In this study, four human AML cell lines—HL-60, THP-1, KG-1, and AML193—were used to cover distinct differentiation stages and molecular subtypes. HL-60 is a type of peripheral blood leukocyte derived from a patient with acute promyelocytic leukemia [21]. Both THP-1 and AML-193 cells exhibit monocytic lineage characteristics and serve as complementary models for monocytic leukemia [22]. KG-1 represents an early myeloblastic leukemia cell line with immature hematopoietic characteristics [23]. Normal human bone marrow stromal cell line HS-5 and AML cell lines (HL-60, AML-193, KG-1, and THP-1) were purchased from ATCC and cultured in RPMI-1640 medium (12633020, Gibco, Grand Island, New York, USA) supplemented with 10% FBS (A5256701, Gibco, Grand Island, New York, USA) and 100 U/mL penicillin/streptomycin (15140122, Gibco, CA, USA). All the cell lines were incubated at 37 °C in a 5% CO₂ humidified atmosphere. All the cell lines were authenticated by STR analysis and regularly tested for mycoplasma contamination using PCR-based methods, with all the results being negative. Detailed methods and results are provided in the Supplementary Material.

Gene overexpression vectors (oe-WNK1 and oe-NC) and short hairpin RNA plasmids targeting WNK1 or PTBP1 (sh-WNK1-1/2/3, sh-PTBP1-1/2/3) were obtained from TranSheepBio (Shanghai, China). These sequences are provided in the Supplementary Material. For transfection, AML cells were seeded into 6-well plates and cultured to approximately 70–80% confluence. Cells were transfected with 2 µg of plasmid DNA per well using Lipofectamine 3000 reagent (L3000015, Invitrogen, Carlsbad, CA, USA). Briefly, plasmid DNA was diluted in Opti-MEM medium and mixed with Lipofectamine 3000 at the ratio recommended by the manufacturer. The transfection mixtures were added to the cells in a dropwise manner, followed by incubation at 37 °C under 5% CO₂. After 6 h, the medium was replaced with fresh complete medium. Cells were collected at 48 h for subsequent analyses and at 24–48 h for functional assays, including migration and invasion.

Proteins from AML cells and liver tumor nodules were extracted using RIPA buffer (P0013B, Beyotime, Shanghai, China) containing protease inhibitor cocktails. Protein concentrations were measured using a BCA kit (P0012, Beyotime, Shanghai, China). Proteins (30 µg) were resolved on SDS-PAGE and electrotransferred to PVDF membranes (IPVH00010, Merck Millipore, Darmstadt, Germany). Following a 2-h block with 5% nonfat milk, blots were incubated overnight at 4 °C with antibodies against WNK1 (ab316279, 1/1000; Abcam, Cambridge, UK), PTBP1 (ab133734, 1/10,000; Abcam, Cambridge, UK), E-cadherin (ab314063, 1/1000; Abcam, Cambridge, UK), N-cadherin (ET1607-37, 1/1000; HUABIO, Hangzhou, Zhejiang, China), Vimentin (ET1610-39, 1/20,000; HUABIO, Hangzhou, China), Snail (ER1706-22, 1/1000; HUABIO, Hangzhou, China), and GAPDH (R1210-1, 1:5000; HUABIO, Hangzhou, China). After being washed with TBST, the PVDF membranes were incubated with secondary antibodies (ab6721, 1/2000; Abcam, Cambridge, UK) for 1 hour. Protein detection was performed using an ECL kit (ab133406, Abcam, Cambridge, UK).

RNA was prepared from cells using TRIzol (Invitrogen), followed by cDNA synthesis with the High-Capacity reverse transcription kit (4374966, Applied Biosystems, Waltham, MA, USA). qPCR amplification was conducted on the LightCycler 480 II system in the presence of ChamQ Universal SYBR Master Mix (Vazyme). Primers are provided in Table 1. Expression values were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reported as fold changes derived from 2^{-Δ⁢Δ⁢Ct} method.

Transwell assays were utilized to assess the migration and invasion abilities of AML cells, as previously described with minor modifications [24]. Briefly, cells were harvested 48 h after transfection and resuspended in serum-free RPMI-1640 medium at a density of 1 $\times$ 10⁵ cells/mL. A total of 200 µL cell suspension was added to the upper chamber of Transwell inserts (8-µm pore size), while 600 µL RPMI-1640 medium supplemented with 20% FBS was added to the lower chamber to establish a chemotactic gradient. For migration assays, the membrane was left uncoated. For invasion assays, the upper surface of the membrane was precoated with 40 µL of VitroGel 3D-RGD solution. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 h. After incubation, cells remaining on the upper surface were removed, and cells that had migrated or invaded to the lower surface were fixed with 4% methanol (15–30 min) and stained with Giemsa working solution for 30 min at room temperature. The number of migrated or invaded cells was counted under a light microscope.

The T7 RiboMAXTM Express Large-Scale RNA Production System (P1320, Promega, Madison, WI, USA) was used to generate abundant WNK1. The Pierce™ RNA 3^′ End Desthiobiotinylation Kit (20163, Thermo Fisher Scientific, Waltham, MA, USA) was used to biotinylate the 3^′ ends of WNK1 RNAs, both sense and antisense, with RNA probes. The mixture was incubated with AML cell lysates, and RNA‒protein interactions were analyzed using the RNA-Protein Pull-Down Kit (20164, Thermo Fisher Scientific, USA).

The RNA-Binding Protein Immunoprecipitation Kit Magna RIPTM (17-700, Merck Millipore, Darmstadt, Germany) was used to perform the RIP assay. Briefly, AML cells were harvested using an anti-PTBP1 antibody. Total RNA was collected using Dynabeads® Protein G (10003D, Thermo Fisher Scientific, USA) and TRIzol. WNK1 level was also determined.

The cells were treated with 10 µg/mL actinomycin D (Act D) once they reached 60–80% confluency. RNA was extracted at 0, 3, and 6 hours post-treatment for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis.

Male NOD/SCID mice (8 weeks) were procured from Gempharmatech Co. Ltd. Each mouse was injected with 1 $\times$ 10⁷ HL-60 cells transfected with sh-WNK1 or sh-NC through the tail vein. Four weeks after the AML model was established. Euthanasia was first induced by CO₂inhalation at a flow rate displacing 30% to 70% of the chamber volume per minute until unconsciousness. Following confirmation of death, the livers, kidneys, and spleens were collected for analysis. The percentage of CD45⁺ cells in mouse BM was measured using flow cytometry with an anti-hCD45-APC antibody (5 µL per million cells in 100 µL staining volume, 304010; BioLegend, San Diego, CA, USA). Briefly, BM cells were harvested from femurs and tibias by flushing with cold PBS, passed through a cell strainer to obtain single-cell suspensions, followed by red blood cell lysis. Cells were then washed and stained with anti-hCD45-APC antibody for flow cytometric analysis. The animal study was approved by the Second Hospital of Hebei Medical University (2025-AE260).

For histology, liver, kidney, and spleen tissues were fixed in 4% paraformaldehyde ($>$24 h), processed for paraffin embedding, and sectioned at 5 µm. After hematoxylin–eosin staining, images were captured using a microscope.

Spleen, liver, and kidney tissues were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose, embedded in OCT, and cryosectioned at 8 µm. The sections were permeabilized with 0.3% Triton X-100, blocked with 5% BSA, and incubated with anti-CD45 antibody (1:100, CL594-60287; Proteintech, Wuhan, Hubei, China) overnight at 4 °C. After being washed, the sections were incubated with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody (1:500, A28180; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Nuclei were counterstained with DAPI, and images were acquired using a fluorescence microscope.

The results are expressed as the mean $\pm$ standard deviation (SD), and all the statistical analyses were performed using GraphPad 10.1.2 software (San Diego, CA, USA). Differences between groups were evaluated using Student’s t-test or ANOVA, with a p value of less than 0.05 considered to indicate statistical significance.

According to the GEPIA database, WNK1 is abnormally expressed in multifarious tumor tissues (Fig. 1A), and its level is abnormally increased in AML patients (Fig. 1B). Moreover, we verified its level in AML cell lines compared with that in HS-5 cells, and WNK1 levels were enhanced in AML cell lines (HL-60, AML-193, KG-1, and THP-1 cells) (Fig. 1C,D). These findings suggest that WNK1 may influence AML progression. HL-60 and THP-1 cell lines were chosen for further experiments.

Fig. 1.

WNK1 abundance was increased in AML. (A) WNK1 expression in a variety of tumor tissues was analyzed through the GEPIA database. (B) Comparison of WNK1 expression levels between AML patients and normal controls on the basis of GEPIA database analysis (AML, n = 173; normal, n = 70). (C) Representative western blot images showing WNK1 protein expression in normal human bone marrow stromal cells (HS-5) and AML cell lines (HL-60, AML-193, KG-1, and THP-1). (D) Relative mRNA expression levels of WNK1 in HS-5 cells and AML cell lines determined by qRT‒PCR. The data are presented as the mean $\pm$ SD. In (B), *p 0.05 vs. normal controls. In (C,D), *p 0.05, **p 0.01, and ***p 0.001 vs. HS-5. AML, acute myeloid leukemia; GEPIA, Gene Expression Profiling Interactive Analysis; qRT‒PCR, Quantitative Reverse Transcription Polymerase Chain Reaction.

The effects of WNK1 on cell proliferation, migration and EMT markers were investigated by constructing a WNK1 knockdown AML cell model. The knockdown efficiency of WNK1-specific short hairpin RNAs (sh-RNAs) (sh-WNK1-1/2/3) in AML cells was validated, as shown in Fig. 2A,B. Follow-up experiments were performed using sh-WNK1-1, which resulted in the highest knockdown efficiency. As depicted in Fig. 2C,D, knockdown of WNK1 hindered the migration and invasion of HL-60 and THP-1 cells. Besides, E-cadherin level was enhanced and N-cadherin, Vimentin, and Snail expressions were decreased in WNK1-knockdown AML cells (Fig. 2E). In summary, WNK1 inhibition curtailed AML cell invasiveness and limited EMT-associated features.

Fig. 2.

Knockdown of WNK1 hindered AML cells migration, invasion, and EMT markers expression. (A) Representative western blot images and quantitative analysis of WNK1 protein levels in HL-60 and THP-1 cells transfected with sh-NC or WNK1-specific sh-RNAs (sh-WNK1-1/2/3). (B) Relative mRNA expression levels of WNK1 in HL-60 and THP-1 cells transfected with sh-NC or sh-WNK1-1/2/3 determined by qRT‒PCR. (C) Representative images and quantification of migrated HL-60 and THP-1 cells in transwell migration assays after WNK1 knockdown (Scale bar, 100 µm). (D) Representative images and quantification of invaded HL-60 and THP-1 cells in Matrigel-coated transwell invasion assays after WNK1 knockdown (Scale bar, 100 µm). (E) Representative western blot images and quantitative analysis of EMT-associated marker proteins (E-cadherin, N-cadherin, Vimentin, and Snail) in HL-60 and THP-1 cells transfected with sh-NC or sh-WNK1-1. The data are presented as the mean $\pm$ SD (n = 3). *p 0.05, **p 0.01, ***p 0.001 vs. sh-NC. EMT, epithelial-to-mesenchymal transition; sh-RNA, short hairpin RNA; WNK1, WNK lysine-deficient protein kinase 1.

By tail vein injection of HL-60 cells transfected with sh-WNK1 or sh-NC, we established a xenogeneic AML model in NOD/SCID mice to explore the function of WNK1 in AML metastasis. After 4 weeks, mice transplanted with sh-WNK1-carrying HL-60 cells had significantly reduced liver and spleen weights (Fig. 3A). Besides, the mice transplanted with WNK1-knockdown HL-60 cells displayed lower proportions of human CD45⁺ cells in the BM (Fig. 3B). The positive signal of human CD45 in the spleen, liver, and kidney was also significantly reduced by WNK1 knockdown. In particular, the spleen, liver, and kidney tissues of the sh-NC group were obviously damaged, which was alleviated by WNK1 knockdown (Fig. 3C). Furthermore, E-cadherin level was elevated and N-cadherin, Vimentin, and Snail expressions were diminished in the liver tumor nodules of the sh-WNK1 group (Fig. 3D). In summary, WNK1 knockdown markedly slowed AML cell metastasis in vivo.

Fig. 3.

WNK1 knockdown markedly restrained the metastasis of AML cells in vivo. The cells were divided into the following two groups: sh-WNK1 or sh-NC. (A) Liver and spleen weights. (B) Statistical analysis of the percentage of human CD45⁺ cells in the BM. (C) H&E and human CD45 staining were used to evaluate the spread of leukemia in the spleen, liver, and kidney (scale bar, 100 µm). (D) E-cadherin, N-cadherin, Vimentin, and Snail protein levels in live tissues were measured by western blot assay. The data are presented as the mean $\pm$ SD (n = 6). *p 0.05, **p 0.01 vs. sh-NC. BM, bone marrow; H&E, Hematoxylin-eosin.

Previously, we noted that PTBP1 modulates AML disease progression [16, 17], but its effect on the migration and invasion of AML cells was unclear. The ENCORI database predicted that PTBP1 and WNK1 had binding sites (Fig. 4A). Therefore, we probed the relationship between WNK1 and PTBP1. An RNA pull-down assay revealed that PTBP1 was distinctly pulled down by WNK1-sense (Fig. 4B). In addition, an RIP assay confirmed the binding between PTBP1 and WNK1 (Fig. 4C). To determine whether PTBP1 could disturb WNK1 levels in AML cells, we first silenced PTBP1 by using PTBP1-specific sh-RNAs (sh-PTBP1-1/2/3). sh-PTBP1 significantly downregulated PTBP1 levels in AML cells, among which sh-PTBP1-1 had the highest knockdown efficiency and was selected for subsequent studies (Fig. 4D,E). As depicted by the results of the actinomycin D assay, PTBP1 knockdown decreased the stability of WNK1 mRNA (Fig. 4F). Furthermore, WNK1 levels decreased in PTBP1-silenced AML cells (Fig. 4G,H). In sum, PTBP1 directly bound to WNK1 and augmented its mRNA stability.

Fig. 4.

PTBP1 bound to WNK1 and augmented its mRNA stability. (A) The ENCORI database predicted that PTBP1 and WNK1 have binding sites. The relationship between PTBP1 and WNK1 was confirmed by an RNA pull-down assay (B) and RNA immunoprecipitation (RIP) (C). (D,E) The transfection efficiency of PTBP1-specific sh-RNAs (sh-PTBP1#1/2/3) into HL-60 and THP-1 cells was validated by qRT‒PCR and western blot assays. (F) After treating cells with actinomycin D for 0, 3, and 6 h, WNK1 mRNA stability was analyzed by qPCR. (G,H) WNK1 mRNA and protein levels were measured by qRT‒PCR and western blot assay, respectively. The data are presented as the mean $\pm$ SD (n = 3). **p 0.01, ***p 0.001 vs. sh-NC or IgG controls, as indicated.

To further explore whether PTBP1 influences AML progression by regulating WNK1, we designed a salvage experiment. WNK1 overexpression (oe-WNK1) increased WNK1 levels in HL-60 and THP-1 cells (Fig. 5A,B). Besides, knockdown of PTBP1 suppressed AML cells migration and invasion, which were abolished by WNK1 overexpression (Fig. 5C,D). As expected, E-cadherin level was augmented and N-cadherin, Vimentin, and Snail expressions were diminished in PTBP1-deficient AML cells, but these changes were reversed by WNK1 expression (Fig. 5E). These results illustrate that PTBP1 facilitates AML cells migration, invasion, and EMT markers expression through WNK1.

Fig. 5.

PTBP1 promoted AML cell migration and invasion while upregulating EMT-associated markers in a WNK1-dependent manner. (A,B) WNK1 mRNA and protein levels were measured by qRT‒PCR and western blot assay, respectively, in WNK1-overexpressing HL-60 and THP-1 cells. The cells were divided into the following groups: sh-NC, sh-PTBP1, sh-PTBP1+oe-NC, and sh-PTBP1+oe-WNK1. (C,D) Cell migration and invasion in AML cells (Scale bar, 100 µm). (E) E-cadherin, N-cadherin, Vimentin, and Snail protein levels were measured by western blot assay in HL-60 and THP-1 cells. The data are presented as the mean $\pm$ SD (n = 3). **p 0.01, ***p 0.001 vs. the sh-NC or sh-PTBP1 groups, as indicated.