A novel candidate tumor biomarker progestin and adipoQ receptor 3 regulates cell metastasis through transforming growth factor-β pathway in hepatocellular carcinoma

Patients and tissue samples

HCC specimens and their corresponding adjacent non-cancerous tissues, located at least 2 cm from the tumor site, were obtained from HCC patients who previously underwent hepatectomy at the General Hospital of Tianjin Medical University. Histopathological examination confirmed the diagnosis, and none of the patients underwent radiotherapy or chemotherapy before surgery. A total of 106 paired formalin-fixed paraffin-embedded specimens were subjected to PAQR3 immunohistochemistry (IHC) and immunoreactivity score (IRS) scoring. For each specimen, three non-overlapping high-power fields (×200) were evaluated, and the scores were averaged. Two board-certified pathologists, blinded to clinical data, independently scored the slides, and any discrepancies were resolved by consensus.

All patients provided written informed consent before sample collection. The study protocol was reviewed and approved by the Institutional Ethics Committee of Tianjin Medical University (Approval No.8217102738). All procedures involving human participants were performed in accordance with the ethical standards of the Institutional Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

IHC

Before the assay, liver tissues were sectioned at 4 μm thickness. The samples were rehydrated through graded ethanol concentrations from 50% to 100% after deparaffinization with xylene. Antigen retrieval was performed using ethylenediaminetetraacetic acid. Sections were then blocked with 10% normal goat serum (Solarbio, SL038) for 1 h at room temperature to reduce non-specific binding. Subsequently, the sections were incubated overnight at 4°C with the PAQR3 primary antibody (1:200; Affinity Biosciences, DF4988, China), followed by incubation with an HRP-conjugated secondary antibody (1:500; Servicebio, GB23303) for 1 h at room temperature. Staining was developed using diaminobenzidine (DAB; Wuhan Golden Wing Industry and Trade Co., Ltd., 91-95-2 Wuhan, Hubei, China). Finally, positive staining was visualized and photographed with an inverted microscope (Leica Microsystems, Wetzlar, Germany). The stained sections were reviewed and scored independently by two pathologists blinded to clinical outcomes. PAQR3 expression was evaluated based on staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the proportion of positive tumor cells (0, <5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, >75%). The final IRS was obtained by multiplying the intensity and proportion scores, yielding a range from 0 to 12. For statistical analysis, IRS scores of 0–4 were classified as low expression, and 5–12 as high expression.^[15]

Cell culture, treatment, and transfection

HCC cell line Huh-7 (SCSP-526) was sourced from authenticated cell cultures’ national collection, Human Hepatic Cell line 5 (HHL-5) (BFN6072012687) normal human hepatocytes and HCC cell line SNU-449 (CVCL_0454) were sourced from American Type Culture Collection, and Hep3B (CL-0102) was purchased from Procell (Wuhan, China). All cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA) in 5% CO₂ at 37°C, added with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin.

Hep3B cells were treated with 2.5 μM of the TGF-b1 inhibitor SB-431542 (AbMole Bioscience, Inc., M57235, Houston, Texas, USA) for 24 h, or with 10 ng/mL TGF-b1 (AbMole Bioscience, Inc., M9391, Houston, Texas, USA) for 24 h.

A PAQR3 overexpressing plasmid DNA (Oe-PAQR3) was used to induce PAQR3 overexpression in Hep3B cells. Cells were transfected with 100 nM of the recombinant constructs using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc., 11668019, Waltham, Massachusetts, USA) for 48 h at 37°C. After a 48-h incubation at 37°C, the transfection efficiency of Oe-PAQR3 was evaluated, and the transfected cells were subsequently used for further experiments.

All HCC cell lines (Huh-7, Hep3B, SNU-449) and the normal hepatocyte line HHL-5 used in this study were authenticated by short-tandem repeat (STR) profiling (performed by [specify service provider, e.g., China Center for Type Culture Collection, Wuhan, China]) to verify their identity and to exclude cross-contamination. Mycoplasma contamination was routinely monitored using a polymerase chain reaction-based mycoplasma detection kit (e.g., Venor^®GeM, Minerva Biolabs, Berlin, Germany), and all cell lines tested negative before experimental use.

Western blotting (WB)

Proteins were isolated carefully from sample cells using RIPA lysis buffer (Shenzhen Ziker Biotechnology Co., Ltd., ZK-L2341, Shenzhen, Guangdong, China) and quantified with a bicinchoninic acid protein assay kit (Shanghai Kuaibo Biotechnology Co., ZK-L2356, Ltd., Shanghai, China) according to the standard protocol. After separation by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were then transferred to polyvinylidene fluoride membranes. The membranes, blocked with 5% non-fat milk, were incubated overnight at 4°C with primary antibodies (Abcam plc, Cambridge, UK) at the following dilutions: GPX4 (1:400, ab262509), SLC7A11 (1:400, ab307601), ACSL4 (1:1500, ab269613), PAQR3 (1:100, ab236798), E-cadherin (1:1000, ab314063), Vimentin (1:2000, ab92547), N-cadherin (1:1000, ab76011), TGF-b1 (1:1000, ab215715), p-Smad3 (1:2000, ab74062), p-Smad2 (1:1000, ab184557), Smad2 (1:2000, ab40855), Smad3 (1:500, ab40854), and b-actin (1:2500, ab8226). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody (Proteintech Group, Inc., S0001, Rosemont, Illinois, USA) at room temperature. Protein bands were detected utilizing an Image Analysis System (Bio-Rad Laboratories, Inc., ChemiDoc^™, Hercules, California, USA) with an Enhanced Chemiluminescence Fluorescence Detection kit (Cytiva, Uppsala, Sweden).

Wound healing assay

Cells were seeded into 24-well plates, which were then cultured overnight. When confluency reached 95%, a linear scratch was made using a 200 μL pipette tip. Images of the wound were captured at 0 and 24 h using a microscope (Leica, Mateo FL, Germany), and the wound closure rate was quantitatively assessed using ImageJ software.

Transwell assay

A 200 μL cell suspension in serum-free DMEM was added into the upper chamber of the Transwell, while 600 μL of DMEM supplemented with 2% FBS was placed into the lower chamber. Following a 24 h incubation at 37°C, the cells were fixed using 4% paraformaldehyde. Cells adhering to the underside of the membrane were then stained with crystal violet solution for 15 min (Beyotime Institute of Biotechnology, Y268091, Haimen, Jiangsu, China), and images were randomly captured using an inverted microscope (Leica Microsystem, Mateo FL, Wetzlar, Germany).

Thiobarbituric acid reactive substance (TBARS) assay

Lipid peroxidation in HCC cells following the indicated treatments was assessed using a TBARS Assay Kit (Elabscience Biotechnology Co., Ltd., E-BC-K298-M, Wuhan, Hubei, China). Briefly, cells were treated with butylated hydroxyanisole (BHA; 10 μL; 500 mM) and trichloroacetic acid (0.5 mL; 15%), followed by centrifugation at 10,000 × g for 10 min at 4°C. The obtained supernatant was thoroughly mixed with 0.5 mL of 0.375% and boiled for 10 min. After cooling, TBARS levels were determined at 532 nm using a microplate reader (Bio-Tek Instruments, Inc., Varioskan ALF, Winooski, Vermont, USA).

Lipid reactive oxygen species (ROS) staining

ROS levels in HCC cells were assessed using C11-BODIPY 581/591 (Thermo Fisher Scientific, Waltham, D3861, Massachusetts, USA). The cell culture medium was supplemented with C11-BODIPY 581/591 to a final concentration of 5 μM. Following incubation in the dark at 37 °C for 30 min and subsequent PBS washes, the cells were imaged using a fluorescence microscope (Leica Microsystems, Mateo FL, Wetzlar, Germany).

Intracellular iron measurement

Following the indicated treatments, HCC cells were incubated with 10 μM Phen Green SK (PGSK, Glpbio, GC40243, Houston, Texas, USA) for 10 min. Excess PGSK was detached and carefully eliminated by washing the cells twice with PBS. The cells were then trypsinized, resuspended in PBS containing 5% FBS, and ferrous iron levels were determined using a flow cytometer (Beckman Coulter, Inc., CytoFLEX S, Brea, California, USA).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, LLC, San Diego, CA, USA). Data are presented as mean±standard deviation. Comparisons between two groups were performed using an unpaired Student‘s t-test, while comparisons among three or more groups were analyzed by one-way analysis of variance, followed by Tukey’s post hoc test for multiple comparisons. Statistical significance was defined as P<0.05. Exact P-values are reported in the Results and figure legends whenever possible; only when P-values were extremely small (< 0.001), thresholds were used for clarity. For paired ordinal data (e.g., IRS for tumor vs. adjacent tissues), the Wilcoxon signed-rank test was applied, and data are displayed as individual values with median and interquartile range unless otherwise stated.

PAQR3 expression is significantly reduced in HCC tissues

IHC analysis of 106 paired HCC and adjacent non-tumor tissues revealed markedly lower PAQR3 expression in HCC tissues compared with adjacent counterparts. Representative IHC staining images are shown [Figure 1a]. Quantitative evaluation using the IRS confirmed this observation, demonstrating a significant reduction of PAQR3 expression in tumor tissues relative to paired adjacent tissues (n = 106, ^✶✶✶P < 0.001, Wilcoxon signed-rank test; [Figure 1b]).

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Figure 1:

PAQR3 expression is reduced in HCC tissues. (a) Representative immunohistochemistry images of PAQR3 expression in adjacent non-tumor liver and HCC tissues (scale bars, 100 μm). (b) Immunoreactivity score of PAQR3 expression in 106 paired HCC and adjacent tissues; each dot represents an individual patient, and lines indicate median and interquartile range. The analysis included 106 patient samples, Statistical analysis was performed using the Wilcoxon signed-rank test. ^✶✶✶P < 0.001, ns: not significant. PAQR3: Progestin and adipoQ receptor 3, HCC: Hepatocellular carcinoma.

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Overexpression of PAQR3 inhibits HCC cell metastasis

PAQR3 expression in HCC cell lines was measured using quantitative polymerase chain reaction and WB. The results indicated that PAQR3 levels were markedly lower in HCC cell lines compared to HHL-5 normal human hepatocytes (P < 0.001, [Figure 2a and b]). To further explore the mechanism of PAQR3, it was overexpressed through transfection with Oe-PAQR3 [Figure 2c and d]. The effects of PAQR3 overexpression on HCC cell invasion and migration were then examined. As shown in Figure 2e-h, PAQR3 overexpression suppressed both invasion and migration of HCC cells. Furthermore, WB results demonstrated that PAQR3 overexpression led to a marked increase in the expression of the epithelial-mesenchymal transition (EMT)-related protein E-cadherin, while the levels of N-cadherin and Vimentin were notably decreased (P < 0.001, [Figure 2i-l]).

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Figure 2:

Overexpression of PAQR3 inhibits HCC cell metastasis. (a, b) Relative PAQR3 expression in HCC cells. (c, d) Relative PAQR3 expression following transfection with Oe-PAQR3 in HCC cells. (e, f) Cell migration was assessed by wound healing assay (scale bars: 200 μm). Data are representative of three independent experiments. (g, h) Cell invasion was assessed using transwell chamber assay (scale bars: 50 μm). Results are from three independent experiments performed in triplicate. (i-l) Protein levels of EMT-related proteins E-cadherin, N-cadherin and Vimentin were estimated by western blotting. Blots are representative of three independent experiments. ^✶✶✶: P < 0.001, ns, not significant. PAQR3: Progestin and adipoQ receptor 3, HCC: Hepatocellular carcinoma, EMT: Epithelial-mesenchymal transition, Oe-NC: Overexpression negative control.

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Overexpression of PAQR3 induces ferroptosis in HCC cells

To investigate the effect of PAQR3 on ferroptosis in HCC cells, lipid peroxidation was first assessed using the TBARS assay. The results showed that PAQR3 overexpression significantly elevated TBARS production compared with the Control and Oe-NC groups (P < 0.001, [Figure 3a]). Consistently, lipid ROS levels assessed by C11-BODIPY staining revealed a marked increase in oxidized C11 signal and a decrease in non-oxidized C11 signal upon PAQR3 overexpression (P < 0.001, [Figure 3b and c]). Intracellular Fe²⁺ levels were also significantly elevated in the Oe-PAQR3 group compared with controls (P < 0.001, [Figure 3d and e]). WB analysis demonstrated substantial alterations in ferroptosis-related proteins following PAQR3 overexpression, including downregulation of SLC7A11 and GPX4 expression [Figure 3f-h] and upregulation of ACSL4 expression (P < 0.001, [Figure 3f and i]). These results indicate that PAQR3 promotes ferroptosis in HCC cells by enhancing lipid peroxidation, ROS and Fe²⁺ accumulation, and by regulating key ferroptosis-associated proteins.

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Figure 3:

Effects of PAQR3 overexpression on ferroptosis in HCC cells. (a) Lipid peroxidation levels measured by TBARS assay. (b, c) ROS levels detected by C11-BODIPY staining (red/green ratio; scale bars: 50 μm). (d, e) Intracellular Fe²⁺ levels assessed by fluorescence intensity (scale bars: 50 μm). (f) Representative western blot bands of ferroptosis-related proteins (SLC7A11, GPX4, ACSL4, and b-actin). (g) Quantification of relative SLC7A11 expression. (h) Quantification of relative GPX4 expression. (i) Quantification of relative ACSL4 expression. ^✶✶✶P < 0.001, ns: Not significant. Data in (a, g, h, i) are presented as mean±standard deviation from three independent experiments. Data in (b-e) and blot in (f) are representative of three independent experiments. PAQR3: Progestin and adipoQ receptor 3, HCC: Hepatocellular carcinoma, TBARS: Thiobarbituric acid reactive substance, ROS: Reactive oxygen species, Oe-NC: Overexpression negative control.

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Overexpression of PAQR3 inhibits the TGF-β pathway

To further explore the effect of PAQR3 on the TGF-b pathway, the expression of key pathway-related proteins was examined. Western blot analysis showed that overexpression of PAQR3 led to a significant reduction in TGF-b1, p-Smad2, and p-Smad3 levels in HCC cells [Figure 4a]. Quantitative analysis further confirmed a marked decrease in TGF-b1 expression in the Oe-PAQR3 group compared with the control and Oe-NC groups (P < 0.001, [Figure 4b]). Similarly, p-Smad2 [Figure 4c] and p-Smad3 [Figure 4d] expression levels were markedly reduced upon PAQR3 overexpression (P < 0.001). Collectively, these findings indicate that PAQR3 overexpression suppresses the TGF-b/Smad signaling pathway in HCC cells.

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Figure 4:

Effects of PAQR3 overexpression on the TGF-b pathway in HCC cells. (a) Representative western blot analysis of TGF-b1, p-Smad2, p-Smad3, Smad2, and Smad3 expression in HCC cells. (b) Quantification of relative TGF-b1 protein expression. (c) Quantification of relative p-Smad2 protein expression. (d) Quantification of relative p-Smad3 protein expression. ^✶✶✶P < 0.001, ns: Not significant. The blot in (a) is representative of three independent experiments. Quantitative data in (b-d) are presented as mean±standard deviation from three independent experiments. PAQR3: Progestin and adipoQ receptor 3, HCC: Hepatocellular carcinoma, TGF-b: Transforming growth factor-b, Oe-NC: Overexpression negative control.

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PAQR3 regulates HCC cell metastasis and ferroptosis through the TGF-β pathway

To investigate whether the effects of PAQR3 on HCC cells are mediated by the TGF-b pathway, cells were treated with TGF-b1 stimulation and the TGF-b inhibitor SB-431542. Wound healing and Transwell assays demonstrated that TGF-b1 treatment significantly promoted cell migration and invasion, whereas SB-431542 attenuated these abilities compared with the Oe-PAQR3 group (P < 0.001, [Figure 5a-d]). WB analysis further revealed that PAQR3 overexpression increased E-cadherin expression and decreased N-cadherin and Vimentin levels; these effects were partially reversed by TGF-b1 stimulation and further strengthened by SB-431542 treatment (P < 0.001, [Figure 5e-h]). Next, we examined ferroptosis-related changes. TBARS assays showed that lipid peroxidation levels were markedly elevated by PAQR3 overexpression, partially suppressed by TGF-b1, and enhanced by SB-431542 (P < 0.05, [Figure 5i]). Consistently, ROS accumulation, assessed by C11-BODIPY staining and red/green fluorescence ratio, followed the same trend. Similarly, intracellular Fe²⁺ levels were increased by PAQR3, reduced by TGF-b1, and further elevated by SB-431542 (ROS: P < 0.05; Fe²⁺: P < 0.001, [Figure 5j-m]). Western blot analysis of ferroptosis-related proteins showed that PAQR3 overexpression downregulated SLC7A11 and GPX4 [Figure 5n-p]) while upregulating ACSL4 [Figure 5q]; these changes were reversed by TGF-b1 and reinforced by SB-431542. Collectively, these results demonstrate that PAQR3 regulates both EMT-mediated metastasis and ferroptosis in HCC cells through the TGF-b/Smad signaling pathway.

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Figure 5:

PAQR3 regulates HCC cell metastasis and ferroptosis through the TGF-b pathway. (a) Wound healing assay images showing cell migration in different groups (scale bars: 200 μm). (b) Quantification of cell migration rates from wound healing assay. (c) Transwell invasion assay images of HCC cells in different groups (scale bars: 50 μm). (d) Quantification of invaded cell counts from the Transwell assay. (e) Representative Western blot images of EMT-related proteins (E-cadherin, N-cadherin, Vimentin, b-actin). (f) Quantification of relative E-cadherin expression. (g) Quantification of relative N-cadherin expression. (h) Quantification of relative Vimentin expression. (i) TBARS assay showing lipid peroxidation levels. (j) C11-BODIPY staining showing ROS levels (merge, oxidized C11, non-oxidized C11, scale bars: 50 μm). (k) Representative images of intracellular Fe²⁺ fluorescence (scale bars: 50 μm). (l) Quantification of red/green fluorescence ratio for ROS levels. (m) Quantification of intracellular Fe²⁺ fluorescence intensity. (n) Representative western blot images of ferroptosis-related proteins (SLC7A11, GPX4, ACSL4, b-actin). (o) Quantification of relative SLC7A11 expression. (p) Quantification of relative GPX4 expression. (q) Quantification of relative ACSL4 expression. ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001, ns: Not significant. Images in (a, c, e, j, k, n) are representative of three independent experiments. Quantitative data in (b, d, f-h, i, l, m, o-q) are presented as mean±SD from three independent experiments. PAQR3: Progestin and adipoQ receptor 3, HCC: Hepatocellular carcinoma, EMT: Epithelial-mesenchymal transition, TBARS: Thiobarbituric acid reactive substance, Oe-NC: Overexpression negative control.

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