Reduction in the hepatocellular carcinoma antioncogene SLC39A14 during the malignant progression of hepatocellular carcinoma

Abstract

BACKGROUND

Hepatocellular carcinoma (HCC) is characterized by high morbidity and mortality owing to its mechanistic complexity and individual heterogeneity, making early diagnosis challenging. The role of SLC39A14, a biomarker for multiple tumors, in HCC remains to be elucidated.

AIM

To determine the tumor-suppressive role of SLC39A14 in HCC and its underlying molecular mechanisms.

METHODS

The expression pattern of SLC39A14 in HCC was evaluated using quantitative reverse transcriptase PCR and western blotting, and its association with tumor malignancy was further validated in C57BL/6J mouse HCC model. A series of functional assays, including cell counting kit-8, colony formation, apoptosis, scratch, and invasion tests, were conducted to assess how alterations in the SLC39A14 expression affect the oncological behavior of HCC cells. In addition, bioinformatics analysis was performed to investigate the potential regulatory mechanism underlying SLC39A14 expression in HCC.

RESULTS

The protein expression and RNA abundance of SLC39A14 in human HCC tissues were significantly lower than those in paracancerous tissues (P < 0.01). In HCC model mice, the SLC39A14 expression decreased gradually as the disease progressed. The overexpression of SLC39A14 significantly inhibited the proliferation, migration, and invasion of Huh7 HCC cells while promoting their apoptosis. The knockdown of SLC39A14 exerted the opposite effect. Bioinformatics analysis suggested the involvement of the transcription factor STAT3 in regulating the SLC39A14 expression.

CONCLUSION

SLC39A14 dysregulation promotes HCC progression through BCL-2/BAX/Caspase-3 apoptotic axis and epithelial–mesenchymal transition transformation axis, suggesting potential therapeutic targets.

Key Words: SLC39A14; Apoptosis; Epithelial–mesenchymal transition; Hepatocellular carcinoma

Core Tip: This study's findings associate SLC39A14 downregulation with hepatocellular carcinoma (HCC) progression. SLC39A14 overexpression inhibits HCC cell proliferation, migration, and invasion while promoting apoptosis via the BCL-2/BAX/caspase-3 pathway. Bioinformatics analysis suggests that STAT3 regulates the expression of SLC39A14. Our findings together highlight SLC39A14-BCL-2/BAX/caspase-3 and SLC39A14-EMT axes as potential therapeutic targets for HCC.

INTRODUCTION

Hepatocellular carcinoma (HCC) is the third-most common cause of cancer-related death globally and the most common malignant tumor in Asia[1,2], with approximately 830000 patients dying from the disease annually[3]. Alpha-fetoprotein (AFP), a biomarker routinely used to screen for HCC, has low sensitivity and specificity and is therefore not supportive of early intervention[4]. In most patients, the tumors have already progressed to an untreatable stage by the time of diagnosis, and despite surgical resection, the overall 5-year survival rate of patients with HCC is only 50%-70%[5,6]. In contrast, if HCC is detected early, the tumor can be completely resected, and recurrence is rare, enhancing the possibility of a complete cure[7]. Therefore, identifying specific biomarkers and effective therapeutic targets is crucial for improving patient prognosis and their quality of life.

SLC39A14, a member of the ZIP metal ion transporter family, encodes the ZIP14 protein. This transmembrane transporter protein has eight transmembrane structural domains that function as a transporter of divalent metal ions, such as zinc, iron, and manganese[8,9]. Abnormal alterations in SLC39A14 disrupt iron uptake in the body, causing hereditary hemochromatosis[10]. Abnormal manganese uptake results in a wide range of neurotoxic effects, culminating in Parkinson’s disease[11,12]. The expression of SLC39A14 is tissue-specific, and its abundance in humans occurs in the following order: Liver > duodenum > kidney > testis > brain[13]. Previous studies have established that SLC39A14 serves as a potential biomarker for prostate[14], cervical[15], and breast[16] cancers and esophageal squamous cell carcinoma[17] and is associated with patient survival. However, its role in HCC has not been adequately investigated. Clinical trials have demonstrated that dual-targeted nanocontrast agents directed at SLC39A14 can enhance the detection rate of early-stage microscopic HCC to 92%[18]. However, the value of SLC39A14 as a specific biomarker for HCC warrants further exploration.

STAT3 is transiently activated in normal cells, undergoes phosphorylation, enters the nucleus, and regulates gene expression by binding to specific target gene sequences[19]. Abnormal activation of STAT3 in cancer cells leads to the massive transcription of antiapoptotic and other molecules, resulting in increased tumor malignancy[20]. STAT3 promotes tumor angiogenesis and upregulates the expression of matrix metalloproteinases, which stimulate tumor growth and metastasis, resulting in a poor prognosis[21-23]. The aberrant expression of STAT3 in various tumors, such as breast cancer[24], lung cancer[25], and HCC[26], is associated with tumor metastasis and poor prognosis. In addition, STAT3 can act as a transcription factor to regulate the expression of solute carrier family 39 (Slc39) in various species. STAT3 functions as a transcription factor that upregulates the expression of myocardial ZIP transporters, particularly ZIP5, providing protection against cardiac ischemia-reperfusion injury[27]. In zebrafish, STAT3 activation promotes the transcription of ZIP6 and ZIP10, facilitating the formation of a ZIP6 homodimer, which is essential for proper EMT during embryonic development[28]. In the freshwater teleost yellow catfish (Pelteobagrus fulvidraco), the STAT3 binding site located at -1383 bp/-1375 bp on the ZIP14 promoter plays a key role in the transcriptional regulation of ZIP14[29]. Additionally, ChIP experiments have confirmed STAT3 binding to the promoter region of the mouse SLC39A14[30]. Nonetheless, the possibility that SLC39A14 is a target of STAT3 in humans warrants further examination.

Therefore, to investigate the clinical significance of SLC39A14 in patients with HCC and its role in the malignant phenotype of HCC, the protein and RNA expression patterns of SLC39A14 were analyzed in tissues from patients with HCC. Subsequently, the regulation of SLC39A14 expression in HCC cells via transfection was explored, revealing the effects of its expression on the biological characteristics of HCC. Finally, possible mechanisms were studied using bioinformatics analysis. The findings confirmed the regulatory role of SLC39A14 in HCC development and provided a novel strategy for its early prediction and targeted therapy. The schematic representation of the study protocol is shown in Figure 1.

Figure 1 Schematic representation of the study protocol. HCC: Hepatocellular carcinoma; DEN: Diethylnitrosamine; CCl₄: Carbon tetrachloride; qRT-PCR: Quantitative reverse transcriptase PCR; CCK-8: Cell counting kit-8; H&E: Hematoxylin and eosin.

MATERIALS AND METHODS

Bioinformatic analysis

The expressions of SLC39A14 and the corresponding clinical information on survival were obtained from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov) for HCC patients. A total of 369 HCC samples and 160 normal tissue samples were obtained. The TCGA database was updated on August 28, 2024.

Multiple databases were used to jointly predict transcription factors that may regulate key genes using the following data platforms: HTF targe (http://bioinfo.life.hust.edu.cn/hTFtarget/), Chip-Atlas (https://chip-atlas.org/), KnockTF 2.0 (https://bio.liclab.net/KnockTF/index.php), ENCODE (https://maayanlab.cloud/Harmonizome/dataset/ENCODE+Transcription+Factor+Targets), GTEx (https://www.genome.gov/Funded-Programs-Projects/Genotype-Tissue-Expression-Project), TCGA. The transcription factors of the target genes were identified in each platform, and the transcription factors that acted with the target genes in all datasets were selected as the transcription factors that potentially regulate the target genes.

Patients and tissue samples

HCC tissues and the corresponding paracancerous tissue samples were collected from 10 patients with HCC at the Second Hospital of Nanjing (Nanjing, China) and 3 patients with HCC at Nanjing Drum Tower Hospital (Nanjing, China).

The patient inclusion criterion was as follows[31]: Age-matched and lifestyle-matched patients with a diagnosis of primary HCC by blood tests, imaging tests, and liver examinations.

The subject exclusion criteria were as follows[32]: (1) Presence of other tumors; (2) Having received preoperative radiotherapy and chemotherapy; (3) Having vascular thrombus, lymphatic infiltration, or metastasis; and (4) Missing critical clinical information.

The study was approved by the Research Ethics Committee of The Second Hospital of Nanjing. Informed consent was obtained from all patients. All samples were handled anonymously in accordance with the ethical and legal standards. The tissue samples were stored in liquid nitrogen for subsequent use.

Animals

Eighty specific pathogen-free C57BL/6J male mice (age: 2 weeks, weight: 8-10 g) were obtained from Jiangsu Huachuang Sino Pharmaceutical Company. To maintain statistical power and minimize experimental animal attrition, mice were randomly assigned to 10 groups of 8 mice each. The mice were maintained in temperature-controlled (24 °C), 12-hour light-dark cycle plastic cages, and provided with standard rodent food and tap water. The animals were gently immobilized during sample collection, and their blood was extracted from the orbits, followed by immediate euthanization via decapitation of the cervical vertebrae[33,34]. The experimental protocol was approved by the Animal Care and Utilization Committee of Nanjing University of Chinese Medicine (Nanjing, China).

Cell culture

The human HCC cell line Huh7 was stored at -80 °C in the laboratory. The cells were cultured in DMEM (HyClone, America) supplemented with 10% fetal bovine serum (Viva Cell, China) and a 1% penicillin-streptomycin mixture (Solarbio, China) and incubated under a 5% CO₂ atmosphere at 37 °C and passaged every 2 days.

Construction of overexpression plasmids and siRNAs targeting SLC39A14

The SLC39A14-overexpression plasmid was constructed by ligating the SLC39A14 coding region cDNA to the pcDNA3.1(-) vector via homologous recombination. The siRNA was synthesized by Sangon Biotech (Shanghai, China) and consisted of the following three targeted sequences: HSLC39A14-858: GAAUCUUCAAGAUCUUCUCdTdT; hSLC39A14-1438: AAGAACAUUCCUCCAGCUAdTdT; and hSLC39A14-1523: AAAUGGAAUCAAGAUGCUGdTdT.

The Huh7 cell line was passaged into six-well plates at a density of 5 × 10⁵ cells/well, and when the cells reached 70%-80% growth, the medium was substituted with an antibiotic-free DMEM containing 10% fetal bovine serum. The cells were transfected using Lipofectamine^TM 3000 transfection reagent, which was mixed with the overexpression plasmid and siRNA and incubated at room temperature for 10 minutes away from direct light. After dropwise addition to six-well plates, the following overexpression groups were prepared: Con, pcDNA3.1(-), and SLC39A14. Meanwhile, the following knockdown groups were prepared: The NC group, the 858 group, the 1438 group, and the 1523 group. The cultures were incubated at 37 °C under a 5% CO₂ atmosphere for 48 hours and used for subsequent analyses.

Protein extraction and western blotting

The tissue and cell samples were lysed in RIPA buffer and centrifuged to obtain the total protein in the supernatant. The obtained protein concentration was determined with a BCA protein assay kit, and protein denaturation was performed by boiling the sample in 5 × loading buffer. A total of 20 μg of denatured protein was separated via SDS-PAGE and transferred onto a PVDF membrane. The PVDF membrane was blocked for 2 hours at room temperature with 5% skim milk powder. The membrane was then incubated overnight in a 4 °C refrigerator with specific primary antibodies (i.e., those against ZIP14, BCL2, BAX, caspase 3, cleaved caspase 3, E-cadherin, N-cadherin, vimentin, and GAPDH). Subsequently, the PVDF membrane was incubated for 2 hours at room temperature with specific secondary antibodies (i.e., those against HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG). Finally, the protein bands were visualized by using a chemiluminescence detection kit. After exposure, the bound antibody was eluted with stripping buffer, and the PVDF membrane was blocked with 5% skim milk powder for 2 hours at room temperature before being treated with other antibodies. GAPDH was used as an internal reference, and ImageJ software was used to analyze and quantify the grayscale values of the protein bands.

RNA extraction and quantitative reverse transcriptase PCR

Total RNA was extracted from the tumor tissues via the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China), and the concentration and purity of the RNA were determined by using a spectrophotometer. Then, the RNA was reverse-transcribed to cDNA by using the HiScript II Q RT SuperMix for qPCR. Quantitative reverse transcriptase PCR (qRT-PCR) was performed by using ChamQ SYBR qPCR Master Mix. The relative-abundance SLC39A14 RNA was calculated by using the 2^-ΔΔCT method, with GAPDH used as an internal reference. The following primers were synthesized by Sangon Biotech (China) and used in the study SLC39A14-F: 5′TGGACACAGCCATTATGCCTC-3′, SLC39A14-R: 5′- GAGTAGCGGACACCTTTCAGC-3′; GAPDH-F: 5′- GGAGCGAGATCCCTCCAAAAT-3′, and GAPDH-R: 5′- GGCTGTTGTCATACTTCTCATGG-3′.

Hematoxylin and eosin staining

The liver tissues were collected and fixed with 4% paraformaldehyde for 24 hours, followed by treatment in a dehydrated gradient series of ethanol. Next, the tissues were placed in xylene for 30 minutes for transparency and dipped in wax to complete tissue embedding. The paraffin sections obtained were cut into 4-μm-thick sections and baked for staining. Before staining, the paraffin sections were deparaffinized with xylene and treated with a gradient series of ethanol, followed by hematoxylin and eosin (H&E) staining. Gradient ethanol and xylene were used to dehydrate the sections, and neutral resin was used to seal the sections. Pathological changes in the sections were observed by light microscopy.

Measurement of alanine aminotransferase and aspartate aminotransferase

Blood samples were collected from mouse orbits, left at room temperature for 1 hour, and then centrifuged at 3000 rpm for 10 minutes to separate the serum. The OD values of the samples were determined by using an alanine aminotransferase (ALT) test kit and an aspartate aminotransferase (AST) test kit. The ALT and AST viabilities of each sample were determined with reference to a standard curve.

Cell counting kit-8 assay

After 48 hours of transfecting the Huh7 cells, the cells were inoculated into a 96-well plate at a density of 2 × 10³ cells/well. Then, after 24, 48, and 72 hours of culture, the cells were incubated with 100 μL of 10% cell counting kit-8 (CCK-8) solution for 1 hour at 37 °C in a 5% CO₂ incubator. After incubation, the OD value at 490 nm was determined using an enzyme meter.

Colony formation experiment

After 48 hours of transfecting the Huh7 cells, the cells were inoculated into a 6-well plate at a density of 500 cells/well. The cells were cultured in an incubator for 14 days, and the medium was refreshed every 3 days. The cells were then fixed with 4% paraformaldehyde for 15 minutes at the end of the culture period and rinsed with phosphate-buffered saline (PBS) and stained with crystal violet for 10 minutes. The entire six-well plate and each well were individually photographed after washing with PBS.

Cell apoptosis assay

After 48 hours of transfection, Huh7 cells were seeded into 6-well plates at a density of 5 × 10⁵ cells/well. Following an additional 48 hours of culture, the cells were harvested, stained using the Annexin V-FITC/PI Apoptosis Detection Kit, and analyzed for apoptosis by flow cytometry.

Scratch assay

After 48 hours of transfection, Huh7 cells were seeded into a 6-well plate at a density of 5 × 10⁵ cells/well. Once the cells reached confluence, a scratch was made using a pipette tip, and the detached cells were removed by washing with PBS. Serum-free DMEM was then added, and images of the initial (0 hour) scratch area were captured using an inverted microscope. The plates were incubated, and images of the same scratch area were taken 24 hours later. Cell migration rate was calculated as: (0 hour scratch spacing - 24 hours scratch spacing)/0 hour scratch spacing × 100%.

Transwell assay

The matrix gel was diluted in a 1:8 ratio with serum-free DMEM, spread in the upper chamber of the Transwell system, and incubated at 37 °C for 2 hours. A total of 2 × 10⁴ cells were placed in 200 µL of serum-free medium and spread in the upper chamber. The chambers were then placed into a 24-well plate supplemented with 10% serum-containing DMEM. After 48 hours of culture at 37 °C, the cells were fixed with 4% paraformaldehyde for 10 minutes, and the chambers were washed with PBS and stained with crystal violet for 10 minutes. After the upper chambers were wiped clean with a cotton swab, cell migration was imaged in three fields of view under a 20 × microscope.

Statistical analysis

All experiments were repeated thrice. The experimental unit for analysis was defined as the animal group. Statistical analysis was performed using ImageJ and GraphPad Prism 9.5.1. The data are presented as the mean ± SD. Student's t-test was performed to compare the differences between two groups, and one-way analysis of variance (ANOVA) was applied to compare the differences among multiple groups. P < 0.05 was considered to indicate statistical significance.

RESULTS

The downregulation of SLC39A14 in HCC

To determine the expression pattern of SLC39A14 in HCC, total protein and RNA were extracted from cancerous and paracarcinomatous tissues of patients with HCC and analyzed using Western blotting and qRT-PCR. The results revealed that the relative expression of ZIP14 protein (Figure 2A) and the relative abundance of RNA (Figure 2B) were significantly lower in HCC tissues than in relatively normal paracancerous tissues (P < 0.05). In addition, the TCGA database showed that the expression of SLC39A14 in human cancer tissues was significantly lower than that in normal tissues (Figure 3A). Subsequently, stratified analysis of patients with HCC with different tumor grades was performed. The results revealed that the expression of SLC39A14 decreased gradually with increasing tumor malignancy (Figure 3B), but the difference was not significant because of the small number of patients in stage 4. Kaplan-Meier survival analysis signified that the overall survival of male patients with low SLC39A14 levels was significantly lower than that of patients with high SLC39A14 levels (P = 0.017; Figure 3C). However, the difference was not significant in female patients (P = 0.2; Figure 3D). These findings suggest that decreased SLC39A14 expression is intricately linked to poor prognosis in patients with HCC and that it plays a role in disease development.

Figure 2 SLC39A14 expression in clinical hepatocellular carcinoma samples. A: Differences in SLC39A14 expression between cancer tissues and paracancerous tissues from hepatocellular carcinoma (HCC) patients were detected by western blotting (n = 13); B: Differences in SLC39A14 mRNA expression in the cancerous and paracancerous tissues of HCC patients were detected by quantitative reverse transcriptase PCR (n = 3). ^aP < 0.01. ^bP < 0.0001.

Figure 3 SLC39A14 expression and survival analysis in The Cancer Genome Atlas cohorting. A: SLC39A14 expression in cancer patients (T) and normal individuals (N) (P < 0.05); B: Changes in SLC39A14 expression across cancer stages; C: Overall survival in male patients with high and low SLC39A14 expression (P = 0.017); D: Overall survival in female patients with high and low SLC39A14 expression (P = 0.2).

The decrease in SLC39A14 expression with increasing tumor malignancy

To further analyze the changes in SLC39A14 abundance during the pathogenesis of HCC, the disease was induced in mice using diethylnitrosamine (DEN) in combination with carbon tetrachloride (CCl₄). Two-week-old mice were injected intraperitoneally with DEN, and, thereafter, CCl₄ was administered intraperitoneally every week until 22 weeks of age. Starting at 6 weeks of age, eight mice were sacrificed every 2 weeks, and their blood and liver tissues were collected and photographed (Figure 4). The effect of HCC induction was evaluated, revealing that the liver surface of the induction group was covered with numerous nodules, in contrast to the smooth surface of the control group. H&E staining showed that the hepatocytes in the control group were neatly arranged, with no abnormal proliferating cells. In contrast, the mice in the induction group exhibited disorganized hepatocytes, large and deeply stained nuclei, and considerable nuclear solidification and abnormal mitosis (Figure 5A). In the induction group, the liver-to-body ratio was greater (Figure 5B; Supplementary material), and the levels of ALT (Figure 5C) and AST (Figure 5D) were significantly elevated. These observations suggested that HCC was successfully induced. Subsequently, we performed Western blotting on the liver tissues (Figure 6). The results revealed that the expression of ZIP14 decreased with increasing degree of induction in mice, implying a close association between SLC39A14 and HCC development, which agrees with the findings in patient samples.

Figure 4 In vivo experimental workflow. Diethylnitrosamine and carbon tetrachloride-induced hepatocellular carcinoma in C57BL/6J mice. DEN: Diethylnitrosamine; CCl₄: Carbon tetrachloride.

Figure 5 Evaluation of hepatocellular carcinoma model induction efficacy. A: Appearance and hemotoxylin and eosin staining of liver lobules in the control and model groups. Abnormal mitosis of hepatocellular carcinoma cells is indicated by black arrows; magnification, 200 ×; B: Liver body ratios for the control and model mice; C: Alanine aminotransferase for the control and model mice; D: Aspartate aminotransferase for the control and model mice. H&E: Hemotoxylin and eosin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase. ^aP < 0.001, ^bP < 0.01.

Figure 6 Changes in SLC39A14 expression during hepatocellular carcinoma progression. A batch of mice was collected every 2 weeks during the induction process, and western blotting was performed to detect the changes in ZIP14 protein expression during the development of hepatocellular carcinoma (n = 8). ^aP < 0.05, ^bP < 0.01, ^cP < 0.0001.

Identification of the SLC39A14-overexpression plasmid and siRNA transfection efficiency

To investigate the biological function of SLC39A14 in HCC, Huh7 cells were transfected with SLC39A14 overexpression plasmids, siRNAs, and corresponding controls. The transfection efficiency was analyzed using western blotting. When SLC39A14 was overexpressed, the expression of SLC39A14 in the Huh7 cell line was 2.15-fold greater than that in the control (Figure 7A). For siRNA transfection, all three siRNA targets downregulated the expression of SLC39A14, of which siRNA-1438 exerted the highest inhibitory effect, with a 75.9% reduction in the SLC39A14 expression. Therefore, siRNA-1438 was selected for subsequent experiments (Figure 7B).

Figure 7 Checking transfection efficiency in Huh7 cells. A: ZIP14 expression after overexpression plasmid transfection; B: ZIP14 expression after siRNA transfection. ^aP < 0.05, ^bP <0.01.

The regulation of the oncological properties of HCC cells by SLC39A14

A CCK-8 assay was performed on transfected Huh7 cells. SLC39A14 overexpression effectively inhibited the proliferation of these cells (Figure 8A), whereas the knockdown produced the opposite effect (Figure 8B). Subsequent plate colony formation assay revealed that the number of clones in the SLC39A14 overexpression group decreased by 47.1% compared with that in the control group (Figure 8C). In contrast, the number of clones in the siRNA-1438 group increased by 76.0% when SLC39A14 was knocked down (Figure 8D). These findings demonstrated that SLC39A14 effectively inhibits HCC cell proliferation.

Figure 8 SLC39A14 in cell proliferation. A: Cell counting kit-8 (CCK-8) assay in Huh7 cells after SLC39A14 overexpression; B: CCK-8 assay in Huh7 cells after SLC39A14 knockdown; C: Colony formation experiment in Huh7 cells after SLC39A14 overexpression; D: Colony formation experiment in Huh7 cells after SLC39A14 knockdown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Subsequently, the alterations in the apoptosis of HCC cells after transfection were investigated using flow cytometry. The expressions of the apoptosis-related genes BCL2/BAX and cleaved caspase 3/caspase 3 were detected using Western blotting. The results revealed that the percentage of apoptotic cells increased from 5.28% to 21.73% after SLC39A14 overexpression (Figure 9A). By contrast, the percentage of apoptotic cells decreased from 7.18% to 5.53% after SLC39A14 knockdown (Figure 9B). Compared with those in the control group, the ratio of the apoptosis-associated proteins BCL2/BAX decreased by 52.7% (Figure 10A), and the cleaved caspase 3/caspase 3 ratio increased by 1.63-fold (Figure 10B) after SLC39A14 overexpression. In addition, SLC39A14 knockdown resulted in a 1.27-fold increase in the BCL2/BAX ratio (Figure 10C), and the cleaved caspase 3/caspase 3 ratio decreased by 71.7% (Figure 10D). These observations demonstrate that SLC39A14 can downregulate the expression of antiapoptotic proteins and promote the initiation of the apoptotic program, thus stimulating apoptosis in HCC cells.

Figure 9 SLC39A14 in cell apoptosis. A: Apoptosis detection by flow cytometry after SLC39A14 overexpression; B: Apoptosis detection by flow cytometry after SLC39A14 knockdown. ^aP < 0.05, ^bP < 0.01.

Figure 10  Alterations in the BCL-2/BAX/caspase-3 pathway. A: Pathway proteins BCL2/BAX expression after overexpression plasmid transfection; B: Pathway proteins cleaved caspase 3/caspase 3 expression after overexpression plasmid transfection; C: BCL2/BAX expression after siRNA transfection; D: Cleaved caspase 3/caspase 3 expression after siRNA transfection. ^aP < 0.01, ^bP < 0.05.

Finally, scratch and invasion assays were performed. These results demonstrated that the overexpression of SLC39A14 could significantly inhibit cell migration (Figure 11A) and invasion (Figure 11B), whereas its knockdown promoted these processes (Figure 11C and D). Subsequent western blotting was performed to examine the expression of EMT-related proteins. Compared with that in the control group, the expression of the EMT marker epithelial intercellular adhesion protein E-cadherin was upregulated by 2.02-fold (Figure 12A). Moreover, the expression of the mesenchymal intercellular adhesion protein N-cadherin was decreased by 62.4% (Figure 12B). In addition, the expression of the intermediate filament protein vimentin was reduced by 51.1% (Figure 12C) after SLC39A14 overexpression. Meanwhile, SLC39A14 knockdown downregulated E-cadherin expression by 51.9% (Figure 12D) and upregulated N-cadherin and vimentin expression by 97.5% (Figure 12E) and 65.5% (Figure 12F), respectively. These findings suggest that SLC39A14 inhibits the invasion and migration of HCC cells by suppressing their EMT.

Figure 11 SLC39A14 in cell metastasis. A: Cell metastasis detection by scratch assay after SLC39A14 overexpression; B: Cell metastasis detection by Transwell assay after SLC39A14 overexpression; C: Cell metastasis detection by scratch assay after SLC39A14 knockdown; D: Cell metastasis detection by Transwell assay after SLC39A14 knockdown. ^aP < 0.05, ^bP < 0.01.

Figure 12  Alterations in epithelial-mesenchymal transition. A: Epithelial–mesenchymal transition (EMT)-related protein E-cadherin expression after overexpression plasmid transfection; B: EMT-related protein N-cadherin expression after overexpression plasmid transfection; C: EMT-related protein vimentin expression after overexpression plasmid transfection; D: E-cadherin expression after siRNA transfection; E: N-cadherin expression after siRNA transfection; F: Vimentin expression after siRNA transfection. EMT: Epithelial–mesenchymal transition. ^aP < 0.05, ^bP < 0.01.

Therefore, SLC39A14 can play an anticancer role in HCC by inhibiting tumor cell proliferation, migration, and invasion and by promoting tumor cell apoptosis.

The location of the transcription factor STAT3 as an upstream gene of SLC39A14 in HCC

To delve deeper into the mechanism of the reduced expression of SLC39A14 in HCC, the hTFtarge[35], KnockTF[36], ENCODE[37], Chip-Atlas[38], GTEx[39], and TCGA databases were searched for transcription factors that might regulate SLC39A14 transcription in HCC (Figure 13A). Finally, the candidate transcription factor STAT3 was identified. Based on the JASPAR database, STAT3 was predicted to bind to the promoter region of SLC39A14 (Figure 13B and C; Table 1). The STAT3 level exhibited a significant positive correlation with the gene level of SLC39A14 (r = 0.41, P < 0.0001; Figure 13D). Thus, STAT3 can regulate the expression of SLC39A14 in HCC, thereby modulating the oncological characteristics of HCC.

Figure 13 SLC39A14 is regulated by the transcription factor STAT3. A: Bioinformatics analysis identified STAT3 as one of potential transcription factor driving SLC39A14 expression; B and C: Binding sites for STAT3 in the promoter region of SLC39A14 were identified according to the JASPAR website; D: Pearson’s correlational analysis was performed to explore the correlation between the mRNA levels of STAT3 and SLC39A14 in hepatocellular carcinoma patients in The Cancer Genome Atlas dataset (r = 0.41, P < 0.0001).

Table 1 Predicted binding sites of the transcription factor STAT3 to the promoter region of the SLC39A14 gene in humans via the Jasper online website.

Name	Score	Relative score	Sequence ID	Start	End	Strand	Predicted sequence
MA0144.3.STAT3	12.341	0.962	SLC39A14	495	503	+	TGCCTGGAA
MA0144.3.STAT3	8.139	0.908	SLC39A14	740	748	+	TGTCTGGAA

DISCUSSION

In this study, qRT-PCR and western blotting were performed to demonstrate that the RNA and protein levels of SLC39A14 were significantly lower in HCC tissues than in paracarcinoma tissues. These findings agreed with the results based on the mRNA of SLC39A14 from the TCGA database. Obtaining tissues from patients at various stages in the clinic was challenging. Moreover, there was no statistically significant difference in the expression of SLC39A14 among various HCC clinical stages, as determined by the analysis of clinical samples from the TCGA database. Therefore, a time-graded mouse HCC induction experiment was performed. The results supported the idea that SLC39A14 expression decreases with increasing malignancy of HCC. The effect of SLC39A14 on the biological behavior of Huh7 cells was further investigated. SLC39A14 overexpression inhibited the proliferation, migration, and invasion of Huh7 cells while promoting apoptosis; conversely, SLC39A14 knockdown had the opposite effect. These findings highlight the tumor suppressor role of SLC39A14 in HCC, suggesting that it is a novel target for HCC therapy. Moreover, due to the significant differences in SLC39A14 expression among patients with HCC with varying degrees of malignancy, its expression may aid in the diagnosis and clinical staging of the disease.

Improved diagnostic and prognostic biomarkers for human HCC are clinically important for the early detection of the disease and the timely initiation of appropriate therapy. Slc39, a member of the ZIP (ZRT/IRT-like protein) metal ion transporter family, predominantly facilitates the uptake of metal ions and transports metal ion substrates across the cell membrane into the cytoplasm[40-42]. This family is categorized into four subfamilies, namely, I, II, Gufa, and LIV-1, with a total of 14 highly conserved gene members[43-45]. Of these, SLC39A14 is highly expressed in the small intestine and liver[13,46], and it is selectively cleaved in the body to form two products, ZIP14A and ZIP14B[47]. Numerous recent studies have demonstrated that SLC39A14 is aberrantly expressed in various types of tumors. In prostate cancer, SLC39A14 downregulation is linked to a poor clinical prognosis[14]. In cervical cancer, SLC39A14 can slow down disease progression by activating the P38 MAPK-signaling pathway[15]. Furthermore, in breast cancer, higher levels of SLC39A14 mRNA are associated with better overall survival in patients[16]. A similar trend was observed in this study, with SLC39A14 levels being lower in cancerous tissues than in paracancerous tissues in all 13 patients and decreasing with disease progression. The overexpression of SLC39A14 may play a tumor-suppressor role in HCC, and SLC39A14 could become a new therapeutic target for this disease.

Subsequently, STAT3, the upstream transcription factor of SLC39A14, was identified in multiple databases[48] via bioinformatics analysis. STAT3 was found to contain conserved SLC39A14 promoter region binding sites. In HCC, the STAT3 level exhibited a significant positive correlation with the SLC39A14 level. STAT3, a transcription protein, is located on chromosome 17q21 and comprises 770 amino acids, including two isoforms α (p92) and β (p83)[49,50]. When cytokines such as interleukin-6 are activated, STAT3 is phosphorylated and translocated into the nucleus to increase the expression of target genes[51,52]. A previous study reported that the inactivation of STAT3 via miR520c targeting can inhibit EMT in breast cancer[24]. STAT3 promotes EMT and invasion in non-small cell lung cancer by activating the YAP-signaling pathway[25]. In addition, STAT3 can be activated by HBx, which in turn activates the Twist promoter and exerts a tumor-promoting effect on HCC[26]. In this study, SLC39A14 significantly affected the expression of EMT-related proteins and the invasive migration ability of HCC cells. As SLC39A14 is a member of the ZIP family, it was predicted to be regulated by the transcription factor STAT3, which affects the malignant phenotype of HCC.

Our findings regarding the effect of SLC39A14 on the malignant phenotype of HCC cells provide new insights and an experimental basis for predicting and targeting HCC. Our study is the first to perform in vivo experiments to determine the association between SLC39A14 expression and HCC progression. This study addresses the challenge of obtaining samples from late-stage patients in clinical studies, which is crucial for an in-depth understanding of the relationship between SLC39A14 and HCC development. Currently, the detection of SLC39A14 primarily relies on tissue samples through liver biopsy, where its protein expression can be directly quantified via Western blotting of liver tissues. In preclinical studies, dual-targeted nanocontrast agents targeting SLC39A14 have improved the detection rate of early-stage microscopic HCC (≤ 1 cm) to 92%[18], significantly outperforming the traditional marker AFP, which has a sensitivity of only 42%[53]. This highlights the strong translational potential of SLC39A14. However, the invasive nature of tissue-based testing limits its routine clinical use, and current screening practices continue to rely on noninvasive serologic methods. Given SLC39A14’s role as a metal ion transporter, its functional activity can be indirectly assessed through downstream serum markers such as non-transferrin-bound iron or transferrin[9], both measurable via standardized ELISA assays. These provide a potential indirect approach for monitoring patient status. Moreover, the feasibility of directly detecting ZIP14 protein or its associated exosomes in blood warrants further investigation, as it could help overcome the limitations of AFP in early HCC detection and prognostic evaluation.

However, this study has certain limitations. For instance, only Huh7 cells were used as they are widely employed in HCC metabolism/pathway studies and also because the cells have appropriate expressions of SLC39A14, with superior culture stability and transfection efficiency. In addition, the genetic background, metabolic status, and signaling pathway activation of different cell lines may differ, thereby affecting the response to experimental treatments. Subsequent validation of the key findings in primary hepatocytes can be used to enhance generalizability. Second, the in vivo experiments deserve further investigation. HCC induction was used in our experiments to observe the changes in SLC39A14 expression. However, SLC39A14 expression was not directly regulated in in vivo experiments. The use of an SLC39A14 mRNA vaccine or an in situ transplantation mouse model to observe the effects of changes in SLC39A14 expression on tumor progression would be helpful. We are currently constructing an eukaryotic expression plasmid for SLC39A14 using the PVAX1 vector, aiming to drive high in vivo expression of SLC39A14 in mice. This approach will allow us to investigate its potential effects on symptomatic alleviation and tumor suppression in an HCC mouse model. However, the construction of vaccines and mouse models will be time-consuming and will be an important part of our future experimental program. Finally, the mechanism by which STAT3, as an SLC39A14 transcription factor, regulates its expression has only been examined via bioinformatics analysis, and molecular experiments are yet to be performed. ChIP-qPCR will be used to verify STAT3 enrichment in the promoter region of SLC39A14. To assess the regulatory effect of STAT3 on SLC39A14 expression STAT3 overexpression plasmids and siRNAs will be constructed. Additionally, these tools will be used to determine to verify whether STAT3 can rescue the phenotypic alterations induced by SLC39A14, as observed in previous experiments.

CONCLUSION

This study demonstrated that SLC39A14 is significantly downregulated in HCC and revealed its suppressive role in disease progression and malignant phenotypes. Its inhibitory effect appears to be mediated primarily through the regulation of the BCL-2/BAX/caspase-3-signaling pathway and modulation of EMT. These findings suggest that SLC39A14 functions as a tumor suppressor and holds promise as a diagnostic marker or therapeutic target for HCC. Nonetheless, further in-depth molecular investigations are necessary to validate and expand upon these findings.