The clinical study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China; approval no. 2017055). Due to the retrospective nature of the study and the use of anonymized patient data, the requirement for written informed consent was waived by the ethics committee. All animal experiments were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Zhengzhou University (approval no. 2019053), and were carried out in strict accordance with guidelines related to animal welfare.

The present retrospective cohort study included a total of 72 patients with HCC who underwent radical hepatectomy in the hepatobiliary surgery department of The First Affiliated Hospital of Zhengzhou University between January 2018 and May 2019. Among them, there were 37 male patients and 35 female patients, and the median age of the patients was 62 years (age range, 28–82 years). The specific demographic and clinical baseline characteristics are shown in Table I. The inclusion criteria were as follows: i) No prior treatment before surgery, such as transcatheter hepatic arterial chemoembolization, radiofrequency ablation or immunotherapy; ii) complete virological testing data, including HBV infection status; iii) exclusion of patients with HCV infection; iv) postoperative pathological confirmation of primary HCC; iv) no residual cancer at the surgical margin; and vi) complete clinical data available. HCC and adjacent normal tissues (≥2 cm from the tumor margin and histologically confirmed to be free of residual cancer cells) were preserved in liquid nitrogen. Routine and regular postoperative follow-up was conducted via telephone interviews and outpatient visits. Overall survival was calculated from the time of diagnosis, and follow-up continued for 5 years, ending on June 1, 2024.

The human normal liver cell line MIHA, and the human liver cancer cell lines Huh7, HCCLM3, MHCC97H and Hep3B were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cell lines were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. DMEM and FBS were purchased from Gibco; Thermo Fisher Scientific, Inc. All human-derived cell lines were subjected to short tandem repeat identification before the start of the experiment to verify the cell line identity and exclude cross-contamination. The relevant results are shown in Table SI. Cells were plated in culture dishes and maintained in a humidified incubator at 37°C with 5% CO₂. When cells reached 80–90% confluence, they were trypsinized, subcultured and those in the logarithmic growth phase were used for subsequent experiments.

Total RNA was extracted from tissues, as well as the human normal liver cell line MIHA, and the human liver cancer cell lines Huh7, HCCLM3, MHCC97H and Hep3B using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.). The concentration and purity of the extracted RNA were determined using a NanoDrop 2000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc.). Subsequently, cDNA was synthesized from total RNA according to the manufacturers' instructions of the TaqMan MicroRNA Assays Reverse Transcription Primer (cat. no. 4427975; Thermo Fisher Scientific, Inc.) and the PrimeScript RT Reagent Kit (cat. no. RR047A; Takara Bio, Inc.). Primers specific to the target genes were designed and synthesized by Takara Bio, Inc. (Table II). qPCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR reaction system was prepared according to the kit instructions and the qPCR amplification conditions were as follows: 95°C for 10 min for initial denaturation; followed by 40 cycles at 95°C for 10 sec, 60°C for 20 sec and 72°C for 34 sec. miR-5590-3p expression levels were normalized to U6, whereas the mRNA expression levels of the other genes were normalized to β-actin. The relative expression levels of target genes were calculated using the 2^−ΔΔCq method (22).

Huh7 and Hep3B cells were seeded in 6-well plates at a density of 5×10⁵ cells/well and cultured in a 37°C, 5% CO₂ incubator until they reached ~70% confluence for transfection. Transfection was performed using Lipofectamine^® 2000 reagent (cat. no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The final concentration of miR-5590-3p mimic, mimic-negative control (NC), miR-5590-3p inhibitor, inhibitor-NC, small interfering RNA (siRNA)-NC and siRNA-HOXB2 was 50 nM each; whereas the concentration of HOXB2 overexpression plasmid (Oe-HOXB2) and Oe-NC was 2 µg/well. Oe-HOXB2 and Oe-NC were constructed based on the pcDNA3.1(+) vector backbone (Invitrogen; Thermo Fisher Scientific, Inc.), with Oe-NC serving as an empty vector control without HOXB2 insertion. Various nucleic acids and Lipofectamine 2000 were diluted separately with serum-free medium, mixed after standing at room temperature for 5 min, and incubated at room temperature for 20 min to form transfection complexes, which were then added to the cells for transient transfection. After 6 h of transfection at 37°C and 5% CO₂, the medium was replaced with complete DMEM containing 10% FBS, and the cells were cultured for a further 48 h before subsequent experiments. The following groups were established: Inhibitor NC, mimic-NC, miR-5590-3p inhibitor, miR-5590-3p mimic, siRNA-HOXB2, siRNA-NC, miR-5590-3p mimic + Oe-NC and miR-5590-3p mimic + Oe-HOXB2. A total of 48 h post-transfection, the medium was replaced with fresh DMEM containing 10% FBS for subsequent experiments. siRNA-NC, siRNA-HOXB2, Oe-NC and Oe-HOXB2 were obtained from Suzhou GenePharma Co., Ltd. miR-5590-3p mimic, mimic-NC, inhibitor-NC and miR-5590-3p inhibitor were obtained from Guangzhou RiboBio Co., Ltd. The sequences were as follows: miR-5590-3p mimic, sense: 5′-AAUAAAGUUCAUGUAUGGCAA-3′, antisense: 5′-UUGCCAUACAUGAACUUUAUU-3′; miR-5590-3p inhibitor, 5′-UUGCCAUACAUGAACUUUA-3′; mimic-NC, sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′; inhibitor-NC, 5′-CAGUACUUUUGUGUAGUACAA-3′; si-HOXB2-1, sense 5′-AUCAGCAAUAUACAAUUAUAAAG-3′, antisense 5′-CUUUAUAAUUGUAUAUUGCUGAU-3′; si-HOXB2-2, sense 5′-CACCAUUGAAAGCCAUGAAUUUU-3′, antisense 5′-AAAAUUCAUGGCUUUCAAUGGUG-3′; siRNA-NC, sense 5′-UUCUCCGAACGUGUCACGU-3′, antisense 5′-ACGUGACACGUUCGGAGAA-3′. In the pre-experiment, the siRNA with the best interference effect (si-HOXB2-1) was selected for the subsequent experiments.

Huh7 and Hep3B cells in the logarithmic growth phase were detached using 0.25% trypsin to prepare a single-cell suspension. After cell counting, 3,000 cells/100 µl were seeded into each well of a 96-well plate and cultured in a 37°C, 5% CO₂ incubator. A total of 24, 48 and 72 h after transfection, 10 µl CCK-8 reagent (MilliporeSigma) was added to each well, followed by incubation at 37°C for 2 h. The optical density at 450 nm was measured using a microplate reader (Shanghai Woyuan Technology Co., Ltd.).

Huh7 and Hep3B cell apoptosis was analyzed using Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BD Biosciences). Cells from each group were centrifuged at 300 × g for 5 min at 4°C, washed with PBS and centrifuged again. After discarding the supernatant, the cells were resuspended in 250 µl binding buffer at a concentration of 1×10⁶ cells/ml. Subsequently, 100 µl cell suspension was transferred to a 5 ml flow tube and incubated with 5 µl Annexin V-APC and 5 µl PI for 5 min in the dark. The mixture was gently mixed and incubated at room temperature (~25°C) in the dark for 15 min. After adding 400 µl PBS, the samples were analyzed using a flow cytometer (BD FACSCalibur; BD Biosciences), and data were analyzed using FlowJo software (version 10.8.1; FlowJo; BD Biosciences) to calculate the proportion of apoptotic cells. The total apoptosis rate was calculated as the sum of the apoptosis rates observed in the lower right quadrant (early apoptotic cells) and the upper right quadrant (late apoptotic/necrotic cells).

Huh7 and Hep3B cells were seeded in 6-well plates at a density of 5×10⁵ cells/well and cultured in a 37°C, 5% CO₂ incubator until they reached 90–100% confluence for the wound healing assay. Subsequently, a sterile ruler was placed to guide the generation of a vertical scratch with a 200-µl pipette tip. Three independent replicate wells were set up for each group. After scratching, the supernatant was carefully aspirated, and the wells were rinsed three times with PBS. The cells were then cultured in a low-serum medium containing 0.5% FBS to minimize the influence of cell proliferation. Images were captured under an inverted optical microscope (Olympus IX71; Olympus Corporation) at 0 and 24 h after scratching. Cell migration rates were quantified using ImageJ software (version 1.53; National Institutes of Health).

Transwell chambers (pore size, 8 µm; 24-well format; Corning Life Sciences) were used to detect cell invasion. Matrigel (Corning Life Sciences) was removed from a −20°C freezer and thawed on ice. After thawing, Matrigel and DMEM were mixed at a ratio of 1:8, and 40 µl of the mixture was added to the upper chamber. The chamber was incubated at 37°C for 4 h to allow the gel to solidify. Subsequently, cells were detached and resuspended in serum-free medium at a density of 3×10⁵ cells/ml; a 150-µl aliquot of the cell suspension was added to the upper chamber and 600 µl DMEM containing 10% FBS was added to the lower chamber. Each experimental group was prepared in triplicate. The Transwell chambers were incubated at 37°C for 24 h. After culture, non-migrated cells and residual Matrigel on the upper surface of the chamber were gently wiped away with a cotton swab and washed with PBS. Cells were then fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 30 min. After staining, excess dye was removed by washing with PBS. Images of the stained cells were captured under an inverted optical microscope (Olympus IX71) and the number of invading cells was counted.

Total protein was extracted from tissues and cells using Radio Immunoprecipitation Assay Lysis buffer (cat. no. P0013B; Beyotime Biotechnology). The protein concentration was measured using a BCA protein assay kit (cat. no. P0012; Beyotime Biotechnology). Equal amounts of protein (30 µg) were electrophoresed by SDS-PAGE on 10% gels and transferred to PVDF membranes. The membranes were then blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) at room temperature for 2 h. Subsequently, the membranes were incubated with the following primary antibodies: MYC (cat. no. 18583; 1:1,000; Cell Signaling Technology, Inc.), PCNA (cat. no. 13110; 1:1,000; Cell Signaling Technology, Inc.), MMP2 (cat. no. 40994; 1:1,000; Cell Signaling Technology, Inc.), MMP9 (cat. no. 13667; 1:1,000; Cell Signaling Technology, Inc.), Bcl2 (cat. no. 3498; 1:1,000; Cell Signaling Technology, Inc.), HOXB2 (cat. no. ab220390; 1:1,000; Abcam) and GAPDH (cat. no. 2118; 1:1,000; Cell Signaling Technology, Inc.), overnight at 4°C. After three washes in PBS-0.1% Tween 20, the membranes were incubated with goat anti-rabbit secondary antibodies (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) at 37°C for 2 h. ECL solution (cat. no. P0018AM; Beyotime Biotechnology) was then prepared, and the bands were scanned and images were captured in a dark room. ImageJ software was used to semi-quantify the results. GAPDH was used as the internal control.

The interaction between miR-5590-3p and HOXB2 was examined using an RIP Kit (cat. no. 17-700; MilliporeSigma). Cells were washed twice with pre-chilled PBS and the supernatant was discarded. RIP Lysis Buffer supplied with the kit (supplemented with protease and RNase inhibitors) was added to lyse the cells on ice for 5–10 min. The lysate was then centrifuged at 12,000 × g for 10 min at 4°C and the supernatant was collected as the RIP input sample (Input); 10% of the supernatant was reserved as the Input control and the remaining portion was used for immunoprecipitation. Subsequently, 50 µl Protein A/G magnetic beads were washed with RIP Wash Buffer and resuspended in 100 µl RIP Wash Buffer. Subsequently, 5 µg rabbit anti-Ago2 antibody (cat. no. NBP2-67121; Novus Biologicals; Bio-Techne) or normal rabbit IgG (cat. no. 12-370; MilliporeSigma) as a NC were added and incubated at room temperature for 30 min to form bead-antibody complexes. Then, 100 µl cell lysate was added to the bead-antibody complexes, and RIP Wash Buffer was added to bring the reaction system to 1 ml. The mixture was incubated at 4°C overnight with rotation. The bead-protein complex was then collected, digested with proteinase K, and the RNA was extracted for the detection of HOXB2 and miR-5590-3p enrichment levels by RT-qPCR as aforementioned.

Potential target genes of miR-5590-3p were predicted using the TargetScan database (https://www.targetscan.org), and potential binding sites in the HOXB2 3′-UTR region were analyzed. Potential binding sites of HOXB2 in the MYC promoter region were predicted using the JASPAR database (https://jaspar.elixir.no/). Dual-luciferase reporter plasmids containing the wild-type (WT) or mutant (MUT) sequence of the HOXB2 3′-UTR (psiCHECK-2 vector; Promega Corporation) and dual-luciferase reporter plasmids containing the WT or binding site MUT sequence of the MYC promoter (pGL3-basic vector; Promega Corporation) were constructed by Shanghai GenePharma Co., Ltd. To evaluate the interaction between miR-5590-3p and HOXB2, Huh7 and Hep3B cells were co-transfected with WT or MUT HOXB2 reporter plasmids and either miR-5590-3p mimic or mimic-NC using Lipofectamine 2000. After 24 h, luciferase activity was measured using a Dual-Luciferase Reporter Assay Kit (cat. no. E1910; Promega Corporation), with Renilla luciferase activity serving as the internal control.

Huh7 and Hep3B cells were co-transfected with siRNA-NC or siRNA-HOXB2, and WT or MUT firefly luciferase reporters (MYC-WT or MYC-mut) into Huh7 cells to detect the regulatory effect of HOXB2 on MYC promoter transcriptional activity. Transfection was performed using Lipofectamine 2000 according to the manufacturer's instructions. A total of 48 h after transfection, luciferase activity was detected using the Dual-Luciferase Reporter Assay Kit. Firefly and Renilla luciferase activities were sequentially detected using a multifunctional microplate reader (BioTek; Agilent Technologies, Inc.), and the relative activity ratio of Renilla/Firefly luciferase was calculated. Each experiment was set up with three independent replicates.

ChIP assays were performed using a ChIP kit (cat. no. 17-371; MilliporeSigma) to evaluate HOXB2 enrichment at the MYC promoter. Briefly, Huh7 cells at 70–80% confluence were crosslinked with 1% formaldehyde for 10 min at room temperature. Subsequently, 125 mM glycine was added to terminate the cross-linking reaction, and the cells were washed twice with pre-chilled PBS. After cell lysis, chromatin was sonicated to fragments of 200–500 bp using a sonicator (Scientz-IID; Ningbo Xinchi Biotechnology Co., Ltd., China) under conditions of 20 kHz, 4°C, 10 sec per cycle, with 10 sec intervals, for a total of 15 cycles. The lysate was then centrifuged at 4°C, 16,000 × g for 10 min, and the supernatant was collected as the chromatin sample. The supernatant was then incubated overnight at 4°C with an anti-HOXB2 antibody (cat. no. 65130; 1:50; Cell Signaling Technology, Inc.) or control IgG (cat. no. ab172730; 1:100; Abcam). Subsequently, ~40 µl Protein A/G agarose beads (provided in the kit) per reaction system were added and incubated at 4°C for 2 h to capture the antibody-protein-DNA complexes. After extensive washing, crosslinks were reversed at 65°C overnight and DNA was then extracted using the standard phenol/chloroform/isoamyl alcohol [25:24:1 (v/v/v)] method (23), and the enrichment of DNA in the MYC promoter region was detected by RT-qPCR as aforementioned.

A total of 12 6-week-old male BALB/c nude mice (weight, 18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (strain code: 401). All animals were housed in an specific pathogen-free-grade animal laboratory under conditions of 22–25°C temperature, 60–65% relative humidity, and a 12-h light/dark cycle. Animals had free access to standard feed and drinking water. After one week of acclimation, experiments were initiated. All animal experimental procedures were approved by the Animal Ethics Committee of our institution. Huh7 cells in the logarithmic growth phase were resuspended in PBS to prepare a cell suspension of 1×107 cells/ml; subsequently, 1×10⁶ cells in 100 µl PBS were subcutaneously injected were injected into the left flank of each mouse. A tumor volume of ~50 mm³ was selected as the starting point for intervention to avoid the unstable stage in the early period of transplantation, and to enhance the repeatability and consistency of in vivo tumor growth assessment (24,25). The mice were then randomly divided into two groups (n=6/group): i) miR-5590-3p agomir group: Intratumoral injection of 10 nmol miR-5590-3p agomir (Guangzhou RiboBio Co., Ltd.) per mouse, once every 5 days for a total of 3 weeks; ii) agomir-NC group: Intratumoral injection of 10 nmol agomir-NC (Guangzhou RiboBio Co., Ltd.) per mouse, with the same dosing regimen as miR-5590-3p agomir. During the experiment, the long diameter (L) and short diameter (W) of the tumor were measured every 3 days using a vernier caliper, and the tumor volume was calculated according to the formula V=(L × W²)/2. Humane endpoints set during the experiment included: Tumor volume exceeding 2,000 mm³, tumor longest diameter exceeding 20 mm, notable weight loss (>20%), markedly decreased activity or the presence of ulcers. In the present study, the tumor volume of all animals did not exceed 2,000 mm³ (maximum 1,862 mm³) and the longest diameter did not exceed 20 mm (maximum 19 mm). In the current study, no mice exhibited body weight loss >20%, a marked reduction in activity or ulceration, thus no animals met the humane endpoint criteria. On day 24 after inoculation, the nude mice were anesthetized. Anesthesia was administered via inhalation of isoflurane (RWD Life Science Co., Ltd.), with an induction concentration of 3–4% and a maintenance concentration of 1.5–2.0%. Medical-grade oxygen (100%) was used as the carrier gas (flow rate: 1.0 l/min), delivered through a small animal anesthesia machine. After the animals completely lost their righting reflex and toe-pinch reflex, they were euthanized by cervical dislocation. Subsequently, the tumor tissues were rapidly and completely dissected to measure their volumes, and the tumor masses were weighed using an electronic balance (Sartorius AG). Portions of the tumor tissues were stored at −80°C for RT-qPCR and western blot analysis. Each experiment was independently repeated three times.

All data were analyzed using SPSS version 22.0 software (IBM Corp.). Measurement data were presented as the mean ± standard deviation. For comparisons between paired samples (such as HCC tissues and corresponding adjacent tissues), a paired Student's t-test was used; for comparisons between two independent samples, an unpaired Student's t-test was employed. Comparisons among multiple groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. CCK-8 assay and tumor volume data in nude mice were analyzed using two-way or mixed ANOVA test followed by Turkey's or Šidák's post-hoc test, as appropriate. For categorical variables, the χ² test was predominantly employed. Patients were stratified into high and low expression groups based on the median expression levels of miR-5590-3p and HOXB2. Prognostic survival time was assessed using the Kaplan-Meier method, and differences between curves were compared using the log-rank test. To evaluate the correlation between miR-5590-3p and HOXB2 expression levels in HCC clinical tissue samples, Pearson correlation analysis was performed. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis showed that miR-5590-3p expression was decreased in HCC tissues compared with that in paired adjacent tissues (Fig. 1A). In addition, its expression in Huh7, HCCLM3, MHCC97H and Hep3B cells was lower than that in MIHA cells, with the lowest expression observed in Huh7 cells (Fig. 1B). Conversely, the expression of HOXB2 was elevated in HCC tissues relative to adjacent tissues (Fig. 1C), and its expression in Huh7, HCCLM3, MHCC97H and Hep3B cells was higher than that in MIHA cells, with the highest expression observed in Huh7 cells (Fig. 1D).

A total of 72 paired HCC and adjacent tissues were stratified into high- and low-expression groups based on the median expression levels of miR-5590-3p and HOXB2. No significant associations were observed between their expression and sex, age, cirrhosis, Child-Pugh grade (26), α-fetoprotein level, HBV infection, tumor embolism and recurrence (Table I). However, the expression of miR-5590-3p and HOXB2 in patients with HCC was associated with tumor-node-metastasis (TNM) stage (27) and pathological grade. Patients with advanced TNM stage and poorer pathological grade exhibited lower miR-5590-3p and higher HOXB2 expression, indicating that both markers are closely associated with HCC malignancy. Kaplan-Meier survival analysis further revealed that low miR-5590-3p expression was associated with poor prognosis in HCC (Fig. 1E).

RT-qPCR confirmed that transfection with the miR-5590-3p mimic markedly increased miR-5590-3p expression in Huh7 and Hep3B cells, whereas transfection with the miR-5590-3p inhibitor markedly decreased endogenous miR-5590-3p levels (Fig. 2A), indicating successful establishment of overexpression and inhibition models.

To examine the role of miR-5590-3p in regulating HCC cell proliferation, CCK-8 assays were conducted to quantify changes in cell viability. The results showed that ectopic overexpression of miR-5590-3p markedly suppressed proliferation in both Huh7 and Hep3B cells, whereas inhibition of miR-5590-3p significantly enhanced the proliferative activity of Huh7 and Hep3B cells (Fig. 2B). These findings indicate that miR-5590-3p functions as a negative modulator of HCC cell proliferation.

Cell migratory and invasive behaviors were further evaluated using scratch test and Transwell assays, respectively. miR-5590-3p overexpression led to a pronounced reduction in the number of cells penetrating the Transwell membrane and delayed wound closure in Huh7 and Hep3B cells (Fig. 2C and D). By contrast, silencing miR-5590-3p markedly increased invasive cell counts and accelerated scratch healing. These results suggest that miR-5590-3p restrains HCC cell motility and invasiveness, whereas its suppression promotes acquisition of a more aggressive phenotype.

In addition, flow cytometric analysis demonstrated that miR-5590-3p overexpression significantly increased apoptosis rate in Huh7 cells, while miR-5590-3p inhibition decreased apoptotic rates (Fig. 2E). This indicates that miR-5590-3p facilitates apoptotic progression, whereas its downregulation enables apoptotic resistance.

Taken together, miR-5590-3p overexpression inhibits HCC cell proliferation, migration and invasion while enhancing apoptosis, whereas its suppression produces opposite effects, supporting a tumor-suppressive role of miR-5590-3p in HCC progression.

To clarify the regulatory relationship between miR-5590-3p and HOXB2, RT-qPCR was first performed to evaluate changes in HOXB2 expression in Huh7 and Hep3B cells following transfection with the miR-5590-3p mimic or inhibitor. Compared with in the NC-transfected cells, suppression of miR-5590-3p notably increased HOXB2 mRNA levels, whereas overexpression of miR-5590-3p markedly reduced HOXB2 expression (Fig. 3A). These expression trends were consistent with the reciprocal patterns observed in clinical HCC tissues, indicating a potential negative regulatory interaction between miR-5590-3p and HOXB2.

Subsequently, bioinformatics prediction using the TargetScan database identified a putative binding site for miR-5590-3p within the 3′-UTR region of HOXB2 that was highly complementary to the miR-5590-3p seed sequence (Fig. 3B). Pearson correlation analysis performed on clinical HCC specimens further demonstrated a significant inverse correlation between miR-5590-3p and HOXB2 expression (Fig. 3C), supporting their antagonistic regulatory relationship in vivo.

To validate the direct targeting effect, dual-luciferase reporter constructs containing HOXB2-WT or HOXB2-MUT 3′-UTR sequences were co-transfected into cells with the miR-5590-3p mimic. The luciferase activity of the HOXB2-WT reporter was reduced by miR-5590-3p overexpression, whereas no notable change was observed in the HOXB2-MUT group (Fig. 3D), confirming that miR-5590-3p specifically binds to the 3′-UTR of HOXB2.

Furthermore, RIP assays demonstrated that both miR-5590-3p and HOXB2 mRNA were enriched in Ago2-associated immunoprecipitates (Fig. 3E), indicating that they coexist within the RNA-induced silencing complex and supporting a direct molecular interaction. Survival analysis further revealed that patients with HCC and elevated HOXB2 expression exhibited significantly shorter overall survival (Fig. 3F), suggesting that aberrant HOXB2 upregulation may be closely associated with an unfavorable prognosis.

Collectively, these multi-level experimental findings indicate that HOXB2 is a direct downstream target of miR-5590-3p, and that miR-5590-3p participates in HCC progression by negatively regulating HOXB2 expression.

To further elucidate the functional role of HOXB2 in HCC, siRNA-HOXB2 or siRNA-NC was transfected into Huh7 and Hep3B cells. RT-qPCR confirmed that HOXB2 mRNA levels were markedly reduced following siRNA-HOXB2 transfection in both cell lines (Fig. 4A), indicating efficient knockdown. Cell proliferation assays using CCK-8 demonstrated that suppression of HOXB2 decreased the proliferative capacity of Huh7 and Hep3B cells (Fig. 4B). Scratch and Transwell assays further revealed that HOXB2 knockdown markedly delayed scratch closure in Huh7 cells, and significantly reduced invasive activity in both Huh7 and Hep3B cells (Fig. 4C and D), indicating that HOXB2 contributes to HCC cell migration and invasion. In addition, flow cytometric analysis showed that HOXB2 silencing increased the proportion of apoptotic Huh7 cells (Fig. 4E), suggesting that inhibition of HOXB2 facilitates apoptosis in HCC cells.

Taken together, these results demonstrate that HOXB2 promotes proliferation, migration and invasion while inhibiting apoptosis in HCC cells, supporting its tumor-promoting role during HCC progression.

To further determine the influence of HOXB2 expression on malignant behaviors of HCC cells, first, RT-qPCR was performed to verify the transfection efficiency of Oe-HOXB2. The results showed that the mRNA expression levels of HOXB2 were significantly increased in the Oe-HOXB2 group compared with those in the Oe-NC group (Fig. 5A). Subsequently, Huh7 and Hep3B cells were co-transfected with the miR-5590-3p mimic and Oe-HOXB2, followed by evaluation of the malignant behaviors of cells in each group, including cell proliferation, migration, invasion and apoptosis. Cell proliferation was detected using the CCK-8 method. The results showed that, compared with in the miR-5590-3p mimic + Oe-NC group, the proliferation of Huh7 and Hep3B cells was significantly increased in the miR-5590-3p mimic + Oe-HOXB2 group (Fig. 5B). Cell invasion was further evaluated using the Transwell assay. The results indicated that, compared with in the miR-5590-3p mimic + Oe-NC group, the membrane-penetrating invasion of Huh7 and Hep3B cells was significantly enhanced in the miR-5590-3p mimic + Oe-HOXB2 group (Fig. 5C). Furthermore, the results of the wound healing assay showed that, compared with in the miR-5590-3p mimic + Oe-NC group, the migration of Huh7 cells was significantly promoted in the miR-5590-3p mimic + Oe-HOXB2 group (Fig. 5D). In addition, flow cytometric analysis of cell apoptosis revealed that, compared with in the miR-5590-3p mimic + Oe-NC group, the proportion of apoptotic Huh7 cells was significantly lower in the miR-5590-3p mimic + Oe-HOXB2 group (Fig. 5E), suggesting that reduced HOXB2 expression may promote apoptosis in HCC cells.

Collectively, these findings indicate that alterations in HOXB2 expression modulate multiple malignant phenotypes of HCC cells, including proliferation, migration, invasion and apoptosis.

HOXB2 is known to function as a transcriptional regulator and has been implicated in controlling downstream gene expression (28). To explore whether HOXB2 mediates HCC cell progression through transcriptional mechanisms, potential HOXB2 binding sites were first predicted using the JASPAR database. A highly conserved binding motif (GCTAATTAAA) was identified within the MYC promoter region (Fig. 6A), suggesting that MYC may be a direct transcriptional target of HOXB2. MYC, as a classic proto-oncogene, serves a crucial role in the occurrence and development of various tumors (29). RT-qPCR analysis showed that MYC expression was significantly elevated in HCC tissues compared with that in adjacent non-tumorous tissues (Fig. 6B). Moreover, MYC mRNA levels were markedly increased in Huh7, HCCLM3, MHCC97H and Hep3B cells relative to normal MIHA cells, with Huh7 cells exhibiting the highest level of expression (Fig. 6C), suggesting aberrant MYC activation in HCC. To verify the regulatory relationship between HOXB2 and MYC, HOXB2 was silenced in Huh7 cells. Downregulation of HOXB2 significantly decreased MYC mRNA expression (Fig. 6D). Dual-luciferase reporter assays further demonstrated that HOXB2 silencing markedly reduced MYC promoter-driven luciferase activity, whereas mutation of the predicted binding site abolished this effect (Fig. 6E), indicating site-specific transcriptional regulation. ChIP assays further confirmed that HOXB2 was directly enriched at the MYC promoter region, and this enrichment was significantly reduced following HOXB2 silencing (Fig. 6F), supporting direct binding of HOXB2 to the MYC promoter at the chromatin level.

To evaluate the functional significance of the HOXB2-MYC axis, Huh7 cells were co-transfected with Oe-HOXB2 and siRNA-MYC. RT-qPCR showed that HOXB2 overexpression increased MYC expression, as well as the levels of proliferation-associated PCNA, migration-associated MMP2 and invasion-associated MMP9, while reducing the expression of the apoptosis-related gene Bcl2; these effects were reversed upon MYC knockdown (Fig. 6G). Western blot analysis yielded consistent results at the protein level (Fig. 6H).

Collectively, these data demonstrate that HOXB2 directly binds to the MYC promoter and activates its transcription, which may regulate gene programs governing proliferation, migration, invasion and apoptosis in HCC cells.

To further validate the regulatory role of miR-5590-3p in HCC progression at the in vivo level, a nude mouse subcutaneous xenograft model was established. Dynamic monitoring of tumor growth demonstrated that, compared with in the control group, xenografts derived from miR-5590-3p-overexpressing cells exhibited markedly attenuated growth (Fig. 7A). Tumor growth curves further showed that the increase in tumor volume was markedly delayed in the miR-5590-3p overexpression group (Fig. 7B). At the experimental endpoint, both tumor volume and tumor weight were significantly reduced compared with those in the control group (Fig. 7C), suggesting that miR-5590-3p effectively inhibits the growth of HCC xenografts in vivo.

To further investigate molecular alterations within the miR-5590-3p/HOXB2/MYC axis in xenograft tissues, RT-qPCR and western blot analysis were performed. RT-qPCR results demonstrated that miR-5590-3p expression was increased in xenografts from the overexpression group, whereas the mRNA levels of HOXB2 and MYC were markedly decreased (Fig. 7D). Consistently, western blot analysis confirmed that HOXB2 and MYC protein levels were substantially reduced in tumors overexpressing miR-5590-3p (Fig. 7E).

Collectively, these in vivo findings further corroborate that miR-5590-3p overexpression suppresses HCC xenograft growth, and that its tumor-inhibitory effect is accompanied by coordinated downregulation of HOXB2 and MYC, in agreement with the in vitro observations.