Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2026 Jun 18;16:27887. doi: 10.1038/s41598-026-57828-0

LINC00222 regulates FOXO3 to interfere with β-catenin signaling pathway and suppresses prostate cancer progression

Hui Li 1,2,✉, Wenfeng Zhao 2, Yuyou Deng 2, Zhongcheng Liu 3,✉, Jiameng Li 3, Shuoqi Tian 3
PMCID: PMC13547367  PMID: 42315881

Abstract

FOXO3 has been confirmed to be closely involved in the occurrence and progression of prostate cancer. In this study, we aimed to investigate the potential mechanisms of FOXO3 in prostate cancer progression. The expression of LINC00222 and FOXO3 in non-cancerous tissues and cancer tissues was detected by RT-qPCR. LINC00222 roles in regulating cell function in vitro as well as tumorigenesis in vivo were detected by gain-of-function and loss-of-function assays. The key protein expression was detected by western blotting assay. Dual luciferase reporter assay, bioinformatics analysis and immunoprecipitation assay were used to analyze the relationship between LINC00222 and miR-19a, miR-19a and FOXO3, or FOXO3 and APC. The results showed that the expression levels of both LINC00222 and FOXO3 were downregulated in prostate cancer tissues. In PC-3 cells, upregulation of FOXO3 significantly inhibited vimentin expression and promoted E-cadherin expression, as well as enhanced cell sensitivity to docetaxel. Compared to the control group, upregulation of LINC00222 in prostate cancer significantly inhibited cell proliferation and metastasis, and promoted cell apoptosis. Furthermore, LINC00222 competitively bound to miR-19a, enhanced FOXO3 expression, and then increased APC expression and inhibited the β-catenin signaling. Consistently, the in vivo experiments further confirmed the cancer inhibitory role of LINC00222 in prostate cancer. The present study revealed that LINC00222 adsorbed miR-19a to modulate FOXO3/APC/β-catenin signaling cascades and thereafter inhibited prostate cancer progression, which provided valuable insights into prostate cancer treatment.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1038/s41598-026-57828-0.

Keywords: Prostate cancer, FOXO3, β-catenin, LINC00222, miR-19a

Subject terms: Urological cancer, Cell biology, Chemical biology

Introduction

Prostate cancer is the second most common cancer among men worldwide, and a leading cause of cancer-related deaths1,2. Prostate cancer is a highly heterogeneous disease, ranging from inert or slow-developing forms to highly aggressive or potentially fatal variants. Approximately 75% of patients present with localized prostate cancer, which has a 5-year survival rate of nearly 100%. However, approximately 10% of prostate cancer patients have metastatic spread, with a 5-year survival rate of only 30%3,4. In the early stage, prostate cancer is usually androgen-dependent prostate cancer (ADPC), and androgen deprivation therapy (ADT) is the mainstay of treatment5. Regrettably, the majority of prostate cancer patients eventually progress to androgen-independent prostate cancer (AIPC), which is resistant to ADT6,7. Recent studies have indicated that prostate cancer showed a shift towards higher grade and higher stage, along with an increased incidence of metastasis, most likely due to changes in screening guidelines8,9. This increased prevalence of metastatic prostate cancer and the dismal prognosis of these cases highlight the urgent need to elucidate the cancer pathogenesis and identify biomarkers for diagnosis, prognosis, and patient-specific treatment.

Currently, several biomarkers are employed in clinical practice to facilitate risk stratification, prognostic evaluation, and the guidance of personalized treatment strategies in prostate cancer. For example, galectin-3 (Gal-3), a β-galactoside-binding protein, has multiple biological functions. It has been demonstrated to play important roles in tumor development processes through regulating cell proliferation, apoptosis, cell invasion, and metastasis10,11. Gal-3 expression typically increases during the progression of prostate cancer, specifically, Gal-3 was negatively expressed in ADPC (e.g. LNCaP) cells, whereas highly expressed in AIPC (e.g. DU-145 and PC-3)12,13. The enhanced expression of Gal-3 led to the suppression of immune response and increased tumor invasiveness and metastatic potential14–16. In addition, evidence has demonstrated that Gal-3 enhances anchorage-independent cell growth and xenograft tumor growth even after castration13. Overall, Gal-3 plays an important role in prostate cancer and serves as an important therapeutic target17, implying that the integration of clinically relevant prognostic and predictive biomarkers are crucial for preventing metastatic disease and developing individualized treatment strategies1.

FOXO3, a transcription factor of the FOXO (forkhead box class O) family, plays a critical role in regulating cell biological processes, including proliferation, apoptosis, cell cycle regulation, and DNA damage18–20. FOXO3a is frequently inactivated in cancer cell lines via gene mutation or cytoplasmic sequestration of FOXO3a protein. Its inactivation is associated with the initiation and progression of different types of cancers21,22, including prostate cancer18,23–26. Specifically, the inactivation of FOXO3 is associated with initiation and progression of prostate cancer, suggesting its role as a tumor suppressor20,24. Moreover, reduced FOXO3a expression, increased FOXO3a phosphorylation and diminished FOXO-responsive promoter activity may contribute to the progression of ADPC to AIPC26. Furthermore, FOXO3 is involved in regulating the treatment response of prostate cancer18,27. For example, FOXO3 serves as an interesting potential treatment target as it is regulated during treatment by very common anti-cancer drugs such as paclitaxel, cisplatin, docetaxel, and doxorubicin18. Overexpression of FOXO3 abated the glycolysis level and alleviated the radio-resistance caused by enhancing miR-223-3p to a certain extent27. Given the crucial roles of FOXO3 in prostate cancer, deciphering its upstream and downstream molecular regulatory networks is of great significance for fully elucidating the pathogenesis and developing novel therapeutic strategies.

The network of competing endogenous RNA (ceRNA) constructed among long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs has been evidenced to be involved in the pathoetiology of various cancers28,29. Identifying such interactions among different RNA molecules facilitates a more profound understanding of the mechanisms driving cancer pathogenesis30,31. For example, LINC00472 inhibited by ZEB1 regulated the miR-23a-3p/FOXO3 axis to suppress the progression of pancreatic cancer32,33; lncRNA GAS5 inhibited cell proliferation via the miR-182-5p/FOXO3 axis in colorectal cancer34. However, lncRNA-miRNA-FOXO3 interactions have not been clearly reported in prostate cancer. Wnt/β-catenin signaling pathway has been reported to be deregulated in various cancers and is closely implicated in cancer occurrence and progression35, including prostate cancer36. Previous study showed that FOXO3 regulated Wnt/β-catenin signaling pathway and inhibited the epithelial-mesenchymal transition (EMT) of prostate cancer cells37. However, the mechanism by which FOXO3 acts on β-catenin in prostate cancer is not yet completely clarified.

In this study, we first explored FOXO3 expression in prostate cancer and its correlation with different lncRNAs through clinical sample analysis and bioinformatic tools. Subsequently, the functional roles and regulatory mechanisms of lncRNA-miRNA-FOXO3 in prostate cancer were explored in vitro and/or in vivo. Finally, the study tried to explore whether FOXO3 regulates β-catenin signaling in prostate cancer. This study aimed to provide new theoretical and experimental basis for the mechanism research and therapeutic strategy for prostate cancer.

Materials and methods

Clinical specimens

Eight prostate cancer tissue samples and the paired non-cancerous tissues from patients with prostate carcinoma were collected at Peking University International Hospital. This project was approved by the Ethics Committee of Peking University International Hospital (Approval no. 2022-KY-0070-01), and followed the rules of the Helsinki Declaration. All tissues involved in this study were secondary uses of previous clinical samples, so an informed consent waiver was approved by the Ethics Committee.

Cell culture and transfection

The prostate carcinoma cell lines (PC-3 and LNCaP) were purchased from Wuhan Pricella Biotechnology Co., Ltd. (Hubei, China). Cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Beijing Solarbio Science & Technology Co., Ltd.) and 1% antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin) in a humidified atmosphere containing 5% CO2 at 37˚C. The short hairpin RNA (shRNA) against LINC00222 (Sh-LINC00222), shRNA negative control (Sh-NC), pcDNA3.1-LINC00222 (OE-LINC00222), pcDNA3.1 vector (OE-NC), miR-19a mimics, miR-19a inhibitor, miRNA negative controls (NC), shRNA against FOXO3 (Sh-FOXO3) and pcDNA3.1-FOXO3 (OE-FOXO3) were purchased and synthesized from Shanghai GenePharma, Co., Ltd. (Shanghai, China). These plasmids were transfected into LNCaP and PC-3 cells using Lipofectamine® 3000 (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. In brief, a mixture of Lipofectamine® 3000 with 50 nM plasmid or mimic was added to the cells and incubated at 37˚C for 6 h. Subsequently, the medium was replaced with fresh RPMI-1640 medium for 24 h. The transfection efficiency was assessed by reverse transcription-quantitative PCR (RT-qPCR).

RT-qPCR assay

Total RNA was extracted from tissues or cells using TRIzol® reagent (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. A total of 2 µg RNA was reversely transcribed into the complementary DNA using PrimeScript RT Reagent kit (Invitrogen, Thermo Fisher Scientific, Inc.). qPCR was performed on a Step One Plus Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Inc.). GAPDH or U6 was used as a normalization control for mRNA or miRNA, respectively. The relative expression levels were calculated via the 2‑ΔΔCt method38.

Western blotting assay

Total protein was collected from cells by radioimmunoprecipitation assay (RIPA) lysis buffer and quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Inc.). The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 30 µg protein/lane was performed with 10–12% resolving gel. Then, the proteins were transferred into polyvinylidene difluoride (PVDF) membranes and the membranes were blocked with 5% skimmed milk at room temperature for 1 h. GAPDH or lamin B1 was utilized as an internal control. The membranes were incubated at 4˚C overnight with the following diluted primary antibodies: anti-E-cadherin (1:500, Abcam, cat. no. ab76055), anti-vimentin (1:1000, Abcam, cat. no. ab92547), anti-FOXO3 (1:1000, Cell Signaling Technology, Inc., cat. no. #2497), anti-β-catenin (1:5000, Abcam, cat. no. ab32572), anti-TCF4 (1:1000, Abcam, cat. no. ab130014), anti-APC (1:1000, Abcam, cat. no. ab40778), anti-GAPDH (1:10000, Abcam, cat. no. ab181602) or anti-lamin B1 (1:2000, Abcam, cat. no. ab133741). Next, the membranes were incubated with horseradish peroxidase (HRP) labeled secondary antibody (1:3000, Bioss, cat. no. bs-0295G-HRP and bs-0296G-HRP) at room temperature for 1 h. Finally, bands were visualized using enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Inc.), and then quantified with Image Lab 4.1 software (Bio-Rad Laboratories, Inc.).

Cell proliferation assay

The cell counting kit-8 (CCK-8) was used to determine cell proliferation. The transfected cells were seeded in 96‑well plates with 3000 cells/well. After 24, 48–72 h of incubation, 10 µL CCK-8 stain (Beyotime Institute of Biotechnology) was added for per well. After incubation for 2 h, the absorbance at 450 nm was measured to draw cell proliferation curves.

Cell invasion and migration

Cell invasion was detected using the transwell chambers (EMD Millipore) coated with Matrigel® (BD Biosciences) to form a matrix barrier. The lower chambers were filled with RPMI-1640 medium supplemented with 10% FBS. The cells were loaded into upper well without FBS and incubated at 37˚C for 24 h. The cells in the lower side were sequentially fixed with methanol and stained with 0.1% crystal violet. The wound healing assay was used to determine cell migration. The cells were cultured in culture plates to 90% confluence. The denuded zone was created with a micropipette tip, followed by removing exfoliated cells. The cells were then cultured in serum‑free RPMI-1640 medium at 37˚C for 48 h. Image analysis was obtained by using ImageJ software.

Cell apoptosis assay

The flow cytometry was used to detect apoptosis. According to the Annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology) instructions, the cells were resuspended in binding buffer. Then, the cells were incubated with Annexin V‑FITC in the dark at room temperature for 15 min, followed by continuous incubation with propidium iodide (PI) at 4 ℃. Finally, the flow cytometry (Bio‑Rad Laboratories, Inc.) was used to measure the cellular fluorescence intensity after 72 h.

Luciferase reporter assay

A luciferase reporter assay was used to confirm the targeting association between miR-19a and the 3’-untranslated region (3’-UTR) of FOXO3. The reporter vector pmirGLO-FOXO3-wild type (WT) was constructed by amplifying the WT 3’-UTR fragment of FOXO3 mRNA containing the predicted miR-19a binding sites, and inserting into a pmirGLO dual-luciferase miRNA target expression vector (Promega Corp.). 293T cells were co-transfected with luciferase plasmids as well as miR-19a or control miRNA. After transfection, the luciferase activities of firefly and renilla were measured with a Dual-Luciferase Reporter Assay System (Promega Corp.). The values were normalized to the renilla luciferase transfection control.

RNA pull-down assay

The cells were transfected with biotinylated miR-19a probe and control probe for 48 h, and incubated with the specific lysis buffer (Ambion, USA) for 10 min. The lysate was incubated with the M-280 streptavidin magnetic beads (Sigma-Aldrich, USA) that were pre-coated with RNase-free and yeast tRNA (Sigma-Aldrich, USA) for 3 h at 4 °C, and then rinsed twice with cold lysis buffer. The expression profile of LINC00222 was detected by RT-qPCR.

Co-immunoprecipitation (Co-IP) assay

The cells were lysed with RIPA buffer (Thermo Fisher Scientific, Inc.) on ice, and protein A/G agarose beads (Thermo Fisher Scientific, Inc.) were used to pre-clean protein from the whole cell extracts for 2 h at 4˚C. Subsequently, the protein was incubated with IgG (control antibody, 1:1000, Abcam, cat. no. ab172730) or TCF4 antibody (1:50, Cell Signaling Technology, Inc., cat. no. #2569) with gentle shaking at 4˚C for 24 h. The beads were washed with RIPA buffer, and the immunocomplexes were eluted by boiling at 100˚C for 5 min in sample buffer. Then, the beads were washed with PBS and immunoprecipitants were assessed via western blotting.

Chromatin immunoprecipitation (Ch-IP) assay

Following 70%-80% cell confluence, the cells were fixed in 1% formaldehyde for 10 min at room temperature and ultrasonicated to produce chromatin fragments. Subsequently, the cells were subject to centrifugation at 4 °C. The supernatants were incubated at 4 °C overnight with the positive control antibody and rabbit anti-FOXO3 (1:50, Cell Signaling Technology, Inc., cat. no. #12829). Protein Agarose/Sepharose was added to precipitate endogenous DNA-protein complex. After centrifugation, the DNA-protein complexes were incubated overnight at 65 °C to terminate cross-linking. Then, phenol/chloroform was included to extract and to purify the isolated DNA fragments. Finally, the expression pattern of APC promoter was detected by RT-qPCR.

Immunostaining assay

Paraffin-embedded femoral sections were kept in a 60°C oven for 24 h, then deparaffinized with xylene and hydrated with an ethanol gradient (100%-70%). After 30 min of continuous incubation with antigen recovery solution and 3% H2O2, the slides were rinsed with water and incubated with the primary antibody (anti-FOXO3, 1:100, Abcam, cat. no. ab314007) overnight at 4°C. Negative controls were performed with nonimmunized serum instead of primary antibody. Next, the slides were incubated with the corresponding secondary antibody for 30 min, followed by 3,3’-diaminobenzidine (DAB) and hematoxylin staining, respectively. The slides were then examined and photographed with a light microscope. On the other hand, the prostate carcinoma cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and then blocked by 2% bovine serum albumin (BSA) for 2 h at room temperature. Cells were sequentially incubated with the anti-FOXO3 antibody (1:100, Abcam, cat. no. ab314007) and the corresponding secondary antibody. Then, cells were stained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma, Merck KGaA) for 15 min. Fluorescent staining was visualized under a laser scanning confocal microscope.

In vivo xenograft model of mice

Male BALB/c nude mice (4–5 weeks) were provided from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were placed in a specific pathogen-free (SPF) environment under isothermal conditions with regular photoperiods (12-h light/dark cycle). Water and food were provided to all animals ad libitum. All animal studies were approved by the Ethics Committee of Peking University International Hospital (Approval no. 2022-KY-0070-01). The mice were randomly divided into 3 groups (n = 15) and subcutaneously inoculated in the right flank with transfection OE-LINC00222 or Sh-LINC00222 PC-3 cells. Tumor volume was measured with a digital caliper (length × width2), and body weight was periodically recorded.

Bioinformatics analysis

The Ualcan database (https://ualcan.path.uab.edu/index.html) was used to analyze the effect of FOXO3 expression and Gleason score on prostate cancer patient survival. The Ensembl database (https://asia.ensembl.org/index.html) was used to find lncRNAs near the FOXO family FOXO3. The starBase/ENCORI website (https://rnasysu.com/encori/) was used for analyzing correlations between genes and predict potential binding sites. The JASPAR database (http://jaspar.genereg.net/) was used to identify the FOXO3-binding motif in the promoter region of APC.

Statistical analysis

All experiments at least three times were performed. Unless otherwise stated, data were expressed as the mean ± standard deviation. Comparisons between multiple or two groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test or Student’s t-test, respectively. Results were statistically significant at *P < 0.05, **P < 0.01 and ***P < 0.001.

Results

Expression pattern of FOXO3 and LINC00222 in prostate tumor tissues

Gleason score is an important prognostic indicator for prostate cancer patients39. The study collected biopsy samples from prostate cancer patients who have Gleason score 7–9 to investigate FOXO3 expression. Immunohistochemistry results showed that tumor tissues with higher Gleason score had a relatively lower expression level of FOXO3 (Fig. 1A). Analysis of the prostate adenocarcinoma (PRAD) dataset from the cancer genome atlas program (TCGA) via the Ualcan online data platform revealed that patients with low/medium expression of FOXO3 in the same Gleason score had lower survival rate than that of patients with high expression of FOXO3 (Fig. 1B). This finding suggested that the expression level of FOXO3 may serve as a potential prognostic biomarker for improving risk stratification in prostate cancer, supporting further explorations toward the function and underlying mechanisms of FOXO3. In order to more precisely quantify the expression of FOXO3 in prostate cancer, we also examined the FOXO3 expression in prostate cancer tissues by RT-qPCR, and the results demonstrated that the expression of FOXO3 in tumor tissues was significantly decreased compared to the paired non-cancerous tissues (Fig. 1C). In addition, we searched the lncRNAs near the FOXO3 genome by Ensembl database and quantified them to further explore the potential mechanism underlying FOXO3 in prostate cancer. The expression of LINC00222 was obviously different in the non-cancerous tissues and tumor tissues (Fig. 1D). Meanwhile, the ENCORI database displayed a closely positive correlation between LINC00222 and FOXO3 expression in 499 PRAD samples (P < 0.001, Fig. 1E). It is evident that LINC00222 might be a potential molecule regulating the expression of FOXO3.

Fig. 1.

Fig. 1

Open in a new tab

Expression patterns of FOXO3 and LINC00222 in prostate cancer tissues.

(A) Expression of FOXO3 in prostate cancer tissues was detected by immunohistochemistry, with non- cancerous tissues as controls; (B) Effect of FOXO3 expression and Gleason score on the prognosis of prostate cancer patients; (C) Expression of FOXO3 mRNA in prostate tumor tissues was detected by RT-qPCR (*P < 0.05); (D) Expression of LINC00598, LINC00222, LINC00261 and FENDRR in prostate tumor tissues were detected by RT-qPCR; (E) Correlation between LINC00222 and FOXO3 was analyzed by starBase/ENCORI website.

FOXO3 inhibited EMT, promoted tumor cell apoptosis and improved drug resistance in prostate cancer

Then, the gain/loss-of-function assay of FOXO3 was used to further investigate its potential role on EMT and drug resistance in prostate cancer cells. FOXO3 expression was significantly increased when the cells were transfected with OE-FOXO3, whereas it was decreased while the cells were transfected with Sh-FOXO3 (Fig. 2A and B). Overexpression of FOXO3 increased the expression of E-cadherin and decreased the expression of vimentin (OE-FOXO3 vs. Control, P < 0.05); conversely, silencing of FOXO3 inhibited E-cadherin expression and promoted vimentin expression (Sh-FOXO3 vs. Control, P < 0.05) (Fig. 2B). Next, we employed docetaxel to examine FOXO3 effect on drug resistance of prostate cancer cells. With the increase of docetaxel treatment concentration, cell viability in OE-FOXO3 group had a significant decreasing trend, while cell viability in Sh-FOXO3 group was superior to that of the control group (OE-FOXO3 vs. Control, P < 0.01; Sh-FOXO3 vs. Control, P < 0.01; Fig. 2C). Similarly, overexpression of FOXO3 triggered a higher rate of apoptosis, whereas silencing FOXO3 decreased the apoptosis rate of PC-3 cells (OE-FOXO3 vs. Control, P < 0.01; Sh-FOXO3 vs. Control, P < 0.01; Fig. 2D). The above results indicated that FOXO3 had a potential role in inhibiting prostate cancer progression.

Fig. 2.

Fig. 2

Open in a new tab

FOXO3 affected cell EMT, apoptosis and drug resistance in prostate cancer.

(A) Regulation of FOXO3 expression in PC-3 cells; (B) Effect of FOXO3 on expression of vimentin and E-cadherin detected by western blotting; (C) Effect of OE-FOXO3 and Sh-FOXO3 on PC-3 cell proliferation was evaluated by CCK-8 kits; (D) Effect of OE-FOXO3 and Sh-FOXO3 on PC-3 cell apoptosis was detected by flow cytometry. *P < 0.05 and **P < 0.01.

LINC00222 adsorbed miR-19a to regulate FOXO3 in prostate cancer

To investigate the relationship between LINC00222 and FOXO3, cell transfection was used to regulate the expression of LINC00222 in prostate cancer cells. The RT-qPCR results revealed that compared with the control group, the expression of LINC00222 in LNCaP and PC-3 cells transfected with Sh-LINC00222 was significantly decreased (Sh-LINC00222 vs. Control, P < 0.001), while the expression of LINC00222 in cells transfected with OE-LINC00222 was significantly increased (OE-LINC00222 vs. Control, P < 0.01) (LNCaP cells in Fig. 3A, PC-3 cells in Fig. 3B). Next, FOXO3 expression was examined in different groups, and the results showed that FOXO3 expression was significantly increased in OE-LINC00222-transfected LNCaP and PC-3 cells (OE-LINC00222 vs. Control, P < 0.05 for mRNA level and P < 0.01 for protein level), and significantly decreased in Sh-LINC00222-transfected cells (Sh-LINC00222 vs. Control, P < 0.05 for both mRNA and protein levels) at both mRNA (Fig. 3C) and protein levels (Fig. 3D). The results of immunofluorescence and fractionation assays showed that overexpression of LINC00222 promoted FOXO3 translocation into cell nucleus (Fig. 3E).

Fig. 3.

Fig. 3

Open in a new tab

LINC00222 adsorbed miR-19a to regulate FOXO3. (A) Regulation of LINC00222 expression in LNCaP cells; (B) Regulation of LINC00222 expression in PC-3 cells; (C) Effect of LINC00222 expression on FOXO3 mRNA expression in LNCaP and PC-3 cells; (D) Effect of LINC00222 expression on FOXO3 protein expression in LNCaP and PC-3 cells; (E) Effect of OE-LINC00222 and Sh-LINC00222 on FOXO3 expression was detected by immunofluorescence (Scale bar, 20 μm) and fractionation assays; (F) Predicted binding sites of miR-19a in LINC00222 and FOXO3; (G) The interaction of miR-19a with LINC00222 was detected by RNA pull down; (H) The interaction of miR-19a with FOXO3 was analyzed by luciferase reporter assay; (I) The effect of OE-LINC00222 on FOXO3 expression was reversed by miR-19a; (J) The effect of Sh-LINC00222 on FOXO3 expression was reversed by miR-19a. *P < 0.05, **P < 0.01 and ***P < 0.001.

The miR-19a, a member of the miR-19 family, is widely recognized as an important oncogenic microRNA40. To further investigate the correlation between miR-19a, LINC00222 and FOXO3, we predicted the binding sites of miR-19a towards LINC00222 and FOXO3 using the starBase/ENCORI database (Fig. 3F). The results displayed that miR-19a could interact with LINC00222 (Fig. 3G). The luciferase reporter assay showed that miR-19a could interact with FOXO3 (Fig. 3H). LINC00222 overexpression significantly increased FOXO3 expression (OE-LINC00222 vs. Control, P < 0.01), whereas OE-LINC00222 + miR-19a mimics significantly downregulated FOXO3 expression (OE-LINC00222 + miR-19a mimics vs. OE-LINC00222 + miR-NC, P < 0.01) (Fig. 3I). On the contrary, knockdown of LINC00222 induced a significant decrease in FOXO3 expression (Sh-LINC00222 vs. Control, P < 0.01), whereas Sh-LINC00222 + miR-19a inhibitor significantly upregulated FOXO3 expression (Sh-LINC00222 + miR-19a inhibitor vs. Sh-LINC00222 + miR-NC, P < 0.01) (Fig. 3J). Thus, it is suggested that LINC00222 might be able to regulate FOXO3 through adsorbing miR-19a.

Overexpression LINC00222 inhibited cell proliferation, invasion and migration, and promoted cell apoptosis in prostate cancer

A series of cell function experiments were performed following the upregulation and downregulation of LINC00222 in prostate cancer. The results illustrated that compared with the control group, overexpression of LINC00222 significantly inhibited cell proliferation, and knockdown of LINC00222 significantly promoted the proliferation of LNCaP cell after 48 h of cell transfection (OE-LINC00222 vs. OE-NC, P < 0.01; Sh-LINC00222 vs. Sh-NC, P < 0.01; Fig. 4A). A similar phenomenon was observed in PC-3 cells (OE-LINC00222 vs. OE-NC, P < 0.01; Sh-LINC00222 vs. Sh-NC, P < 0.05; Fig. 4B). The wound healing assay demonstrated that knockdown of LINC00222 significantly increased the migration rate of LNCaP and PC-3 cells (Sh-LINC00222 vs. Sh-NC, P < 0.01), while overexpression of LINC00222 significantly inhibited cell migration ability (OE-LINC00222 vs. OE-NC, P < 0.01) (LNCaP cells in Fig. 4C, PC-3 cells in Fig. 4D). Besides, compared to the control group, the count of invasion cells was significantly increased in the Sh-LINC00222-transfected cells (Sh-LINC00222 vs. Sh-NC, P < 0.05), while it was significantly decreased in the OE-LINC00222-transfected cells (OE-LINC00222 vs. OE-NC, P < 0.05) (Fig. 4E). In addition, the flow cytometry assay showed that cell apoptosis in Sh-LINC00222 group was significantly inhibited (Sh-LINC00222 vs. Sh-NC, P < 0.05), while it was significantly increased in the OE-LINC00222 group (OE-LINC00222 vs. OE-NC, P < 0.01) (LNCaP cells in Fig. 4F, PC-3 cells in Fig. 4G). The above results illustrated that overexpression of LINC00222 could inhibit prostate cancer cell proliferation, migration and invasion, and promote apoptosis.

Fig. 4.

Fig. 4

Open in a new tab

LINC00222 regulated FOXO3 to affect tumor cell proliferation, invasion, migration, and apoptosis.

(A) LNCaP cell proliferation was evaluated by CCK-8 kits; (B) PC-3 cell proliferation was evaluated by CCK-8 kits; Sh-LINC00222 vs. Sh-NC *P < 0.05 and **P < 0.01, OE-LINC00222 vs. OE-NC #P < 0.05 and ##P < 0.01; (C) LNCaP cell migration ability was evaluated by wound healing assay (Scale bar, 100 μm); (D) PC-3 cell migration ability was evaluated by wound healing assay (Scale bar, 100 μm); (E) Tumor cell invasion ability was evaluated by transwell assays (Scale bar, 100 μm); (F) LNCaP cell apoptosis was detected by flow cytometry; (G) PC-3 cell apoptosis was detected by flow cytometry. *P < 0.05 and **P < 0.01.

FOXO3 inhibited β-catenin signaling pathway by initiating APC expression in prostate cancer cells.

The Co-IP results displayed that FOXO3 interacted with β-catenin and competed for binding to TCF4 (Fig. 5A), which form a transcriptional regulatory complex with β-catenin through direct interaction, serves as a mainstay in Wnt/β-catenin signaling41,42. Overexpression of FOXO3 significantly reduced the expression level of β-catenin in the nucleus of PC-3 cells at protein level (OE-FOXO3 vs. Control, P < 0.01; Fig. 5B), but not the mRNA level (Fig. 5C). And, silencing of FOXO3 induced an opposite trend (Fig. 5B and C). Notably, ENCORI database analysis displayed a strongly positive correlation between APC and FOXO3 expression in PRAD samples (P < 0.001, Fig. 5D). The JASPAR database predicted the potential binding sites in APC promoter region for FOXO3 (Fig. 5E). In addition, Ch-IP assay confirmed that FOXO3 could bind to APC (Fig. 5F). Overexpression of FOXO3 significantly increased the mRNA and protein expression of APC, and silencing of FOXO3 significantly downregulated the mRNA and protein expression of APC (OE-FOXO3 vs. Control, P < 0.01; Sh-FOXO3 vs. Control, P < 0.05; Fig. 5G and H). Subsequently, to verify the direct functional dependence of LINC00222, FOXO3, APC, and β-catenin, we conducted rescue experiments. Western blot analysis confirmed that overexpression of LINC00222 significantly increased the protein expression of both FOXO3 and APC. Conversely, the knockdown of FOXO3 markedly reduced APC expression. Notably, silencing FOXO3 in the context of LINC00222 overexpression markedly reversed the upregulatory effect of LINC00222 on APC expression, suggesting that the regulation of APC by LINC00222 may be FOXO3-dependent (Fig. 5J). Similarly, simultaneous overexpression of FOXO3 and silencing of APC obviously attenuated the inhibitory effect of FOXO3 on β-catenin expression, indicating that APC may serve as a critical mediator in the regulation of β-catenin by FOXO3 (Fig. 5K). Collectively, these two rescue experiments verified that LINC00222 positively modulated APC expression by upregulating FOXO3, thereby suppressing the activation of the β-catenin signaling in prostate cancer cells.

Fig. 5.

Fig. 5

Open in a new tab

FOXO3 interfered with APC to affect β-catenin signaling pathway. (A) Correlation of FOXO3, β-catenin and TCF4 detected by Co-IP; (B) Expression of FOXO3 and β-catenin in cell nucleus was detected by western blotting; (C) Effect of FOXO3 on β-catenin protein expression was detected by western blotting; (D) Effect of FOXO3 on β-catenin mRNA expression was detected by RT-qPCR; (E) Correlation between APC and FOXO3 was analyzed by starBase/ENCORI website; (F) Identification of FOXO3 binding motifs in the APC promoter region by JASPAR database; (G) FOXO3 binding to APC promoter region was detected by Ch-IP; (H) Effect of FOXO3 on APC mRNA expression was detected by RT-qPCR; (I) Effect of FOXO3 on APC protein expression was detected by western blotting. (J) Rescue experiments involving LINC00222 overexpression + FOXO3 knockdown. (K) Rescue experiments involving FOXO3 overexpression + APC knockdown. *P < 0.05 and **P < 0.01.

LINC00222 regulated FOXO3 to interfere with β-catenin signaling pathway to inhibit prostate cancer progression

Also, LINC00222 effect on the tumorigenesis of prostate cancer was examined using the xenograft models. After 4 weeks of tumor growth, six mice in each group were randomly selected for euthanasia and their tumor tissues were stripped for analysis. The stripped tumor tissues are shown in Fig. 6A. Compared with the control group, tumor growth was slower in the OE-LINC00222 group and significantly increased in the Sh-LINC00222 group (OE-LINC00222 vs. Control, P < 0.05; Sh-LINC00222 vs. Control, P < 0.05; Fig. 6B-D). Furthermore, significant differences in survival rates were observed among the 3 groups, with the highest rate in the Sh-LINC00222 group, followed by the control group, and the lowest in the OE-LINC00222 group (Fig. 6E). Immunohistochemical analysis of tissue sections revealed that FOXO3 expression was obviously increased, while the expression of Ki67 and β-catenin was obviously decreased in the OE-LINC00222 group compared with that of the control group. On the contrary, FOXO3 expression was obviously decreased, and the expression of Ki67 and β-catenin was obviously increased in the Sh-LINC00222 group (Fig. 6F). As a result, upregulation of LINC00222 expression could promote FOXO3 expression and inhibit β-catenin signaling, which in turn suppressed prostate cancer progression.

Fig. 6.

Fig. 6

Open in a new tab

LINC00222 inhibited prostate cancer progression in vivo. (A) Images of the tumors in the OE-LINC00222 and Sh-LINC00222 at 4 weeks after inoculation; (B) The tumor weights after 4-week inoculation; (C) The expression levels of LINC00222 in the control, OE-LINC00222, and Sh-LINC00222 group. (D) The tumor volumes measured every week after cell inoculation; (E) Effect of LINC00222 on the survival percentage of mice; (F) Expression of FOXO3, Ki67 and β-catenin in the tumor tissues was detected by immunohistochemistry. *P < 0.05 and **P < 0.01.

Discussion

Prostate cancer is the most common malignant tumor type for males and the second leading cause of cancer-related mortality worldwide43. Prostate cancer is distinctly heterogeneous and could be subdivided into many intermediate clinical states44. However, there is still uncertainty in the current research on the molecular mechanisms of prostate cancer progression. Herein, this study reveals that LINC00222 suppresses prostate cancer progression by a mechanism in which it competitively binds miR-19a to elevate FOXO3 expression, leading to the transcriptional activation of APC and subsequent inhibition of the β-catenin signaling pathway.

FOXO3 as a tumor suppressor has been demonstrated in several kinds of tumors20,45,46. The Ualcan database analysis showed that PRAD patients with low expression of FOXO3 in the same Gleason grade had a poor prognosis. Also, it has been reported that reduced FOXO3a expression, increased FOXO3a phosphorylation and diminished FOXO-responsive promoter activity may contribute to the progression of ADPC to AIPC26. These findings indicated that FOXO3 expression holds significant clinical relevance in prostate cancer, supporting further explorations toward the function and underlying mechanisms of FOXO3. Notably, the lncRNA-miRNA-RNA, as a ceRNA network, has been found to contribute to the pathoetiology of different cancers, including prostate cancer. Interestingly, the ENCORI database confirmed the positive correlation between LINC00222 and FOXO3 in prostate cancer. Consistently, this study found that both FOXO3 and LINC00222 were lowly expressed in prostate cancer tissue samples, and upregulation of LINC00222 in prostate cancer cells resulted in a subsequent increase in FOXO3 expression, which was hypothesized that FOXO3 and LINC00222 might be involved in prostate cancer progression. In fact, it has been revealed that LINC00222 functioned as a tumor suppressor in lung adenocarcinoma47. However, the function of LINC00222 in prostate cancer has not been reported yet. In order to uncover the role of LINC00222 and FOXO3 in prostate cancer, the project conducted gain-of-function experiments. The results showed that FOXO3 overexpression inhibited cell proliferation and induced apoptosis in prostate cancer. Furthermore, our data indicated that upregulation of LINC00222 inhibited prostate cancer cell progression by promoting FOXO3 expression.

Further, extensive studies have revealed the fundamental biological functions and clinical applications of miRNAs in many types of cancers48,49. miR-19a serves as an oncogenic miRNA in colorectal cancer, indicating its significance in tumorigenesis and development50. Meanwhile, miR-19a has been reported to be aberrantly expressed in various types of cancers and has been observed to be possibly associated with the prognosis of cancer patients51. In addition, Wang et al. found that miR-19a promoted the migration, invasion and EMT ability by binding to CUL5 in prostate cancer52. miR-19a targets downstream target genes and thus regulates tumor progression40, which was consistent with our findings. Herein, we revealed that LINC00222 could competitively bind miR-19a to upregulate FOXO3 expression in prostate cancer. Through starBase database prediction, we hypothesized that LINC00222 might regulate prostate cancer progression through integrative binding to miR-19a, which was verified by the RNA pull down experiment. In addition, the dual luciferase reporter assay confirmed the binding relationship between FOXO3 and miR-19a.

The Wnt/β-catenin signaling pathway is a conserved signaling axis involved in a wide variety of physiological processes such as cell proliferation, differentiation, apoptosis, migration, invasion and tissue homeostasis53. In the Wnt/β-catenin pathway, aberrant regulation of the transcription factor β-catenin, a key component of the Wnt signaling pathway, can lead to early events in carcinogenesis35.

Previous studies have confirmed that downregulation of β-catenin can inhibit the activity of the Wnt/β-catenin signaling pathway and markedly reduce the expression of its downstream Wnt target genes (e.g., c-Myc, Cyclin D1)54–56. It has been found that a derivative of ivermectin inhibits the growth and metastatic phenotypes of prostate cancer cells by targeting the β-catenin pathway57. Notably, APC gene deletion drives β-catenin protein activation58. Meanwhile, it has been reported that Wnt/β-catenin signaling pathway activation in colorectal cancer is associated with loss of function of the tumor regulator APC53. More importantly, a strong association was found between APC gene methylation and prostate cancer59. In our study, the results of ENCORI, JASPAR database predictions and Ch-IP experiments indicated that FOXO3 could target the APC promoter region and induce its expression. Excitingly, this regulatory axis was validated both in vivo and in vitro. In detail, our data suggested that silencing LINC00222 led to the upregulation of β-catenin, a mechanism which, through miR-19a-mediated suppression of FOXO3, ultimately promoted prostate cancer cell proliferation and inhibited apoptosis. To enhance the generalizability of the findings in this study, further validation is required using clinical tissue samples or patient-derived xenograft models.

Conclusion

Taken together, our results indicate that LINC00222, a lncRNA that can adsorb miR-19a to regulate the expression of FOXO3 in prostate cancer, functions as a tumor suppressor in prostate cancer. In essence, our findings demonstrate that LINC00222 competitively binds to miR-19a, thereby upregulating FOXO3 and inhibiting the APC/β-catenin signaling pathway, and then inhibits prostate cancer progression. Collectively, this study reveals the significance of LINC00222/miR-19a/FOXO3/APC/β-catenin axis as a therapeutic target for prostate cancer.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (2.5MB, pdf)

Author contributions

Hui Li: Conceptualization, Data curation, Methodology, Project administration, Writing – original draft; Wenfeng Zhao: Formal analysis, Methodology, Writing – original draft; Yuyou Deng: Formal analysis, Methodology; Zhongcheng Liu: Conceptualization, Data curation, Project administration, Supervision, Writing – review and editing; Jiameng Li: Validation, Visualization; Shuoqi Tian: Validation, Visualization. The final version of the manuscript was read and approved by all the authors.

Data availability

The datasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study has been approved by the Ethics Committee of Peking University International Hospital (Approval no. 2022-KY-0070-01).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Hui Li, Email: urolgyxec@163.com.

Zhongcheng Liu, Email: liuzc@hbu.edu.cn.

References

  • 1.1 Am, K., Rajan, S., Alkhamees, P., Holley, M., Lakshmanan, V. K. & M. & Prostate cancer theragnostics biomarkers: An update. Investig Clin. Urol.65, 527–539. 10.4111/icu.20240229 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.2 Agbetuyi-Tayo, P., Gbadebo, M., Rotimi, O. A. & Rotimi, S. O. Advancements in Biomarkers of Prostate Cancer: A Review. Technol. Cancer Res. Treat.23, 15330338241290029. 10.1177/15330338241290029 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.3 Raychaudhuri, R., Lin, D. W. & Montgomery, R. B. Prostate Cancer: A Review. JAMA333, 1433–1446. 10.1001/jama.2025.0228 (2025). [DOI] [PubMed] [Google Scholar]
  • 4.4 Shen, M. et al. Identification of a 5-gene signature panel for the prediction of prostate cancer progression. Br. J. Cancer. 131, 1748–1761. 10.1038/s41416-024-02854-w (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.5 Luo, P. et al. The human positive cofactor 4 promotes androgen-independent prostate cancer development and progression through HIF-1α/β-catenin pathway. Am. J. Cancer Res.9, 682–698 (2019). [PMC free article] [PubMed] [Google Scholar]
  • 6.Lv, S. et al. 6 Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer. J Clin Invest 131, (2021). 10.1172/jci141335 [DOI] [PMC free article] [PubMed]
  • 7.7 Siu, M. K. et al. TCF7 is suppressed by the androgen receptor via microRNA-1-mediated downregulation and is involved in the development of resistance to androgen deprivation in prostate cancer. Prostate Cancer Prostatic Dis.20, 172–178. 10.1038/pcan.2017.2 (2017). [DOI] [PubMed] [Google Scholar]
  • 8.8 Siegel, R. L., Giaquinto, A. N. & Jemal, A. Cancer statistics, 2024. CA Cancer J. Clin.74, 12–49. 10.3322/caac.21820 (2024). [DOI] [PubMed] [Google Scholar]
  • 9.9 Borregales, L. D. et al. Grade Migration of Prostate Cancer in the United States During the Last Decade. J. Natl. Cancer Inst.114, 1012–1019. 10.1093/jnci/djac066 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.10 Souza, D. S., Macheroni, C., Pereira, G. J. S., Vicente, C. M. & Porto, C. S. Molecular regulation of prostate cancer by Galectin-3 and estrogen receptor. Front. Endocrinol. (Lausanne). 14, 1124111. 10.3389/fendo.2023.1124111 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lima, T. et al. 11 Gal-3 Protein Expression and Localization in Prostate Tumours. Curr Oncol 30, 2729–2742, (2023). 10.3390/curroncol30030206 [DOI] [PMC free article] [PubMed]
  • 12.12 Wang, Y. et al. The significance of galectin-3 as a new basal cell marker in prostate cancer. Cell. Death Dis.4, e753. 10.1038/cddis.2013.277 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.13 Dondoo, T. O. et al. Galectin-3 Is Implicated in Tumor Progression and Resistance to Anti-androgen Drug Through Regulation of Androgen Receptor Signaling in Prostate Cancer. Anticancer Res.37, 125–134. 10.21873/anticanres.11297 (2017). [DOI] [PubMed] [Google Scholar]
  • 14.14 Sharma, S. et al. Use of Glycoproteins-Prostate-Specific Membrane Antigen and Galectin-3 as Primary Tumor Markers and Therapeutic Targets in the Management of Metastatic Prostate Cancer. Cancers (Basel). 14. 10.3390/cancers14112704 (2022). [DOI] [PMC free article] [PubMed]
  • 15.15 Caputo, S. et al. Galectin-3 in Prostate Cancer Stem-Like Cells Is Immunosuppressive and Drives Early Metastasis. Front. Immunol.11, 1820. 10.3389/fimmu.2020.01820 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.16 Farhad, M., Rolig, A. S. & Redmond, W. L. The role of Galectin-3 in modulating tumor growth and immunosuppression within the tumor microenvironment. Oncoimmunology7, e1434467. 10.1080/2162402x.2018.1434467 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.17 Tiraboschi, C. et al. Combining inhibition of galectin-3 with and before a therapeutic vaccination is critical for the prostate-tumor-free outcome. J. Immunother Cancer. 8 10.1136/jitc-2020-001535 (2020). [DOI] [PMC free article] [PubMed]
  • 18.18, Habrowska-Górczyńska, D. E., Kozieł, M. J., Kowalska, K. & Piastowska-Ciesielska A. W. FOXO3a and Its Regulators in Prostate Cancer. Int. J. Mol. Sci.22 10.3390/ijms222212530 (2021). [DOI] [PMC free article] [PubMed]
  • 19.19 Donlon, T. A. et al. FOXO3 longevity interactome on chromosome 6. Aging Cell.16, 1016–1025. 10.1111/acel.12625 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.20 Jiramongkol, Y. & Lam, E. W. FOXO transcription factor family in cancer and metastasis. Cancer Metastasis Rev.39, 681–709. 10.1007/s10555-020-09883-w (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Liu, Y. et al. 21 Critical role of FOXO3a in carcinogenesis. Mol Cancer 17, 104, (2018). 10.1186/s12943-018-0856-3 [DOI] [PMC free article] [PubMed]
  • 22.22 et al. FOXO transcription factors as therapeutic targets in human diseases. Trends Pharmacol. Sci.43, 1070–1084. 10.1016/j.tips.2022.09.010 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.23 Shen, Z., Zhou, L., Zhang, C. & Xu, J. Reduction of circular RNA Foxo3 promotes prostate cancer progression and chemoresistance to docetaxel. Cancer Lett.468, 88–101. 10.1016/j.canlet.2019.10.006 (2020). [DOI] [PubMed] [Google Scholar]
  • 24.24 Lv, Z., Rao, P. & Li, W. MiR-592 represses FOXO3 expression and promotes the proliferation of prostate cancer cells. Int. J. Clin. Exp. Med.8, 15246–15253 (2015). [PMC free article] [PubMed] [Google Scholar]
  • 25.25 Li, R., Quan, Y. & Xia, W. SIRT3 inhibits prostate cancer metastasis through regulation of FOXO3A by suppressing Wnt/β-catenin pathway. Exp. Cell. Res.364, 143–151. 10.1016/j.yexcr.2018.01.036 (2018). [DOI] [PubMed] [Google Scholar]
  • 26.26 Lynch, R. L. et al. The progression of LNCaP human prostate cancer cells to androgen independence involves decreased FOXO3a expression and reduced p27KIP1 promoter transactivation. Mol. Cancer Res.3, 163–169. 10.1158/1541-7786.MCR-04-0163 (2005). [DOI] [PubMed] [Google Scholar]
  • 27.27 Zhou, K., Wei, Y., Li, X. & Yang, X. MiR-223-3p targets FOXO3a to inhibit radiosensitivity in prostate cancer by activating glycolysis. Life Sci.282, 119798. 10.1016/j.lfs.2021.119798 (2021). [DOI] [PubMed] [Google Scholar]
  • 28.28 Qi, X. et al. ceRNA in cancer: possible functions and clinical implications. J. Med. Genet.52, 710–718. 10.1136/jmedgenet-2015-103334 (2015). [DOI] [PubMed] [Google Scholar]
  • 29.29 Taheri, M., Safarzadeh, A., Hussen, B. M., Ghafouri-Fard, S. & Baniahmad, A. LncRNA/miRNA/mRNA Network Introduces Novel Biomarkers in Prostate Cancer. Cells11 10.3390/cells11233776 (2022). [DOI] [PMC free article] [PubMed]
  • 30.30 Shetty, A., Venkatesh, T., Kabbekodu, S. P., Tsutsumi, R. & Suresh, P. S. LncRNA-miRNA-mRNA regulatory axes in endometrial cancer: a comprehensive overview. Arch. Gynecol. Obstet.306, 1431–1447. 10.1007/s00404-022-06423-5 (2022). [DOI] [PubMed] [Google Scholar]
  • 31.31 Zhou, R. S. et al. Integrated analysis of lncRNA-miRNA-mRNA ceRNA network in squamous cell carcinoma of tongue. BMC Cancer. 19, 779. 10.1186/s12885-019-5983-8 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.32 Bi, C. & Wang, G. LINC00472 suppressed by ZEB1 regulates the miR-23a-3p/FOXO3/BID axis to inhibit the progression of pancreatic cancer. J. Cell. Mol. Med.25, 8312–8328. 10.1111/jcmm.16784 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.33 Zhou, Y. et al. LncRNA UCA1 impacts cell proliferation, invasion, and migration of pancreatic cancer through regulating miR-96/FOXO3. IUBMB Life. 70, 276–290. 10.1002/iub.1699 (2018). [DOI] [PubMed] [Google Scholar]
  • 34.34 Cheng, K., Zhao, Z., Wang, G., Wang, J. & Zhu, W. lncRNA GAS5 inhibits colorectal cancer cell proliferation via the miR–182–5p/FOXO3a axis. Oncol. Rep.40, 2371–2380. 10.3892/or.2018.6584 (2018). [DOI] [PubMed] [Google Scholar]
  • 35.35 Zhang, Y. & Wang, X. Targeting the Wnt/beta-catenin signaling pathway in cancer. J. Hematol. Oncol.13, 165. 10.1186/s13045-020-00990-3 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.36 Liu, Y. M. et al. Gastrodin inhibits prostate cancer proliferation by targeting canonical Wnt/beta-catenin signaling pathway. Med. Oncol.41, 32. 10.1007/s12032-023-02254-9 (2023). [DOI] [PubMed] [Google Scholar]
  • 37.37 Liu, H. et al. FOXO3a modulates WNT/beta-catenin signaling and suppresses epithelial-to-mesenchymal transition in prostate cancer cells. Cell. Signal.27, 510–518. 10.1016/j.cellsig.2015.01.001 (2015). [DOI] [PubMed] [Google Scholar]
  • 38.38 Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408, (2001). 10.1006/meth.2001.1262 [DOI] [PubMed]
  • 39.39 Varma, M., Berney, D., Oxley, J. & Trpkov, K. Gleason score assignment is the sole responsibility of the pathologist. Histopathology73, 5–7. 10.1111/his.13472 (2018). [DOI] [PubMed] [Google Scholar]
  • 40.40 Touchaei, A. Z., Vahidi, S. & Samadani, A. A. Decoding the interaction between miR-19a and CBX7 focusing on the implications for tumor suppression in cancer therapy. Med. Oncol.41, 21. 10.1007/s12032-023-02251-y (2023). [DOI] [PubMed] [Google Scholar]
  • 41.41 Yan, M., Li, G. & An, J. Discovery of small molecule inhibitors of the Wnt/β-catenin signaling pathway by targeting β-catenin/Tcf4 interactions. Exp. Biol. Med. (Maywood). 242, 1185–1197. 10.1177/1535370217708198 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.42 Liu, L. et al. RNF6 Promotes Colorectal Cancer by Activating the Wnt/β-Catenin Pathway via Ubiquitination of TLE3. Cancer Res.78, 1958–1971. 10.1158/0008-5472.Can-17-2683 (2018). [DOI] [PubMed] [Google Scholar]
  • 43.43 Pizzato, M. et al. The epidemiological landscape of thyroid cancer worldwide: GLOBOCAN estimates for incidence and mortality rates in 2020. Lancet Diabetes Endocrinol.10, 264–272. 10.1016/S2213-8587(22)00035-3 (2022). [DOI] [PubMed] [Google Scholar]
  • 44.Teo, M. Y., Rathkopf, D. E. & Kantoff, P. Treatment of Advanced Prostate Cancer. Annu. Rev. Med.70, 479–499. 10.1146/annurev-med-051517-011947 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.45 Hou, T., Li, Z., Zhao, Y. & Zhu, W. G. Mechanisms controlling the anti-neoplastic functions of FoxO proteins. Semin Cancer Biol.50, 101–114. 10.1016/j.semcancer.2017.11.007 (2018). [DOI] [PubMed] [Google Scholar]
  • 46.46 Tsuji, T. et al. FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells. Oncogene40, 3072–3086. 10.1038/s41388-021-01757-x (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.47 Zhang, H., Wang, Y., Lu, J. & Zhao, Y. Long non-coding RNA LINC00222 regulates GSK3beta activity and promotes cell apoptosis in lung adenocarcinoma. Biomed. Pharmacother. 106, 755–762. 10.1016/j.biopha.2018.06.165 (2018). [DOI] [PubMed] [Google Scholar]
  • 48.48 Hill, M. & Tran, N. miRNA interplay: mechanisms and consequences in cancer. Dis. Model. Mech.14 10.1242/dmm.047662 (2021). [DOI] [PMC free article] [PubMed]
  • 49.49 He, B. et al. miRNA-based biomarkers, therapies, and resistance in Cancer. Int. J. Biol. Sci.16, 2628–2647. 10.7150/ijbs.47203 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.50 Fan, H. et al. MiR-19a suppresses ferroptosis of colorectal cancer cells by targeting IREB2. Bioengineered 13, 12021–12029, (2022). 10.1080/21655979.2022.2054194 [DOI] [PMC free article] [PubMed]
  • 51.51 Peng, Y., Huang, D., Ma, K., Deng, X. & Shao, Z. MiR-19a as a prognostic indicator for cancer patients: a meta-analysis. Biosci. Rep.39 10.1042/BSR20182370 (2019). [DOI] [PMC free article] [PubMed]
  • 52.52 Wang, Y., Hu, J., Qi, G., Wang, S. & Gao, J. miR-19a promotes the metastasis and EMT through CUL5 in prostate cancer cell line PC3. J. BUON. 25, 2028–2035 (2020). [PubMed] [Google Scholar]
  • 53.53 Krishnamurthy, N. & Kurzrock, R. Targeting the Wnt/beta-catenin pathway in cancer: Update on effectors and inhibitors. Cancer Treat. Rev.62, 50–60. 10.1016/j.ctrv.2017.11.002 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.54 Wu, X., Que, H., Li, Q. & Wei, X. Wnt/β-catenin mediated signaling pathways in cancer: recent advances, and applications in cancer therapy. Mol. Cancer. 24, 171. 10.1186/s12943-025-02363-1 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.55 Yao, H. et al. KCTD9 inhibits the Wnt/β-catenin pathway by decreasing the level of β-catenin in colorectal cancer. Cell. Death Dis.13, 761. 10.1038/s41419-022-05200-1 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Dev, A. Jr, Vachher, M., Prasad, C. & 56, & P. β-catenin inhibitors in cancer therapeutics: intricacies and way forward. Bioengineered14, 2251696. 10.1080/21655979.2023.2251696 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.57 Samy, A., Hussein, M. A. & Munirathinam, G. Eprinomectin: a derivative of ivermectin suppresses growth and metastatic phenotypes of prostate cancer cells by targeting the beta-catenin signaling pathway. J. Cancer Res. Clin. Oncol.149, 9085–9104. 10.1007/s00432-023-04829-5 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.58 Kennell, J. & Cadigan, K. M. APC and beta-catenin degradation. Adv. Exp. Med. Biol.656, 1–12. 10.1007/978-1-4419-1145-2_1 (2009). [DOI] [PubMed] [Google Scholar]
  • 59.59 Chen, Y. et al. APC gene hypermethylation and prostate cancer: a systematic review and meta-analysis. Eur. J. Hum. Genet.21, 929–935. 10.1038/ejhg.2012.281 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (2.5MB, pdf)

Data Availability Statement

The datasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request.

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES