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. 2025 Aug 13;15:29739. doi: 10.1038/s41598-025-15581-w

HOPX regulates the invasion and migration abilities of hepatocellular carcinoma by targeting SNAIL

Rong Wang 2,#, Haizhou Xu 1,#, Changhuan Hu 1, Kun Chen 1, Shaowen Tang 1, Xiaoli Wu 1, Qing Zhang 3,5,✉, Qingfeng Xiang 1,4,✉
PMCID: PMC12350657  PMID: 40804282

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.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-15581-w.

Keywords: HOPX, EMT, Metastasis, Hepatocellular carcinoma

Subject terms: Cancer, Cell biology

Introduction

Worldwide, hepatocellular carcinoma (HCC) was reported as the sixth most frequent cancer and the fourth most frequent cause of cancer-related death1. Despite intensive search for new treatment strategies, the prognosis of HCC is still poor2. HCC cell metastatic and invasive behavior is the main cause of unsuccessful therapy and death of HCC patients3. 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 heart4,5. 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 migration6. 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 cancer7 colorectal cancer8 gastric cancer9 uterine endometrial cancer10 and pancreatic cancer11. 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 cancer12 thyroid cancer13 and sarcoma14 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, expression15,16. The improper induction of EMT can increase the migration and invasion capabilities of tumor cells, initiating the tumor metastasis cascade17.

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 CO2 incubator at 37 °C in a controlled 5% CO2 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 protocol18 was used to conduct Real-time PCR analysis. Supplementary Table 1 shows the primer sequences used.

Western blot

Cells were washed with PBS to remove residual culture medium and lysed using RIPA buffer supplemented with 1% PMSF. The resulting cell lysate was homogenized by ultrasonication. Subsequently, the lysate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected for protein quantification using a BCA Protein Assay Kit. Proteins were then separated via SDS-PAGE (10% acrylamide gel), transferred onto NC membranes, and blocked with 5% non-fat milk. Membranes were incubated overnight at 4 °C with primary antibodies against HOPX(Cat#5670), ECADHERIN-1(Cat#3195), α-CATENIN(Cat#3236), VIMENTIN(Cat#5741), FIBRONECTIN(Cat#26836), SNAIL(Cat#3879), and GAPDH(Cat#2118) (all purchased from CST). This was followed by a 2-hour incubation with secondary antibodies (goat anti-rabbit and goat anti-mouse). Proteins were detected using chemiluminescence, and blots were visualized with an enhanced chemiluminescence detection system (Millipore, USA) and imaged using a Syversen chemiluminescence imaging system. Notably, due to limitations in the white light function of the imaging system, some band marker positions appeared less clear; however, this did not interfere with the statistical analysis or interpretation of the results.

Cell proliferation assay

CCK-8 assay was used as previously detailed to explore cell proliferation. In each well in a 96-well plate, 1 × 103 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 × 102 cells per well. Two weeks later, 4% paraformaldehyde was applied for 15 min 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 × 104 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% CO2 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 × 104 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% CO2 and the cells were permitted for 48 h 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. 1A). Similar results were observed in primary HCC tumor tissues (Fig. 1B). Compared with samples of non-malignant liver tissues, protein and mRNA level HOPX expression was decreased in primary HCC tumor tissues.

Fig. 1.

Fig. 1

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HOPX 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. *, p < 0.05 vs. the control group.

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. 2A). 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. 2B,C).

Fig. 2.

Fig. 2

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HOPX 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. (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. *, p < 0.05 vs. the control group.

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. 3A). 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. 3B). 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. 3C, 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. 3D–F).

Fig. 3.

Fig. 3

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HCC cell migration and invasion is suppressed in vitro 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. *, p < 0.05 vs. the control group.

HOPX inhibits EMT in HCC cells

EMT is crucial for tumor progression and plays an important role in tumor migratory and invasive behaviors17. 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).

Fig. 4.

Fig. 4

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HOPX 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.

SNAIL is essential for the invasion and migration Inhibition of HOPX

EMT is regulated by several transcription factor families19,20. 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. 5A, 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. 5B).

Fig. 5.

Fig. 5

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HOPX 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. *, p < 0.05 vs. the control group.

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. 5C). Moreover, significant elimination of the inhibitory effect produced by HOPX on HCC cell invasion (Fig. 6C) and migration (Fig. 6A,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.

Fig. 6.

Fig. 6

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SNAIL 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. *, p < 0.05 vs. the control group.

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).

Fig. 7.

Fig. 7

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HOPX inhibits HCC cell metastasis in vivo. 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. *, p < 0.05 vs. the control group.

Discussion

Although in a variety of normal tissues, HOPX expression is ubiquitous, similar expression is not seen in malignant tissues of the same type9. HOPX has been found to have an effect on cancer prognosis. For example, HOPX downregulation in breast cancer is linked to a poor clinical outcome7 and stable transfection with a HOPX expressing vector was found to suppress lung cancer cell growth8. HOPX has been found to be downregulated in nasopharyngeal carcinoma and head and neck cancer21 and it can inhibit cancer cells growth, invasion, and tumorigenesis. Conversely, HOPX is upregulated in invasive pancreatic cancer12 and sarcoma14 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 cancer22,23. 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 elusive14,21. The results of many studies show that EMT is a crucial process involving epithelial tumor cell dissociation, as well as dissemination to distant sites15,16. 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 process24–26. 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 cancers27–29. 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.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (12KB, docx)
Supplementary Material 2 (11.7MB, docx)

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.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rong Wang and Haizhou Xu contributed equally to this work.

Contributor Information

Qing Zhang, Email: xhnkzjs@163.com.

Qingfeng Xiang, Email: xiangqf171@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (12KB, docx)
Supplementary Material 2 (11.7MB, docx)

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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