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Knockdown of PIMREG modulates the Wnt/β-catenin pathway to suppress proliferation and stemness in endometrial cancer
1Department of Gynecology, Longyan First Affiliated Hospital of Fujian Medical University, 364000 Longyan, Fujian, China
2Department of Gynecology, The Second Hospital of Longyan, 364000 Longyan, Fujian, China
*Corresponding Author(s):rh21_q5@163.com (Ronghua Qiu)
| History | Submitted: 22 May 2025 | Accepted: 23 June 2025 | Published: 15 July 2025 |
| Copyright: | ©2025 The Author(s). Published by MRE Press. |
Background: Endometrial cancer is the most common gynecological malignancy, and poses serious threat to health. PIMREG (PICALM interacting mitotic regulator also known as FAM64A), has been uncovered in promoting tumorigenesis in multiple cancers. But, its regulatory impacts in endometrial cancer remain poorly understood. Methods: Cell viability was assessed by the Cell Counting Kit-8 (CCK-8) assay. Protein levels were inspected by Western blotting. Cell proliferation was evaluated by colony formation assay, while stemness was examined by sphere formation assays. Results: PIMREG expression was found to be upregulated in endometrial cancer. Suppression of PIMREG significantly reduced cell viability and suppressed proliferative and stemness capacities. Furthermore, silencing PIMREG retarded the evoking of the Wnt/β-catenin signaling pathway. Conclusions: PIMREG knockdown suppressed proliferation and stemness by modulating the Wnt/β-catenin pathway in endometrial cancer. This project hinted that PIMREG may be one useful therapeutic target for endometrial cancer treatment.
Cite this article
Zhiqing Hu, Qing Wang, Jinyong Wang, Ronghua Qiu. Knockdown of PIMREG modulates the Wnt/β-catenin pathway to suppress proliferation and stemness in endometrial cancer.European Journal of Gynaecological Oncology,2025,46(7):88-94 DOI:10.22514/ejgo.2025.099
1. Introduction
Endometrial cancer is one prevalent gynecological malignancies and is associated with a significant mortality rate [1]. Recently, the occurrence rate of endometrial cancer has shown a rapid upward trend globally [2, 3]. Despite considerable progress in therapeutic strategies, clinical outcomes remain suboptimal for many patients [4]. Therefore, identifying novel molecular targets and elucidating the underlying mechanisms of endometrial cancer progression is imperative to improve treatment options.
Numerous proteins have been identified as potential therapeutic targets in endometrial cancer. For example, EGF-like domain, multiple 6 (EGFL6) has been shown to promote cell migration and proliferation [5]. Proline, glutamate and leucine rich protein 1 (PELP1) suppression impairs ribosomal biogenesis, thereby inhibiting tumor progression [6]. Similarly, myosin IIIB (MYO3B) can facilitate tumor growth [7]. Additionally, silencing of S100 calcium binding protein A2 (S100A2) has been reported to activate the stimulator of interferon genes (STING) pathway, leading to reduced aggressiveness of endometrial cancer cells [8].
PIMREG, also known as FAM64A, is a clathrin assembly lymphoid myeloid leukemia gene (CALM) interactor [9]. It has been identified as a key promoter of tumor formation and progression in many cancers. For example, PIMREG activates the nuclear factor kappa-B (NF-κB) pathway to enhance tumor aggressiveness in breast cancer [10], modulates the DNA damage response to promote glioblastoma progression [11], and is associated with poor survival outcomes and altered immune cell infiltration in lung adenocarcinoma [12]. Notably, high PIMREG expression in uterine corpus endometrial carcinoma (UCEC) is linked to reduced overall survival [13]. However, the precise molecular mechanisms through which PIMREG contributes to endometrial cancer progression hold unclear.
In this project, we demonstrate that knockdown of PIMREG suppresses proliferation and stemness through restraining the Wnt/β-catenin signaling pathway in endometrial cancer. This finding supplies useful insights into the oncogenic role of PIMREG and suggest it may serve as a novel therapeutic target in endometrial cancer.
2. Materials and methods
2.1 Database analysis
The mRNA expression levels of PIMREG (FAM64A) in UCEC tumor tissues and corresponding normal tissues were analyzed using data from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov). The mRNA expression profiles of PIMREG in UCEC tumor tissues and normal tissues were confirmed through the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn) where PIMREG mRNA expression was assessed both in UCEC and across various cancer types compared to normal tissues. The protein expression of PIMREG in endometrial cancer tumor tissues and normal tissues was evaluated using immunohistochemistry (IHC) data available from the Human Protein Altas (https://www.proteinatlas.org). Prognostic relevance of PIMREG expression in endometrial cancer patients was verified through the KMplot database (https://kmplot.com/analysis), comparing survival outcomes between patients with high and low PIMREG expression levels.
2.2 Cell lines and culture
Human endometrial epithelial cells (hEEC) and endometrial cancer cell lines Ishikawa, HEC-1B, KLE and RL-952 were obtained from the American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle Medium (DMEM) (12800017, Invitrogen, Carlsbad, CA, USA) was employed for culturing cells. Cells were maintained in one incubator. Cell lines were authenticated by short tandem repeat (STR) profiling and were confirmed to be free of mycoplasma contamination.
2.3 Cell transfection
Short hairpin RNAs targeting PIMREG (sh-PIMREG#1 and sh-PIMREG#2) and a negative control (sh-NC) were bought from GenePharma company (Shanghai, China). Transfections were performed in Ishikawa and KLE cells by Lipofectamine 2000 reagent (11668019, Invitrogen, Carlsbad, CA, USA).
2.4 Western blotting
Total proteins from endometrial cancer cells were drew through radioImmunoprecipitation assay (RIPA) lysis buffer, and protein concentrations were quantified through the bicinchoninic acid (BCA) Protein Assay Kit (P0011, Beyotime, Shanghai, China). Proteins were suffered to Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) for division. Next, putting proteins to polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China). The primary antibodies were placed into membranes for 12 h, next the secondary antibodies for 2 h. Protein bands were confirmed through the chemiluminescence detection kit.
The primary antibodies: PIMREG (1:1000; ab251896; Abcam, Shanghai, China), c-Myc (1:1000; ab32072), CyclinD1 (1:200; ab16663), β-catenin (1:5000; ab32572), Sex Determining Region Y Box Protein 2 (SOX2) (1:1000; ab92494) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; ab8245).
2.5 CCK-8 assay
Endometrial cancer cells were placed into 96-well plate at an appropriate density. CCK-8 solution with 10 μL was put into each well. Cell viability (at 450 nm) was inspected by the spectrophotometer (ND-ONE-W, Thermo Fisher Scientific, Waltham, MA, USA).
2.6 Colony formation assay
Endometrial cancer cells were put in to the 6-well plate. Incubation for 14 days, colonies were got. The fixation and dyeing for colonies were performed. Eventually, colonies were counted.
2.7 Sphere formation assay
Endometrial cancer cells were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 20 ng/mL fibroblast growth factor (FGF), 20 ng/mL epidermal growth factor (EGF), and 1% fetal bovine serum (FBS) was adopted for cell incubation in the ultra-low-attachment culture dishes (Corning, USA). After 14 days, the formed spheres were observed and imaged using a light microscope (CX23, Olympus, Tokyo, Japan).
2.8 Statistical analysis
Data were demonstrated as mean ± standard deviation (SD). The statistical analysis was executed through GraphPad Prism Software 9 (GraphPad Software, San Diego, CA, USA). The data follow a normal distribution. Three repetitions were performed in each cell experiment. One-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) test was employed for comparisons. The statistically significant was affirmed at p < 0.05.
3. Results
3.1 PIMREG expression was elevated in endometrial cancer
Analyses of the TCGA and GEPIA databases revealed that PIMREG expression was augmented in endometrial cancer tumor tissues (Fig. 1A–C). Consistent with this, IHC data from the Human Protein Atlas further confirmed elevated PIMREG protein levels in tumor tissues (Fig. 1D). Moreover, survival analysis using KMplot demonstrated that high PIMREG expression can cause poorer overall survival (Fig. 1E). Next, it was verified that PIMREG owned the lifted protein expression in endometrial cancer cell lines (Fig. 1F). In general, PIMREG expression was elevated in endometrial cancer.
Fig. 1.PIMREG expression was elevated in endometrial cancer. (A) The mRNA expression of PIMREG (FAM64A) in UCEC tumor tissues and normal tissues was verified through the TCGA database. (B) The mRNA expression of PIMREG in UCEC tumor tissues and normal tissues was verified through GEPIA database. (C) The mRNA expression of PIMREG in multiple cancers’ tumor tissues and normal tissues was confirmed through the GEPIA database. (D) The protein expression of PIMREG in endometrial cancer tumor tissues and normal tissues was examined through IHC assay from the Human Protein Altas online website. (E) The prognosis of endometrial cancer patients with high or low PIMREG expression was obtained from the KMplot tool. (F) The protein expression of PIMREG in human endometrial epithelial cell line hEEC and endometrial cancer cell lines (Ishikawa, HEC-1B, KLE and RL-952) through Western blot. *p < 0.05, **p < 0.01, ***p < 0.001. PIMREG: PICALM interacting mitotic regulator; UCEC: uterine corpus endometrial carcinoma; GEPIA: Gene Expression Profiling Interactive Analysis; IHC: Immunohistochemistry; TCGA: The Cancer Genome Atlas; TPM: Transcripts Per Million; hEEC: Human Esophageal Epithelial Cells; HR: Hazard Ratio.
3.2 Suppression of PIMREG inhibited cell viability in endometrial cancer
The knockdown efficiency of PIMREG by shRNA was confirmed by Western blot analysis (Fig. 2A). Moreover, the cell viability was cut down after PIMREG suppression (Fig. 2B). The cell proliferation was restrained after silencing PIMREG (Fig. 2C). These results suggest that suppression of PIMREG impaired cell proliferation.
Fig. 2.Suppression of PIMREG inhibited cell viability in endometrial cancer. Groups were separated into the sh-NC, sh-PIMREG#1 and sh-PIMREG#2 groups. (A) The protein expression of PIMREG was tested through western blot. (B) The cell viability was examined through the CCK-8 assay. (C) The cell proliferation was determined through colony formation assay. ***p < 0.001. PIMREG: PICALM interacting mitotic regulator; sh-NC: Short hairpin-negative control; OD: optical density.
3.3 Silencing of PIMREG repressed stemness in endometrial cancer
Sphere formation assays showed that stemness capacity was significantly reduced in endometrial cancer cells following PIMREG knockdown (Fig. 3A). Consistently, SOX2 protein expression, a key marker of stemness, was also downregulated upon PIMREG silencing (Fig. 3B). These findings indicate that knockdown of PIMREG suppresses the stem-like properties of endometrial cancer cells.
Fig. 3.Knockdown of PIMREG repressed stemness in endometrial cancer. Groups were separated into the sh-NC, sh-PIMREG#1 and sh-PIMREG#2 groups. (A) The stemness ability was confirmed through the sphere formation assay. (B) The SOX2 protein expression was measured through Western blot. **p < 0.01, ***p < 0.001. PIMREG: PICALM interacting mitotic regulator; sh-NC: Short hairpin-negative control; SOX2: Sex Determining Region Y Box Protein 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
3.4 Inhibition of PIMREG attenuated the Wnt/β-catenin pathway
The protein levels of β-catenin, c-Myc and CyclinD1 were lessened after PIMREG inhibition (Fig. 4), suggesting that inhibition of PIMREG weakened the Wnt/β-catenin pathway.
Fig. 4.Inhibition of PIMREG attenuated the Wnt/β-catenin pathway. Groups were separated into the sh-NC, sh-PIMREG#1 and sh-PIMREG#2 groups. The protein expressions of β-catenin, c-Myc and CyclinD1 were inspected through Western blot. **p < 0.01, ***p < 0.001. PIMREG: PICALM interacting mitotic regulator; sh-NC: Short hairpin-negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
4. Discussion
PIMREG has been revealed to own a critical role in tumor formation and progression in multiple cancers [10, 11, 12, 13, 14]. However, its functional significance and regulatory mechanisms in endometrial cancer have remained largely unclear. In this project, we demonstrated that PIMREG expression is significantly upregulated in endometrial cancer. Importantly, elevated PIMREG expression was connected with poor patient prognosis.
Cancer stem cells (CSCs) owns the capacities of anti-apoptosis, self-renewal, tumorigenesis and high metastasis [15]. They are widely regarded as a core driver of tumorigenesis, and their presence has been strongly linked to drug resistance and tumor metastasis [16]. CSCs have been clarified in many solid tumors, and targeting CSCs is considered as the key strategy for effective cancer treatment [17]. In endometrial cancer progression, recent studies have highlighted the importance of stemness regulation. For example, minichromosome maintenance complex component 2 (MCM2) has been shown to enhance CSC-like traits in endometrial cancer [18], while phosphocholine alleviates stemness by suppressing the mammalian target of rapamycin (mTOR)/c-Myc signaling pathway [19]. Furthermore, chromodomain helicase DNA binding protein 4 (CHD4) mutations can stimulate transforming growth factor (TGF)-beta signaling to aggravate endometrial cancer stemness [20]. Additionally, Sirtuin 2 promotes stemness and reduces chemosensitivity, further complicating treatment outcomes [21]. Similarly, in this work, it was also testified that PIMREG knockdown significantly reduced sphere formation and downregulated SOX2 expression, indicating that silencing of PIMREG repressed stemness.
The Wnt/β-catenin pathway exhibits importance in the maintenance of CSCs [22]. The accumulated β-catenin can move into the nucleus, and then combine with T-cell factor (TCF) transcription factors, next evoking downstream genes, thereby strengthening stemness and accelerating tumor progression [23]. Thus, focusing on the Wnt/β-catenin pathway and stemness becomes pivotal. For instance, lysine (K)-specific demethylase 1 (KDM1A) evokes Wnt/β-catenin signaling to promote stemness in thyroid cancer [24], while Sec62 enhances CSC properties in colorectal cancer via Wnt/β-catenin activation [25]. In contrast, forkhead box F2 (FOXF2) negatively regulates this pathway to suppress stemness in breast cancer [26]. It has also been clarified that kinesin family member 15 (KIF15) knockdown can weaken stemness to retard the development of endometrial cancer by restraining the Wnt/β-catenin pathway [27]. In line with these findings, PIMREG has been reported to activate the β-catenin pathway in glioma, contributing to tumor development [28]. However, in endometrial cancer, the impacts of PIMREG on the β-catenin pathway remain elusive. Similar to these above previous studies, in this work, it was also manifested that inhibition of PIMREG weakened the Wnt/β-catenin pathway.
5. Conclusions
Our results uncovered that knockdown of PIMREG inhibited cell proliferation and stemness in endometrial cancer via the Wnt/β-catenin pathway. However, this project has several limitations. The in vivo animal models, clinical tissue samples, and comprehensive clinical investigations were not included, which may limit the translational applicability. In future research, further validation using animal models and patient-derived samples, as well as expanded clinical and mechanistic studies, will be essential to fully probe the influences of PIMREG on endometrial cancer progression.
Availability of data and materials
Data sharing is not applicable to this article as no new data were created or analyzed in this study.
Author contributions
ZQH, RHQ—designed the study and carried them out; prepared the manuscript for publication and reviewed the draft of the manuscript. ZQH, QW, JYW—supervised the data collection; analyzed the data; interpreted the data. All authors have read and approved the manuscript.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
Acknowledgment
Not applicable.
Funding
This research received no external funding.
Conflict of interest
The authors declare no conflict of interest.
