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The KDM5A/HOXA5 axis regulates osteosarcoma progression via activating the Wnt/β-catenin pathway

European Journal of Medical Research volume 30, Article number: 284 (2025) Cite this article

Abstract

As an oncogenic driver, lysine-specific demethylase 5A (KDM5A) participates in regulating numerous tumor progression-related processes. Moreover, KDM5A functions as a histone demethylase, modulating the expression levels of its target genes by adjusting methylation levels. However, the underlying molecular mechanism of KDM5A in osteosarcoma remains elusive. To elucidate this mechanism, specifically how the KDM5A /Homeobox A5 (HOXA5) axis regulates osteosarcoma progression, we measured the expression levels of KDM5A and HOXA5 genes using reverse transcription–quantitative real-time PCR. The correlation between HOXA5 and KDM5A was analyzed via Pearson correlation analysis and further validated through chromatin immunoprecipitation-quantitative real-time PCR. Immunohistochemistry was conducted to determine the number of KDM5A—or HOXA5-positive cells present in osteosarcoma tissues. Additionally, Western blot analysis was utilized to quantify the protein levels of KDM5A, HOXA5, di- and tri-methylation of lysine 4 on histone H3, and β-catenin. Colony formation assays, wound healing assays and flow cytometry were used to detect cell proliferation, migration and apoptosis. The factors associated with the five-year survival rate of patients were analyzed. Our results illustrated that KDM5A was up-regulated in osteosarcoma and associated with a poor prognosis; KDM5A knockdown inhibited osteosarcoma cell proliferation and migration and promotes apoptosis. Subsequently, KDM5A knockdown induced HOXA5 expression by promoting di- and tri-methylation of lysine 4 on histone H3 demethylation, and HOXA5 overexpression inhibited osteosarcoma cell proliferation and migration, and promoted apoptosis by inhibiting the Wnt/β-catenin pathway. We finally proved that HOXA5 silence weakened the inhibitory effect of sh- KDM5A on osteosarcoma proliferation and migration and promoted apoptosis via activating Wnt/β-catenin pathway in vivo and in vitro. Our study demonstrated that the KDM5A /HOXA5 axis regulates osteosarcoma progression by activating the Wnt/β-catenin pathway.

Highlights

  • KDM5A was up-regulated in osteosarcoma and associated with a poor prognosis.

  • KDM5A knockdown inhibited osteosarcoma cell proliferation and migration and promoted apoptosis.

  • KDM5A knockdown activated HOXA5 expression via promoting H3K4me3 demethylation.

  • KDM5A/HOXA5 axis regulates osteosarcoma progression via activating Wnt/β-catenin pathway.

Introduction

Osteosarcoma is the most prevalent primary malignancy and is characterized by malignant osteogenesis and malignant osteoblast differentiation. Because of its rapid progression and poor prognosis, osteosarcoma has become the main fatal disease in adolescents [1, 2]. The survival rate of patients with osteosarcoma has been improved with improvements in chemotherapy and surgery. However, the survival rate is still not ideal due to the high local invasiveness and the potential of rapid metastasis [3]. Therefore, exploring the pathogenesis of osteosarcoma is beneficial to seek new treatment strategies should be beneficial.

Lysine-specific demethylase 5A (KDM5A) belongs to the family of JUMONJI demethylases [4]. The KDM5A protein serves as a highly specific demethylase, targeting the di- and tri-methylation of lysine 4 on histone H3 (H3K4me2 and me3). These methylation states are hallmarks of active transcription, often found in gene promoters, with H3K4me3 being particularly enriched in active enhancers. Based on these functions, the KDM5A protein is classified as a transcriptional repressor [5, 6]. In recent years, accumulating evidence has implicated KDM5A as an oncogenic driver involved in the regulation of multiple tumor progression. Matthew et al. reported that KDM5A knockout reduced the occurrence and metastasis of SCLC [7]. Shokri et al. verified that downregulation of KDM5A showed potent anti-leukemia effects [8]. Hou et al. found that the activity of KDM5A histone demethylase is related to the proliferation and drug resistance of breast cancer [5]. It has been confirmed that KDM5A was highly expressed in the serum of patients with acute leukemia and in various leukemia cells [9]. Besides, KDM5A was highly expressed in ovarian cancer tissues compared with adjacent normal tissues and promoted proliferation, epithelial-to-mesenchymal transition (EMT), and metastasis in ovarian cancer [10]. KDM5A was significantly upregulated in human lung adenocarcinoma and regulated the proliferation of lung adenocarcinoma cells [11]. Collectively, these findings indicate that KDM5A plays a key role in tumorigenesis, metastasis and drug tolerance, and has become a promising therapeutic target for cancer [12]. Moreover, KDM5A was confirmed to be highly expressed in osteosarcoma tissues, and KDM5A knockdown inhibited osteosarcoma cell proliferation and induced apoptosis [13], indicating that KDM5A plays a promoting role in osteosarcoma progression. However, the underlying molecular mechanism by which KDM5A regulates osteosarcoma remains unclear.

Homeobox A5 (HOXA5) belongs to the HOX family and contains a homeobox domain, which is a conserved DNA-binding domain [14]. HOXA5 encodes an ANTP-like homologous structural protein composed of 270 amino acids, which is located on human chromosome 7 [15]. Ma et al. indicated that HOXA5 acts as a tumor suppressor, inhibiting the proliferation of cervical cancer cells [16]. Peng et al. reported that HOXA5 exerts a tumor suppressor role in gastric cancer progression by inhibiting the abnormal proliferation of cancer cells [17]. Interestingly, HOXA5 proved to be the most interesting candidate gene, showing a significant age-dependent increase in genomic DNA methylation (DNAm) associated with a significant decrease in its gene and protein expression [18]. Besides, studies have shown that H3K4me3 was enriched in the promoter of HOXA5 and promotes the high expression of HOXA5 in breast cancer through methylation regulation [19]. Although there are methylation sites in HOXA5, whether the histone demethylase KDM5A inhibits its expression by regulating HOXA5 demethylation has not been reported. The activation of Wnt/ β-catenin pathway is mainly due to the interaction between secretory protein Wnt and cell surface specific receptors through autocrine or paracrine, resulting in the accumulation of β-Catenin through a series of phosphorylation and dephosphorylation of downstream proteins [20]. Therefore, the classical Wnt signal pathway mainly activates gene transcription through β-Catenin [21]. On this foundation, it is crucial to delve deeper into the impact of the Wnt/β-catenin pathway in cancer [22]. This pathway plays a pivotal role in cancer progression by regulating cell proliferation, migration, and invasion [23]. Aberrant activation of the Wnt/β-catenin pathway has been implicated in various types of cancer, including breast, colon, and liver cancers, where it contributes to the oncogenic transformation and maintenance of the malignant phenotype [24,25,26]. Moreover, there are several research reports highlighting the regulation of Wnt by HOXA5. HOXA5, a member of the homeobox (HOX) gene family, has been shown to negatively regulate the Wnt/β-catenin pathway [16]. By interacting with components of the Wnt signaling machinery, HOXA5 can inhibit the activation of β-catenin and subsequent gene transcription, thereby suppressing cancer growth and progression [27]. In this context, the Wnt/β-catenin pathway emerges as a potential downstream target of HOXA5.

In this study, we focus on the effect of HOXA5 on the activity of β-catenin to regulate the activation of the pathway. It has been reported that HOXA5 participates in cancer progression by regulating the Wnt/β-catenin pathway. For example, the HOXA5 and Wnt/β-catenin pathways form a negative feedback loop to inhibit the dryness of colorectal cancer cells [27]. However, it is not clear whether HOXA5 participates in osteosarcoma progression by regulating the Wnt/β-catenin pathway. Therefore, in this paper, we aimed to prove that whether the KDM5A/HOXA5 axis regulated the progression of osteosarcoma by activating Wnt/ β-Catenin pathway.

Materials and methods

Animal model

To investigate the role of KDM5A and HOXA5 in osteosarcoma progression, we constructed a mouse osteosarcoma model by implanting transduced osteosarcoma cells with lentiviruses carrying shRNA constructs targeting KDM5A (sh-KDM5A), HOXA5 (sh-HOXA5), or a non-targeting control (sh-NC) into immunocompromised mice (nude nude mice, 4–6 weeks) purchased from Slack Jingda (Hunan, China). The MNNG cells (5 × 106 cells/200 μL) treated by subcutaneous injection were selected to form subcutaneous transplanted tumor. Tumor growth was monitored and recorded 15, 19, 23, 27, 31 and 35 days after operation. On the 35th day, mice were killed by dislocation of spine and tumor tissues were collected for western blot and immunohistochemical analysis. All experiments were approved by the Changsha Central Hospital Ethics Committee.

Specimen

Between May 2021 and May 2022, information was collected from 58 osteosarcoma patients at the Affiliated Changsha Central Hospital., and surgical tumor tissues and adjacent non-tumor tissues were collected. Forty-seven patients were not receiving chemotherapy and/or radiation therapy. The samples were immediately frozen in liquid nitrogen for subsequent experiments. According to the average expression multiple value for KDM5A mRNA in osteosarcoma (2.57), the patients were divided into two groups: the high KDM5A expression group (n = 25) and the low KDM5A expression group (n = 22). Furthermore, survival time between groups was evaluated using Kaplan–Meier method or univariate Cox regression analysis. The study was approved by the ethics Committee of the Changsha Central Hospital (No. 2021-S0203), and we have obtained the informed consent form of the patient.

Immunohistochemistry (IHC)

For IHC analysis, 4 μm tissue sections were initially deparaffinized in a hot air oven for 24 h at a temperature range of 60–65 °C. Subsequently, the slides underwent a rehydration process in xylene and a graded ethanol series for approximately 45 min. Afterward, the slides were immersed in Tris buffer (pH = 9, Sigma-Aldrich) and subjected to antigen retrieval via autoclaving at 121 °C for 20 min, followed by rinsing in phosphate-buffered saline (PBS, Sigma-Aldrich) solution. Next, the sections were incubated overnight at 4 °C with the anti-HOXA5 (Abcam, ab140636, diluted 1:500), anti-KDM5A (Abcam, ab92533, diluted 1:500) and anti-Ki67 (Abcam, ab15580, diluted 1:500). Afterward, they were incubated with a peroxidase-conjugated secondary antibody (Bioss, BA-0295 M, Beijing, China) for 1 h at room temperature. The sections were then developed using 3,3'-diaminobenzidine and examined under a microscope. The extent of positive cell staining was scored on a scale of 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). Following independent evaluation by three individuals who were not involved in the experimental procedure, the scoring was averaged three times to ensure accuracy.

Cell culture

The human osteoblast line hFOB1.19 and osteosarcoma cell lines including MNNG (CRL-1547), MG63 (CRL-1427), 143B (CRL-8303) and SaOS2 (HTB-85) cells were provided by the American Center for Typical Culture Depositary (ATCC, Manassas, VA, USA), All cells were cultured in DMEM, (Invitrogen, California, USA) containing 10% fetal bovine serum (FBS, Gibco, NY, USA) and cultured in a humid incubator at 37 ℃ and 5% CO2.

Cell transfection

The plasmids for the short hairpin RNAs targeting KDM5A (sh-KDM5A), HOXA5 (sh-HOXA5), and their respective negative controls (sh-NC) were crafted by Shanghai Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China). ShRNA includes sh-KDM5A and sh-HOXA5 were used to knock down the expression of KDM5A and HOXA5 genes, respectively [28]. To achieve HOXA5 overexpression, the full-length sequence of HOXA5 was precisely inserted into the pcDNA3.1 vector (Invitrogen). The resulting plasmids were then efficiently transfected into MNNG and MG63 cells using the Lipofectamine™ 3000 Transfection Reagent (Invitrogen), ensuring precise and efficient delivery of the genetic material into the target cell lines.

Reverse transcription–quantitative real-time PCR (qRT-PCR)

A minimum of 2 × 106 MNNG or MG63 from treated and control samples were used for RNA extraction. Total RNAs were purified from all samples using TRIzol (Invitrogen) according to the manufacturer’s protocol. The RNA was converted to complementary DNA by using Maxime RT premix (iNtron) with random primers. qPCR was performed according to the instructions and previous reports [29]. GAPDH or β-actin was selected as reference genes using GeNorm (PrimerDesign Ltd., Southampton, UK). The result that β-actin as an internal control were presented in Figure S2. The fold induction was expressed relative to a calibrator sample using the 2−ΔΔCt method. Table 1 displays the primers used in this study.

Table 1 Primer sequences
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Western blot

RIPA cleavage buffer (Beyotime, Nanjing, China) was applied to lyse cells and obtain total proteins. Proteins were separated by 10% SDS-PAGE gel and transferred to PVDF membrane. Subsequently, blocked PVDF membrane incubated with specific antibodies (Cambridge, UK), including KDM5A (ab194286, 1:5000), H3K4me3 (ab8580, 1:2000), Wnt (ab219412, 1:1000), GSK3B (ab227208, 1:1000), p-GSK3B (ab107166, 1:1000), c-myc (ab17355, 1:1000), PI3K (ab302958, 1:1000), p-PI3K (ab125633, 1:1000), AKT (ab8805, 1:1000), p-AKT (ab38449, 1:1000), Notch (ab128076, 1:1000), MAPK (ab308333, 1:1000), JAK (ab108596, 1:1000), p-JAK (ab32101, 1:1000), STAT3 (ab68153, 1:1000), p-STAT3 (ab267373, 1:1000) and β-catenin (ab32572, 1:5000) at 4 °C for overnight. The second day, after incubation with Goat Anti-Rabbit IgG H&L (HRP) (ab6721, 1:5000), the bands were detected by gel imaging system (Bio-Rad), and protein bands were analyzed by ImageJ software for quantification with GAPDH as the endogenous control.

Colony formation assay

Single-cell suspensions of MNNG and MG63 cells were delicately plated onto 15-mm dishes for culturing. After a period of 14 days, to preserve the integrity of cellular morphology, the cells were fixed with 4% paraformaldehyde. Subsequently, they were stained with crystal violet to enhance visibility. For quantitative analysis, colonies comprising more than 50 individual cells were systematically enumerated under a microscope, ensuring accurate counting and data reliability.

Wound healing assay

Wound-healing assay was performed to assess MNNG and MG63 cell migration. In detail, MNNG and MG63 cells were added into 24 well cell culture plates. When the cells reached 80% growth, the corresponding plasmids were introduced into the cells. After 24 h, the cells were sliced through the cell monolayer with the tip of a yellow pipette, creating an artificial wound gap. The migration ability of cells on the cell culture plate (0–48 h) was observed under an inverted microscope.

Flow cytometry

Annexin V-FITC/propidium iodide (PI) double staining was performed to assess apoptosis of MNNG and MG63 cells. After centrifugation, the cells were re-suspended with bound buffer and adjusted to 1 × 106 cell/mL. Subsequently, 100 μL cell suspension was treated with 5 μL Annexin V-FITC and 5 μL PI solution (BD Biosciences, New York, USA). Finally, the suspension was loaded into flow cytometry (Thermo Fisher, Invitrogen Attune Xenith) for computer automatic analysis.

ChIP-qPCR

After 4 days of treatment, MNNG and MG63 cells were collected and sonicated. According to the manufacturer's instructions, The chromatin immunoprecipitation (ChIP) kit (Upstate, cat. no. 17–371) was used for coimmunoprecipitation experiments; the supernatant of sonicated cells was incubated with anti-H3K4me3 (Abcam, ab8580, 1:500) or anti-KDM5A (ab194286, 1:500) antibody respectively, in which A/G magnetic beads were coupled with anti-H3K4me3 or anti-KDM5A antibodies, and mouse immunoglobulin (IgG) was used as a negative control. The enrichment of HOXA5 in DNA sediment was analyzed by qRT-PCR.

Statistical analysis

The data of three replicates were expressed as the mean ± standard deviation (SD). A t-test was used for comparisons between two groups, and one-way analysis of variance and Tukey’s multiple comparison test were used for comparisons between groups. Pearson correlation was used to analyze the correlation between the HOXA5 and KDM5A genes. P < 0.05 was considered to indicate statistical significance.

Results

KDM5A was up-regulated in osteosarcoma and associated with a poor prognosis

Firstly, we analyzed KDM5A levels in osteosarcoma tissues. The results indicated that compared with those in adjacent tissues, KDM5A level was significantly up-regulated in osteosarcoma tissues (Fig. 1A, t-test, P < 0.01). IHC analysis indicated that the number of KDM5A-positive cells in osteosarcoma tissues was significantly increased, compared with that in adjacent tissues (Fig. 1B, t-test, and Figure S1B P < 0.01). As shown in Table 2, compared with the KDM5A low expression group, the KDM5A high expression group had a higher rate of recurrence, metastasis and clinical stage. Five patients were randomly selected, and it was found that the level of KDM5A protein in osteosarcoma tissue was significantly higher than that in adjacent tissues (Fig. 1C, t-test, P < 0.05). Subsequently, we found that the five-year survival rate of patients with high level of KDM5A was significantly lower than that of patients with low level of KDM5A (Fig. 1D, p = 0.0363). Consistently, our results showed that compared with that in hFOB1.19 cells, KDM5A level was significantly up-regulated in osteosarcoma cells (Fig. 1E, one-way analysis, P < 0.05). Consistently, KDM5A protein level was also significantly increased (Fig. 1F, one-way analysis, P < 0.05). The change 217 of KDM5A level was obviously in MNNG and MG63 cells, which were selected for subsequent functional studies. To sum up, these findings suggested that KDM5A was up-regulated in osteosarcoma and associated with poor prognosis.

Fig. 1
figure 1

KDM5A was up-regulated in osteosarcoma and associated with poor prognosis. A qRT-PCR detected KDM5A level in osteosarcoma tissues, adjacent tissues served as the negative control (n = 58). B IHC detected the number of KDM5A-positive cells in osteosarcoma tissues (five fields of vision, n = 3). C KDM5A protein levels were detected by western blot in 5 out of 58 clinical samples. D The correlation between KDM5A level and overall survival rate of patients (KDM5A high level patients n = 29; KDM5A low level patients n = 29). E qRT-PCR detected KDM5A in osteosarcoma cells, including MNNG, MG63, 143B and SaOS2, hfOB1.19 cells served as the negative control. F Western blot measured KDM5A protein level in osteosarcoma cells, hfOB1.19 cells served as the negative control. * P < 0.05, ** P < 0.01, *** P < 0.001

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Table 2 An analysis of the correlation between KDM5A expression and patients' clinical characteristics
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KDM5A knockdown inhibited osteosarcoma cell proliferation and migration and promoted apoptosis

For further investigate the effect of KDM5A on osteosarcoma cell bioactivity, we inhibited the level of KDM5A in MNNG and MG63 cell lines. qRT-PCR and western blot analysis showed that KDM5A level was significantly inhibited (Fig. 2A and B, t-test, P < 0.01). As shown in Figure S1D-E (t-test), KDM5A mRNA and protein expression levels were both significantly decreased after transfection with sh-KDM5A#2. As showing in Fig. 2C, D (t-test), KDM5A knockdown significantly inhibited MNNG and MG63 cell proliferation and migration (P < 0.01). Inversely, the rate of MNNG and MG63 cell apoptosis was significantly increased by KDM5A knockdown (Fig. 2E, t-test, P < 0.01). In total, these findings indicated that KDM5A knockdown inhibited osteosarcoma cell proliferation and migration, and promotes apoptosis.

Fig. 2
figure 2

KDM5A knockdown inhibited osteosarcoma cell proliferation and migration, and promotes apoptosis. sh-KDM5A was transfected into MNNG and MG63 cell for KDM5A knockdown, sh-NC served as the negative control. A qRT-PCR detected KDM5A level. B KDM5A levels were measured by western blot after transfection with sh-KDM5A. C Cell proliferation was detected by colony formation assay. D Cell migration was detected by wound healing assay. E Flow cytometry detected cell apoptosis. * P < 0.05, ** P < 0.01, *** P < 0.001

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KDM5A knockdown activated HOXA5 expression via promoting H3K4me3 demethylation

As a demethylase of H3K4me3, KDM5A may be widely involved in cancer progression regulation [30]. However, it was not clear whether KDM5A regulated HOXA5 level by mediating H3K4me3 demethylation. To determine the potential mechanism of KDM5A accelerating osteosarcoma progression, we detected HOXA5 level in osteosarcoma tissues and cells. Compared with that in adjacent tissues, HOXA5 level was significantly down-regulated in osteosarcoma tissues (Fig. 3A, t -test, P < 0.01). IHC analysis indicated that the number of HOXA5-positive cells in osteosarcoma tissues was significantly decreased, compared with that in adjacent tissues (Figs. 3B and S1C, t-test, P < 0.01). We also found that the five-year survival rate of patients with low level of HOXA5 was significantly lower than that of patients with high level of HOXA5 (Fig. 1D, p = 0.0295). Nest, there was a weak negative correlation between the expression of HOXA5 and KDM5A in osteosarcoma (Fig. 3D, Pearson, r = −0.3658, p = 0.0114). Subsequently, qRT-PCR analysis furtherly showed that KDM5A knockdown promoted HOXA5 expression in MNNG and MG63 cells (Fig. 3E, t-test, P < 0.01). Consistently, KDM5A knockdown promoted the levels of HOXA5 and H3K4me3 proteins (Fig. 3F, one-way analysis, P < 0.01). ChIP-qPCR analysis indicated that KDM5A knockdown reduced the enrichment ability of anti-KDM5A to HOXA5 promoter, but significantly enhanced the enrichment ability of anti-H3K4me3 to HOXA5 promoter (Fig. 3G, one-way analysis, P < 0.01). In conclusion, the above results indicated that KDM5A knockdown activated HOXA5 expression via promoting H3K4me3 demethylation.

Fig. 3
figure 3

KDM5A knockdown activated HOXA5 expression via promoting H3K4me3 demethylation. A qRT-PCR detected HOXA5 level in osteosarcoma tissues, adjacent tissues served as the negative control (n = 58). B IHC detected the number of HOXA5-positive cells in osteosarcoma tissues (five fields of vision, n = 3). C The correlation between HOXA5- level and overall survival rate of patients (HOXA5-high level patients n = 29; HOXA5-low level patients n = 29). D Analysis of correlation between the levels of HOXA5 and KDM5A. E qRT-PCR detected HOXA5 level in KDM5A knockdown-MNNG and MG63 cells. F Western blot measured the levels of HOXA5 and H3K4me3 proteins. G ChIP-qPCR verified the correlation between KDM5A, HOXA5 and H3K4me3. * P < 0.05, ** P < 0.01, *** P < 0.001

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HOXA5 overexpression inhibited osteosarcoma cell proliferation and migration, and promoted apoptosis via inhibiting Wnt/β-catenin pathway

To clarify the potential mechanism of HOXA5 inhibiting osteosarcoma progression, pcDNA3.1-HOXA5 was transfected into MNNG and MG63 cells for HOXA5 overexpression. qRT-PCR and western blot analysis showed that HOXA5 level was significantly up-regulated in MNNG and MG63 cells (Fig. 4A and B, t-test, P < 0.01). Subsequently, we studied the changes in expression levels of proteins related to several classical cancer pathways (PI3K/AKT, WNT/β-catenin, Notch, MAPK, JAK2/STAT3). Results showed that among them the changes in Wnt/β-catenin (β-catenin, Wnt, GSK3B, p-GSK3B, c-myc) were the most different (Figs. 4C and S1A, t-test, P < 0.01). Therefore, we chose the Wnt/β-catenin pathway for subsequent research. As shown in Fig. 4D, E (t-test), HOXA5 overexpression significantly inhibited MNNG and MG63 cell proliferation and migration (P < 0.01). Oppositely, the rate of MNNG and MG63 cell apoptosis was significantly increased (Fig. 4F, t-test, P < 0.01). Therefore, we concluded that HOXA5 overexpression may inhibit osteosarcoma cell proliferation and migration, and promoted apoptosis via inhibiting Wnt/β-catenin pathway.

Fig. 4
figure 4

HOXA5 overexpression inhibited osteosarcoma cell proliferation and migration, and promoted apoptosis via inhibiting Wnt/β-catenin pathway. pcDNA3.1-HOXA5 was transfected into MNNG and MG63 cells for HOXA5 overexpression, pcDNA3.1-NC served as the negative control. A qRT-PCR detected HOXA5 level. B HOXA5 protein levels were detected by western blot. C Western blot measured the levels of β-catenin Wnt, GSK3B, p-GSK3B and c-myc proteins. D Cell proliferation was detected by colony formation assay. E Cell migration was detected by wound healing assay. F Cell apoptosis was detected by flow cytometry. * P < 0.05, ** P < 0.01, *** P < 0.001

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HOXA5 silence weakened the inhibitory effect of sh-KDM5A on osteosarcoma proliferation and migration and promoted apoptosis via activating Wnt/β-catenin pathway

To further investigate the specific molecular mechanism of KDM5A regulating osteosarcoma progression, sh-HOXA5 was transfected into KDM5A knockdown-MNNG and MG63 cells, and the recovery experiment was carried out. Firstly, the knock-down efficiency of sh-HOXA5 was analyzed by qRT-PCR and Western blot. The results showed that compared with the control group, sh-HOXA5#1 and sh-HOXA5#2 significantly reduced the expression level of HOXA5 in cells. Among them, the knock-down efficiency of sh-HOXA5#2 is higher. Then we transfected sh-HOXA5(sh-HOXA5#2) into MNNG and MG63 cells knocked out by KDM5A (Figure S1F, one-way analysis, P < 0.05). The results showed that knocking down HOXA5 at the same time did not affect the expression of KDM5A in cells. However, compared with the control group, the level of HOXA5 in KDM5A knockout MNNG and MG63 cells was partially recovered by HOXA5 silencing (Fig. 5A, one-way analysis, P < 0.01). Western blot analysis indicated that the expression level of KDM5A was noticeably reduced in MNNG and MG63 cells following KDM5A knockdown, compared with the control. Moreover, after inhibition of HOXA5, the expression of HOXA5 decreased, while Wnt, β-catenin and c-myc increased, but p-GSK-3B decreased, and the expression of KDM5A was not affected by inhibition of HOXA5. Besides, the up-regulation of HOXA5 induced by KDM5A knockdown was abolished by HOXA5 silence, whereas low level of Wnt, β-catenin and c-myc inhibited by KDM5A knockdown was rescued by HOXA5 silence, but the expression of p-GSK-3B showed the opposite trend (Fig. 5B, one-way analysis, P < 0.01). Moreover, the proliferation and migration ability of MNNG and MG63 cells decreased by KDM5A knockdown was recovered by HOXA5 silence (Fig. 5C, D, t-test, P < 0.01). Inversely, KDM5A knockdown-induced cell apoptosis was abolished by HOXA5 silence (Fig. 5E, t-test, P < 0.01). Taken together, HOXA5 silence weakened the inhibitory effect of sh-KDM5A on osteosarcoma proliferation and migration and promoted apoptosis via activating Wnt/β-catenin pathway.

Fig. 5
figure 5

HOXA5 silence weakened the inhibitory effect of sh-KDM5A on osteosarcoma proliferation and migration and promoted apoptosis via activating Wnt/β-catenin pathway. sh-HOXA5 was transfected into KDM5A knockdown-MNNG and MG63 cells for HOXA5 silence, sh-NC served as the negative control. A qRT-PCR detected HOXA5 level. B Western blot measured the levels of KDM5A, HOXA5, β-catenin, Wnt, GSK3B, p-GSK3B, c-myc proteins. C Cell proliferation was detected by colony formation assay. D Cell migration was detected by wound healing assay. E Cell apoptosis was detected by flow cytometry. * P < 0.05, ** P < 0.01, *** P < 0.001

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The KDM5A/HOXA5 axis regulates osteosarcoma progression in mice by activating the Wnt/β-catenin pathway

We constructed a mouse osteosarcoma model by implanting transduced osteosarcoma cells with lentiviruses carrying shRNA constructs targeting KDM5A (sh-KDM5A), HOXA5 (sh-HOXA5), or a non-targeting control (sh-NC) into immunocompromised mice, selecting subcutaneous tumor implantation. Our results indicated that knocking down KDM5A slowed the growth of xenograft tumors in mice, while concurrent knocking down of HOXA5 reversed this trend (Fig. 6A, one-way analysis, P < 0.001). Western blot analysis showed that HOXA5 and p-GSK-3β protein in tumor tissues increased after knocking down KDM5A. The expression of GSK-3β remained unaffected by the inhibition of KDM5A. Furthermore, the silencing of HOXA5 abrogated the upregulation of HOXA5 and p-GSK-3β in tumor tissues that had been induced by the knockdown of KDM5A. Additionally, the silencing of HOXA5 rescued the levels of Wnt, β-catenin, and c-myc that had been diminished by the knockdown of KDM5A (Fig. 6B, one-way analysis, P < 0.01). The IHC results demonstrated that knocking down KDM5A suppressed the levels of Ki67 in mouse tumor tissues, while concurrent knocking down of HOXA5 reversed this trend (Fig. 6C, one-way analysis, P < 0.01). In total, the KDM5A/HOXA5 axis regulated osteosarcoma progression in mice by activating the Wnt/β-catenin pathway.

Fig. 6
figure 6

The KDM5A/HOXA5 axis regulates osteosarcoma progression in mice by activating the Wnt/β-catenin pathway. We constructed a mouse osteosarcoma model by implanting transduced osteosarcoma cells with lentiviruses carrying shRNA constructs targeting KDM5A (sh-KDM5A), HOXA5 (sh-HOXA5), or a non-targeting control (sh-NC) into immunocompromised mice, selecting subcutaneous tumor implantation. A Mouse osteosarcoma transplanted tumor and growth curve. B Western blot was used to detect the expression of KDM5A, HOXA5, and Wnt/β-catenin path-related proteins in mouse tumor tissues. C IHC measured the level of Ki67 in tumor tissues. *P < 0.05, **P < 0.01, ***P < 0.001

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Discussion

Osteosarcoma, a prevalent primary bone malignancy among adolescents, is characterized by intricate pathological mechanisms and resistance to conventional therapies. Persistent metastasis is the primary cause of mortality associated with this condition [31]. Currently, the standard treatment regimen for osteosarcoma patients typically comprises surgical resection and multidrug chemotherapy. Nevertheless, due to recurrent disease and drug resistance, the overall survival rate remains suboptimal [32]. Our research demonstrated that the KDM5A/HOXA5 axis regulated the progression of osteosarcoma by activating the Wnt/β-catenin pathway. Specifically, KDM5A activated HOXA5 expression by methylation of H3K4me3, which in turn activated the Wnt/β-catenin pathway and ultimately affected the proliferation, metastasis and apoptosis of osteosarcoma cells (Fig. 7). The objective was to provide insights for clinical studies on osteosarcoma.

Fig. 7
figure 7

Molecular mechanism diagram of research

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KDM5A, a ubiquitous transcriptional corepressor, has been implicated in the oncogenic processes of diverse cancers. Notably, it is overexpressed in breast cancer, contributing to its progression, and in acute myeloid leukemia, where it inhibits differentiation and enhances angiogenesis, metastasis, invasiveness, and proliferation. Furthermore, in lung cancer, KDM5A overexpression facilitates cell proliferation, invasion, and metastasis through modulation of cyclin D1 and ITGB1 expression [33,34,35]. Consistent with these findings, we observed upregulation of KDM5A in osteosarcoma and its association with unfavorable prognosis. Furthermore, we validated that KDM5A knockdown suppressed osteosarcoma cell proliferation and migration, while promoting apoptosis. These observations suggest that KDM5A plays a pivotal role in osteosarcoma progression. Consequently, elucidating the tumor suppression pathway mediated by KDM5A knockdown may yield promising therapeutic targets for osteosarcoma management.

HOXA5, a stem cell-related gene belonging to the homeobox gene cluster [36], has emerged as a pivotal regulator and tumor suppressor involved in embryonic development and cell differentiation [37]. Our research has uncovered a significant downregulation of HOXA5 in osteosarcoma tissues, accompanied by a notable decrease in the number of HOXA5-positive cells. Intriguingly, this downregulation coincided with alterations in the levels of H3K4me3, a histone modification closely linked to transcriptional activation, particularly in proximity to transcriptional start sites [38]. Previous studies have highlighted KDM5A, a Jumonji C domain-containing demethylase, as possessing the ability to remove H3K4me2 and H3K4me3 markers [39]. Importantly, KDM5A has been shown to be recruited to specific gene regions within the genome to remove H3K4me3 methylation [40]. Building on these observations, we postulated a potential regulatory relationship between KDM5A and HOXA5 in the context of osteosarcoma. Through further experiments, we validated our hypothesis, demonstrating that KDM5A exerts a negative regulatory effect on HOXA5 expression upon its knockdown. Consistently, we found that following KDM5A inhibition, the enrichment of anti-KDM5A to the HOXA5 promoter decreased, while the enrichment of anti-H3K4me3 to the HOXA5 promoter increased. For the first time, our study established that KDM5A modulates HOXA5 expression in osteosarcoma by regulating the demethylation of H3K4me3. This finding enriches the regulatory network of HOXA5 in cancer, as previous studies have primarily focused on its indirect regulatory mechanisms [14]. Our investigation into the methylation modification of HOXA5 itself revealed that KDM5A regulates its expression levels in cancer by mediating the methylation status of HOXA5. Additionally, we observed a tenuous negative correlation between the expression levels of HOXA5 and KDM5A in osteosarcoma samples, although this could potentially be attributed to the limited number of samples and individual variability. To further strengthen our research, we are actively collecting additional osteosarcoma tissues from clinical patients. In parallel, our study corroborated previous findings that demonstrated an antagonistic relationship between HOXA5 and the Wnt/β-catenin signaling pathway. Specifically, Ma et al. had discovered that HOXA5 hindered cervical cancer progression by suppressing Wnt/β-catenin pathway activity [16], and similar observations were made in colorectal cancer, where HOXA5 mitigated stem cell-like characteristics by inhibiting Wnt/β-catenin signal transduction [27]. Our research further supported these findings, indicating that HOXA5 overexpression attenuates osteosarcoma cell proliferation, migration, and enhances apoptosis through inhibiting the Wnt/β-catenin pathway. Moreover, we validated that silencing HOXA5 mitigated the suppressive effects of KDM5A knockdown on osteosarcoma proliferation and migration, while promoting apoptosis, through the activation of the Wnt/β-catenin pathway. This was evident from the altered protein expression levels of Wnt, GSK3B, p-GSK3B, and c-myc. Thus, we postulate that the KDM5A/HOXA5 axis modulates osteosarcoma progression via the activation of the Wnt/β-catenin pathway. Recent research has uncovered that HOXA5 was capable of influencing the activity of the JAK/STAT3 classical pathway. In studies of adipocytes, it has been discovered that HOXA5 can inhibit adipocyte proliferation by reducing the phosphorylation levels of the key factors JAK2 and STAT3 within the JAK2/STAT3 signaling pathway. This suggests that HOXA5 negatively regulates the activity of the JAK/STAT3 pathway, thereby affecting cellular proliferation [41]. However, our findings indicate that overexpression of HOXA5 has no impact on the JAK/STAT3 classical pathway. Moreover, it is worth noting that in this study, we employed knockdown rather than knockout, with plans to further refine and enhance our research endeavors in the future.

In summary, our study proved that KDM5A is conspicuously upregulated in osteosarcoma, exerting a detrimental influence on its progression. Functionally, we have demonstrated that silencing HOXA5 diminishes the suppressive effects of KDM5A knockdown on osteosarcoma proliferation and migration, while promoting apoptosis, through the activation of the Wnt/β-catenin pathway. Our investigation into the role of the KDM5A/HOXA5 axis in cell proliferation, migration, and apoptosis offers a novel perspective on the underlying mechanisms of osteosarcoma pathogenesis, adding to the existing body of knowledge and suggesting potential therapeutic targets.

Conclusions

Our study demonstrated that the KDM5A/HOXA5 axis regulated the progression of osteosarcoma by activating the Wnt/β-catenin pathway. In the future, we hoped to further validate our findings from different perspectives and aimed to provide insights for clinical research on osteosarcoma.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

H3K4me2:

Di- and tri-methylation of lysine 4 on histone H3

KDM5A:

Lysine-specific demethylase 5A

HOXA5:

Homeobox A5′

DNAm:

DNA methylation

IHC:

Immunohistochemistry

PI:

Propidium iodide

ChIP:

Chromatin immunoprecipitation

qRT-PCR:

Reverse transcription–quantitative real-time PCR

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Acknowledgements

The authors gratefully acknowledge the financial supports of Changsha Natural Science Foundation general project (kq2202050).

Funding

The authors gratefully acknowledge the financial supports of Changsha Natural Science Foundation general project (kq2202050) and Key Innovation Project of The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China (YNKY202212).

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Authors and Affiliations

  1. Department of Spine Surgery, Hengyang Medical School, The Affiliated Changsha Central Hospital, University of South China, The No.161 of the Shaoshan South Road, Changsha City, Hunan Province, China

    Yi Luo, Youzhi He, Yuxia Xu, Yongfu Wang & Li Yang

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Contributions

Yi Luo:Conceptualization, Methodology, Resources, Writing—Original Draft, Writing—Review & Editing, Youzhi He: Investigation, Formal analysis, Writing—Original Draft & Editing, Supervision. Yuxia Xu: Investigation, Data analysis, Supervision, Yongfu Wang: Investigation,Data analysis, Supervision, Li Yang: Investigation, Data analysis,Supervision.

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Correspondence to Yi Luo.

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Luo, Y., He, Y., Xu, Y. et al. The KDM5A/HOXA5 axis regulates osteosarcoma progression via activating the Wnt/β-catenin pathway. Eur J Med Res 30, 284 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02478-7

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European Journal of Medical Research

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