition effect on invasion and migration induced by HOPX, we re-established SNAIL expression of HOPX overexpressing HCC cells. Co-transfection with the SNAIL expression vector suppressed the expression of E-CADHERIN induced by HOPX and increased the expression of VIMENTIN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, significant elimination of the inhibitory effect produced by HOPX on HCC cell invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B) was achieved by SNAIL expression vector co-transfection. These results indicate that the invasion and migration inhibition effect induced by HOPX definitely requires SNAIL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHOPX prevents metastasis of HCC cells\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the determination whether HOPX can decrease metastasis, a lung colonization model was employed. Eight weeks after injection of Huh7 or SK-HEP1 cells that overexpress HOPX in a stable manner, the mice were sacrificed and the lungs were removed. The results reveal that the formation of lung metastases decreased by 41.98% and 46.61% in Huh7 and SK-HEP1 cell metastatic mouse models, respectively, compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough in a variety of normal tissues, HOPX expression is ubiquitous, similar expression is not seen in malignant tissues of the same type(Ooki et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). HOPX has been found to have an effect on cancer prognosis. For example, HOPX downregulation in breast cancer is linked to a poor clinical outcome(Kikuchi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and stable transfection with a HOPX expressing vector was found to suppress lung cancer cell growth(Katoh et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). HOPX has been found to be downregulated in nasopharyngeal carcinoma and head and neck cancer(Yap et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and it can inhibit cancer cells growth, invasion, and tumorigenesis. Conversely, HOPX is upregulated in invasive pancreatic cancer(Walsh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and sarcoma(Kovarova et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), while it has also been shown to function as a prometastatic gene, suggesting that HOPX may play contrary roles in cancer development. Additionally, certain genes that are similar to HOPX have been shown to play both tumor-suppressing and tumor-promoting roles, depending on the type of cancer(Bosch-Presegue and Vaquero, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Robbs et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In the present study, we reveal the role of HOPX as a tumor suppressor in HCC.\u003c/p\u003e \u003cp\u003eWe first evaluated the expression of HOPX in HCC cells and normal liver LO2 cells. Western blotting and real-time PCR analysis reveal decreased expression of HOPX in HCC cells at both mRNA and protein levels. HOPX is downregulated at protein and mRNA level in primary HCC tumor tissues, compared with corresponding non-cancerous tissues.\u003c/p\u003e \u003cp\u003eWe then investigated whether HOPX is a tumor suppressor in HCC progression. Stable overexpression of HOPX was found to inhibit HCC cell migration and invasion using functional assays. Notably, the stable overexpression of HOPX in HCC cells has a minimal effect on growth but significantly decreases invasion and migration potential, indicating that HOPX impacts cell motility and invasion without affecting proliferation. We further investigated whether HOPX could reduce metastasis \u003cem\u003ein vivo.\u003c/em\u003e Not surprisingly, lungs from mice in the HOPX overexpression group display an apparent decrease in metastatic foci, compared with the control group. Several studies have shown that HOPX inhibits cancer cell invasion and migration, but the molecular mechanisms remain elusive(Kovarova et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yap et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The results of many studies show that EMT is a crucial process involving epithelial tumor cell dissociation, as well as dissemination to distant sites(Kalluri and Weinberg, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lamouille et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Further to this fact, we observed a decrease in mesenchymal markers and enhancement of epithelial marker expression in HOPX overexpressing HCC cells, suggesting that HOPX can inhibit HCC cell invasion and migration through EMT inhibition.\u003c/p\u003e \u003cp\u003eEMT, which is regulated by multiple transcription factors, including ZEB1, ZEB2, SNAIL, TWIST and SLUG, is a complex process(Chaffer and Weinberg, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ell and Kang, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fischer et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among them, SNAIL is the most important transcription factor and is associated with a poor clinical outcome in HCC, melanoma, squamous cell carcinoma, as well as lung, colorectal, ovary and breast cancers (Miyoshi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Peinado et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Smith et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In order to identify the mechanism of HOPX inhibition of EMT, we investigated if the expression of EMT transcription factors are suppressed by HOPX and identified SNAIL to be a target molecule of HOPX. The overexpression of SNAIL abolishes the HCC cell migration and invasion inhibition effect of HOPX, indicating that the inhibition effect of tumor metastasis induced by HOPX definitely requires SNAIL.\u003c/p\u003e \u003cp\u003eIn conclusion, our results demonstrate that HOPX inhibits HCC cell invasion and migration through SNAIL induced EMT modulation. Our findings indicate that HOPX is a potential therapy target for HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e Qingfeng Xiang and Qing Zhang developed the concept and designed the study. Qingfeng Xiang wrote the manuscript. Rong Wang, Haizhou Xu Changhuan Hu and Shaowen Tang performed the experiments and assisted in creating figures. Kun Chen and Xiaoli Wu reviewed and corrected the manuscript. The final manuscript was read and approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThe Natural Science Foundation of Xiaogan City (XGKJ2024010007) supported this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAnsieau, S., Collin, G., Hill, L., 2014. EMT or EMT-Promoting Transcription Factors, Where to Focus the Light? Front Oncol 4, 353.\u003c/li\u003e\n \u003cli\u003eBosch-Presegue, L., Vaquero, A., 2011. The dual role of sirtuins in cancer. 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Snail promotes epithelial mesenchymal transition in breast cancer cells in part via activation of nuclear ERK2. PLoS One 9(8), e104987.\u003c/li\u003e\n \u003cli\u003eTung-Ping Poon, R., Fan, S.T., Wong, J., 2000. Risk factors, prevention, and management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg 232(1), 10-24.\u003c/li\u003e\n \u003cli\u003eValastyan, S., Weinberg, R.A., 2011. Tumor metastasis: molecular insights and evolving paradigms. Cell 147(2), 275-292.\u003c/li\u003e\n \u003cli\u003eWalsh, N., Larkin, A., Swan, N., Conlon, K., Dowling, P., McDermott, R., Clynes, M., 2011. RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation. Cancer Lett 306(2), 180-189.\u003c/li\u003e\n \u003cli\u003eWaraya, M., Yamashita, K., Katoh, H., Ooki, A., Kawamata, H., Nishimiya, H., Nakamura, K., Ema, A., Watanabe, M., 2012. Cancer specific promoter CpG Islands hypermethylation of HOP homeobox (HOPX) gene and its potential tumor suppressive role in pancreatic carcinogenesis. BMC Cancer 12, 397.\u003c/li\u003e\n \u003cli\u003eXiang, Q.F., Zhang, D.M., Wang, J.N., Zhang, H.W., Zheng, Z.Y., Yu, D.C., Li, Y.J., Xu, J., Chen, Y.J., Shang, C.Z., 2015. Cabozantinib reverses multidrug resistance of human hepatoma HepG2/adr cells by modulating the function of P-glycoprotein. Liver Int 35(3), 1010-1023.\u003c/li\u003e\n \u003cli\u003eYamaguchi, S., Asanoma, K., Takao, T., Kato, K., Wake, N., 2009. Homeobox gene HOPX is epigenetically silenced in human uterine endometrial cancer and suppresses estrogen-stimulated proliferation of cancer cells by inhibiting serum response factor. Int J Cancer 124(11), 2577-2588.\u003c/li\u003e\n \u003cli\u003eYap, L.F., Lai, S.L., Patmanathan, S.N., Gokulan, R., Robinson, C.M., White, J.B., Chai, S.J., Rajadurai, P., Prepageran, N., Liew, Y.T., Lopes, V., Wei, W., Hollows, R.J., Murray, P.G., Lambert, D.W., Hunter, K.D., Paterson, I.C., 2016. HOPX functions as a tumour suppressor in head and neck cancer. Sci Rep 6, 38758.\u003c/li\u003e\n\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":"HOPX, EMT, Metastasis, Hepatocellular carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-6140393/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6140393/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHOPX acts as a tumor suppressor in certain cancers, but the function of HOPX, as well as its mechanism of action in hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, using \u003cem\u003ein vitro\u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e animal models, the effect of HOPX on the development of HCC was explored. In our study, the HOPX expression at both protein and mRNA level were found to be downregulated in HCC cells and tumor tissues. Restoration of HOPX expression was found to inhibit HCC cell invasion and migration capabilities, but produced no effect on growth. Moreover, HOPX prevented metastasis in an HCC cell metastatic mouse model. Further investigations showed that HOPX could suppress HCC cell epithelial-to-mesenchymal transition (EMT) by inhibiting SNAIL, an EMT transcription factor that is required for the metastasis-inhibition activity of HOPX to proceed.\u003cstrong\u003e \u003c/strong\u003eOur study identified HOPX as a suppressor of the development of HCC, which implies that HOPX suppresses HCC cells invasion and migration by inhibiting SNAIL.\u003c/p\u003e","manuscriptTitle":"Downregulation of HOPX promotes the invasion and migration abilities of hepatocellular carcinoma cells through EMT-related transcription factor SNAIL","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 10:45:00","doi":"10.21203/rs.3.rs-6140393/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-16T07:01:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T04:27:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-24T02:18:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267963366819200375949147158549322450237","date":"2025-03-23T20:51:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120749493135370840546396401344893922743","date":"2025-03-23T07:07:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-23T07:03:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-23T06:49:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-16T18:32:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-13T12:16:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-02T16:03:16+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":"7d1999b3-6574-4983-86fa-c98aa1294ed3","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46169739,"name":"Biological sciences/Cancer"},{"id":46169740,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-08-18T16:02:40+00:00","versionOfRecord":{"articleIdentity":"rs-6140393","link":"https://doi.org/10.1038/s41598-025-15581-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-13 15:57:51","publishedOnDateReadable":"August 13th, 2025"},"versionCreatedAt":"2025-04-01 10:45:00","video":"","vorDoi":"10.1038/s41598-025-15581-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-15581-w","workflowStages":[]},"version":"v1","identity":"rs-6140393","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6140393","identity":"rs-6140393","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}