Paeoniflorin inhibits colorectal cancer stem cell properties via regulating LINC01711/KMT2D/KLF7 axis

Introduction

Colorectal cancer (CRC) remains the third most commonly diagnosed malignancy and the second leading cause of cancer-related death worldwide, with an estimated 1.9 million new cases and 935,000 deaths in 2020 alone (1-3). Despite incremental improvements in early detection, surgical techniques, and systemic therapies, recurrence and metastasis continue to drive mortality (4,5). The 5-year survival for patients with distant disease stubbornly hovers below 15% (6). One of the most compelling explanations for this therapeutic recalcitrance is the existence of a functionally distinct sub-population of cells-colorectal cancer stem cells (CSCs)-that endow tumors with sustained self-renewal, aberrant differentiation, and resistance to conventional cytotoxic, targeted, and immune-based modalities (7,8). These cells express canonical stemness markers, such as leucine rich repeat containing G protein-coupled receptor 5 (LGR5), CD44v6, aldehyde dehydrogenase (ALDH1), and B-cell-specific Moloney murine leukemia virus insertion region 1 (BMI1), and are enriched after chemotherapy or radiotherapy, ultimately fueling clonal evolution and metastatic dissemination (9,10). Consequently, eradication of the CSC compartment has emerged as an essential, yet still unmet, therapeutic objective.

Natural products have regained prominence as a prolific source of structurally diverse scaffolds that can selectively perturb CSC signaling nodes while sparing normal tissue stem cells (11). Among these botanical leads, paeoniflorin (PF), a water-soluble monoterpene glycoside extracted from the root of Paeonia suffruticosa and Paeonia lactiflora Pall, has attracted considerable attention because of its multifaceted pharmacological profile (12,13). PF has been shown in pre-clinical models to exert anti-inflammatory, anti-oxidant, anti-angiogenic, and neuroprotective activities (14-17). Recently, mechanistic studies in hepatocellular carcinoma, lung cancer, and gastric cancer have demonstrated that PF can suppress tumor-initiating capacity (18-20). However, the direct impact of PF on colorectal CSCs has not been systematically investigated, and the molecular circuitry through which PF might attenuate stemness-associated transcriptional programs in CRC remains largely enigmatic.

Wnt/β-catenin signaling is the canonical gatekeeper of colonic stem cell identity. Hyper-activation of this signaling pathway—either through truncating APC mutations or stabilizing catenin beta 1 mutations—drives constitutive transcription of stemness genes, such as LGR5 and achaete-scute family bHLH transcription factor 2 (21-23). Recent work has highlighted that β-catenin stability is regulated by a phosphorylation-destruction complex comprising glycogen synthase kinase 3 beta (GSK-3β), and casein kinase 1 alpha 1 (24,25). Phosphorylation of β-catenin at Ser33/Ser37/Thr41 by GSK-3β earmarks it for ubiquitin-mediated proteasomal degradation (26). Intriguingly, PF has been reported to activate GSK-3β in neuronal cells, raising the possibility that PF destabilizes β-catenin in CRC cells (27). We therefore postulated that PF triggers a GSK-3β-dependent phosphorylation cascade culminating in β-catenin degradation, thereby silencing Wnt target genes and extinguishing CSC identity.

This study investigated how PF dismantles the molecular and metabolic scaffolding that underpins CRC stemness. Our findings not only expand the therapeutic repertoire against CSCs but also provide a mechanistic blueprint for integrating botanical agents into precision oncology frameworks. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-975/rc).

Methods

Cell culture and transfection

Human CRC cells HCT116 and non-tumor NCM460 cells were purchased from ATCC USA. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 5% CO₂ at 37 ℃. Then cells were passaged for 6 generation.

Cells were treated with 40 µM PF (Yuanye Biotechnology, China).

HCT116 cells were transfected with LINC01711 overexpression plasmids and the empty vector, or shRNA and shNC using Lipofectamine 3000 (Invitrogen, USA). The KMT2D_fusion mutant was created with a site-specific mutagenesis kit from Vazyme Biotech Corp, China.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

RNA was extracted using TRIzol, DNase-treated, and reverse-transcribed (PrimeScript RT). qPCR was performed on a QuantStudio 6 Flex with SYBR Green (Takara, Japan). Relative expression was calculated using the 2^–ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase as reference.

Western blotting and co-immunoprecipitation (Co-IP) assays

Proteins were extracted in radioimmune precipitation assay buffer with protease and phosphatase inhibitors. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to polyvinylidene fluoride, membranes were probed with primary antibodies (overnight, 4 ℃) and HRP-conjugated secondary antibodies (1 h, RT). For co-IP, 1 mg whole-cell lysate was incubated with 2 µg anti-β-catenin or anti- KLF transcription factor 7 (KLF7) overnight, followed by Protein A/G magnetic beads (Pierce). Blots were developed with Enhanced Chemiluminescence Prime and quantified by ImageJ.

Cycloheximide chase (CHX) assay

HCT116 cells were plated in a 24-well plate. CHX (100 µg·mL⁻¹; Sigma-Aldrich, Germany) was added to block new protein synthesis. Then the protein was collected and subjected to Western blot.

Chromatin immunoprecipitation (ChIP)

HCT116 cells (1×10⁷ per IP) were cross-linked with 1% formaldehyde for 10 min, quenched with 125 mM glycine, and sonicated (Covaris S220) to 200–500 bp fragments. Immunoprecipitation was performed with anti-KLF7 or rabbit IgG (Cell Signaling). Peaks were called using MACS2 (q<0.01) and annotated with HOMER. Captured genomic DNA was reverse-crosslinked and purified by ethanol precipitation with Dr. GenTLE Precipitation Carrier (Takara). RT-qPCR was conducted to amplify the precipitated DNA.

Dual-luciferase reporter assay

The binding motif of KLF7 was predicted using JASPAR (https://jaspar.elixir.no/). The binding sites of KLF7 on the promoter of LINC01711 were predicted. The wild type (WT) and mutant type (MUT) of 3’UTR of LINC01711 were inserted into the Luciferase Reporter Vector (Promega, USA). Then cells were transfected with WT/MUT of LINC01711 and KLF7 overexpression plasmids or the vector using Lipofectamine 3000 (Invitrogen). Finally, the luciferase activities were analyzed using Dual-Luciferase Reporter Assay System (Promega).

RNA immunoprecipitation (RIP) assay

RIP assay was conducted by an RIP assay kit (Millipore, USA). Briefly, cells were lysed with RIP Lysis Buffer and centrifuged. The supernatants were incubated with magnetic beads conjugated with antibodies against KMT2D, KLF7 or the control IgG. The immunoprecipitants were resuspended in proteinase K buffer. The RNA was purified and subjected to RT-qPCR.

Cell counting kit-8 (CCK-8) assay

HCT116 cells were seeded at 3×10³ cells/well in 96-well plates. After overnight attachment, PF or vehicle was added in 100 µL complete medium. At 24, 48, and 72 h, 10 µL CCK-8 (CK04; Dojindo, Japan) reagent was added and plates incubated for 2 h at 37 ℃. Absorbance at 450 nm was recorded on a SpectraMax i3x (Molecular Devices, USA).

Colony formation assay

HCT116 cells were seeded in 96-well plates (2×10³ cells/well) and cultured for 14 days. After washed with phosphate-buffered saline (PBS), cells were mounted on with 75% ethanol and stained with 1% crystal violet. Finally, images were captured under an inverted microscope (IX-73; Olympus, Japan).

5-ethynyl-2’deoxyuridine (EdU) assay

HCT116 cells were seeded in 96-well black-wall plates (5×10³ cells/well). After 24 h, medium was replaced with or without PF for indicated durations (6–48 h). EdU solutions (C10337; Thermo Fisher Scientific) was added and cultured for 2 h. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min, and permeabilised with 0.5% Triton X-100 for 20 min. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (2 µg·mL⁻¹). Images were captured on an ImageXpress Micro Confocal (Molecular Devices) at 20× magnification; ≥1,000 cells/condition were analyzed. Proliferation index = (EdU-positive nuclei/total nuclei) × 100%.

Transwell assays

Log-phase cells were serum-starved for 12 h to synchronise the cell cycle and minimise basal motility. Single-cell suspensions (>95% viability by trypan blue exclusion) were obtained by gentle trypsinisation, washed twice in serum-free DMEM, and re-suspended at 2×10³ cells·mL⁻¹. A 24-well Transwell insert with 8-µm pore polycarbonate membranes (Corning 3422) was equilibrated in serum-free medium for 1 h at 37 ℃. 200 µL cell suspension (2×10³ cells) was loaded into the upper chamber, while the lower chamber contained 600 µL complete medium (10% FBS) as chemoattractant. PF was added to both chambers to exclude chemotaxis-independent effects. After 24 h incubation, non-migrated cells on the upper surface were removed with a cotton swab. Migrated cells on the underside were fixed in 4% PFA for 15 min, and stained with 0.1% crystal violet for 15 min. Images were captured at 200× magnification using an Olympus IX-73 inverted microscope equipped with a DP80 CCD camera. Five random fields per insert were quantified using ImageJ (NIH). Each experimental condition was tested in biological triplicate.

Sphere-forming assay

HCT116 cells (1,000 cells/well) were seeded in ultra-low-attachment 96-well plates in serum-free DMEM/F12 supplemented with 20 ng·mL⁻¹ basic fibroblast growth factor and 20 ng·mL⁻¹ epidermal growth factor. After 7 days, spheres >50 µm were counted under an inverted microscope (IX-73; Olympus, Japan).

Flow cytometry

Cell suspensions were stained with the following antibody panel: human: CD44-PE and CD24-FITC. Dead cells were excluded with 7-AAD. Data were acquired on a BD FACSAria III or LSRFortessa and analyzed with FlowJo v10.8. Sorting purity was routinely >95%.

In vivo assay

HCT116 cells (5×10⁶) in PBS or treated with PF (50 mg·kg⁻¹) were subcutaneously injected into the armpits of immunodeficient NOD-SCID nude mice (18,28). After 4 weeks, all mice were sacrificed. All animal experiments were performed under a project license (No. 2024-033-5) granted by The Second Affiliated Hospital of Hunan University of Chinese Medicine, in compliance with the national guidelines for the care and use of animals.

Statistical analysis

Data are presented as mean ± standard deviation. The differences were analyzed by Student’s t-test and ANOVA with Tukey’s post-hoc test. Data analysis was performed in GraphPad Prism 9.5. P<0.05 was deemed statistical significance.

Results

PF inhibits the proliferation of CRC cells

Figure 1A showed the molecular structure of PF. PF significantly reduced the cell viability of HCT116 cells (Figure 1B), whereas showing no significant effects on NCM460 cells (Figure S1). Moreover, PF significantly reduced the number of CRC cell colonies compared to control group (Figure 1C,1D). Furthermore, PF significantly reduced the ratios of EdU positive cells compared with control groups (Figure 1E,1F). These results suggested that PF inhibits the proliferation of CRC cells.

Figure 1 PF inhibits the proliferation of CRC cells. (A) Molecular structure of PF was analyzed by TCMSP. (B) Cell viability was determined by CCK-8 assay. (C,D) Cell proliferation was determined by colony formation assay. (E,F) Cell proliferation was determined by EdU assay (magnification). ***, P<0.001. CCK-8, Cell counting kit-8; CRC, colorectal cancer; EdU, 5-ethynyl-2'deoxyuridine; OD, optical density; PF, paeoniflorin; TCMSP.

PF inhibits CRC cell stem-like properties

Cancer cell stem-like behaviors drive the aggressiveness of CRC. Therefore, we analyzed the effects of PF on CRC cell stem-like properties. We found that PF treatment significantly inhibited the migration of CRC cells (Figure 2A,2B). PF treatment significantly suppressed the sphere formation of CRC cells (Figure 2C,2D). CD133 is the key biomarker of cancer cell stemness. We found that PF treatment significantly reduced the ratios of CD44⁺CD24⁻ cells (Figure 2E,2F). Moreover, PF significantly downregulated CSC markers (Figure 2G,2H). These results indicate that PF treatment inhibits CRC cell stem-like properties.

Figure 2 PF inhibits CRC cell stem-like properties. (A,B) Cell migration was determined using transwell assay. (C,D) CRC cell stem-like properties were analyzed by sphere formation assay. (E,F) CRC cell stem-like properties were analyzed by flow cytometry. (G,H) Protein expression was analyzed using Western blot. **, P<0.05; ***, P<0.001. (A,C) Magnification. CRC, colorectal cancer; PF, paeoniflorin.

PF downregulates LINC01711

LINC01711 functions as an oncogene in various type of cancer. We found that LINC01711 expression was markedly upregulated in CRC patients (Figure 3A). High expression of LINC01711 was associated with advanced stages as well as poor prognosis in long-term run (Figure 3B,3C). LINC01711 expression was significantly increased in HCT116 cells (Figure 3D). LINC01711 expression was significantly downregulated by PF (Figure 3E), suggesting successful transfection. LINC01711 knockdown significantly blocked KLF7/Wnt/β-catenin signaling (Figure 3F), which is the canonical gatekeeper of cancer cell stemness.

Figure 3 PF downregulates LINC01711. (A) LINC01711 expression in CRC patients was analyzed by Starbase3.0. LINC01711 expression in CRC patients (B) and its correlation with overall survival rates of CRC patients (C) were analyzed by GEPIA. (D) LINC01711 expression was analyzed by RT-qPCR. (E) LINC01711 expression in CRC cells was analyzed by RT-qPCR. (F) Protein expression in CRC cells was determined by Western blot. **, P<0.01; ***, P<0.001. CRC, colorectal cancer; FPKM, fragments per kilobase of transcript per million fragments mapped; HR, hazard ratio; NC, negative control; PF, paeoniflorin; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TPM, transcripts per million.

PF inhibits the formation of LINC01711/KMT2D/KLF7 ternary complex

LINC01711 is involved in histone modification. KMT2D promotes histone modification of KLF7, maintaining KLF7 protein stability (29). As shown in Figure S2, KMT2D significantly alleviated the CHX-mediated degradation of KLF7. Therefore, we hypothesized that LINC01711 may be involved in regulating histone modification of KLF7 via KMT2D. RNA pulldown assay showed that KMT2D was significantly enriched by LINC01711 probe (Figure 4A). RIP assay showed that LINC01711 was significantly immunoprecipitated by KMT2D (Figure 4B). Overexpressed LINC01711 significantly increased KLF7 protein expression and methylation (Figure 4C), which, however, was reversed by KMT2D shRNA or PF. LINC01711 overexpression significantly alleviated the effects of PF and promotes the proliferation, sphere formation, and migration of HCT116 cells (Figure 4D-4F), which was reversed by KMT2s inhibitor M-808. The interaction between KLF7 and LINC01711 was further confirmed using RNA pulldown assay (Figure 4G) and RIP (Figure 4H). Furthermore, LINC01711 promoted the binding between endogenous KMT2D and KLF7, while this interaction remarkably inhibited by PF (Figure 4I). Figure 4J showed the schematic diagram of PF regulating LINC01711/KMT2D/KLF7 ternary complex to inhibit CRC stemness. To confirm the role of LINC01711/KMT2D/KLF7 ternary in CRC, we established KMT2D-C and KMT2D-fusion HCT116 cells. We found that LINC01711 significantly proliferation, migration, as well as stemness of KMT2D-C HCT116 (Figure 4K-4M), but showed slightly effects on KMT2D-fusion HCT116 cells, suggesting that LINC01711, at least partially, promotes the proliferation and stemness of CRC via regulating KMT2D/KLF7 axis.

Figure 4 PF inhibits the formation of LINC01711/KMT2D/KLF7 ternary complex. The interaction between LINC01711 and KMT2D was analyzed using RNA pull-down assay (A) and RIP assay (B). (C) KLF7 protein expression was determined by Western blot. (D) Cell viability was determined by CCK-8 assay. (E) CRC cell stem-like properties were analyzed by sphere formation assay. (F) Cell migration was determined using transwell assay. The interaction between LINC01711 and KLF7 was analyzed using RNA pull-down assay (G) and RIP assay (H). (I) The interaction between KMT2D and KLF7 in LINC01711-knockdown cells was analyzed by Co-IP assay. (J) Schematic diagram of PF regulating ternary complex to inhibit CRC stemness. (K) Cell viability was determined by CCK-8 assay. (L) CRC cell stem-like properties were analyzed by sphere formation assay. (M) Cell migration was determined using transwell assay. **, P<0.01; ***, P<0.001. CCK-8, Cell counting kit-8; Co-IP, co-immunoprecipitation; CRC, colorectal cancer; KLF7, KLF transcription factor 7; KMT2D, lysine methyltransferase 2D; NC, negative control; OD, optical density; PF, paeoniflorin; RIP, RNA immunoprecipitation.

Taken together, these results dictated that PF suppresses the formation of LINC01711/KMT2D/KLF7 ternary complex to inhibit CRC stemness.

PF inhibits KLF7-mediated transcription of LINC01711

KLF7 is a key oncogenic transcription factor, which may in turn mediates the transcription of LINC01711. We found that PF-mediated downregulation of LINC01711 was significantly reversed by KLF7 overexpression (Figure 5A). Furthermore, JASPAR was used to predict the binding motif of KLF7 (Figure 5B) and the possible binding sites in the promoter of LINC01711 (not presented in figure). ChIP assay was conducted to design primers targeting the KLF7 binding sites on LINC01711 promoter. We found that KLF7 significantly promoted the enrichment at S1, but not S2 and S3 (Figure 5C,5D). However, PF inhibits the enrichment at S1 (Figure 5E). We found that KLF7 overexpression significantly increased luciferase activities at WT, MUT2, and MUT3 (Figure 5F), whereas showed no significant change in MUT1, suggesting that KLF7 promotes the transcription of LINC01711 through binding to Site1. Moreover, KLF7-mediated transcription of LINC01711 was significantly inhibited by PF (Figure 5G).

Figure 5 PF inhibits KLF7-mediated transcription of LINC01711. (A) LINC01711 expression in CRC cells was analyzed by RT-qPCR. (B) The binding motif of KLF7 was analyzed by JASPAR. (C-E) The interaction between LINC01711 and KLF7 was analyzed using ChIP assay. (F,G) The interaction between LINC01711 and KLF7 was analyzed using luciferase assay. **, P<0.01; ***, P<0.001. ChIP, chromatin immunoprecipitation; CRC, colorectal cancer; KLF7, KLF transcription factor 7; PF, paeoniflorin; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

PF inhibits tumor growth in vivo

In vivo assays were conducted to further detected the effects of PF on CRC. We found that PF treatment significantly reduced the size, volume, and weight of CRC tumor (Figure 6A-6C), whereas showing no significant effects on body weight (Figure 6D).

Figure 6 PF inhibits tumor growth in vivo. In vivo assays were conducted to further detected the effects of PF on CRC. We found that PF treatment significantly reduced the size, volume, and weight of CRC tumor (A-C). The tumor size (A), volume (B), weight (C), and body weight (D) were analyzed using in vivo assay. ***, P<0.001. CRC, colorectal cancer; PF, paeoniflorin.

Discussion

In this study, PF exerted an anti-tumor effect in CRC. PF inhibited the proliferation and stem-like properties of CRC cells. Mechanistically, PF inhibited the interaction between LINC01711 and KMT2D and suppressed histone methylation of KLF7, which, in turn, impeded the transcription of LINC01711. Furthermore, PF inhibited CRC cell stem-like properties in vivo, suppressing tumor growth of CRC.

Accumulating studies have demonstrated that PF inhibits the progression of gastrointestinal cancer (30,31). For instance, PF suppresses the migration and invasion of gastric cancer associated fibroblasts (32). PF inhibits the progression of colitis-associated CRC via blocking interleukin 6/signal transducer and activator of transcription 3 pathway (33). Yue et al. (34) also evidence that PF suppresses cell growth as well as mediates cell cycle arrest of CRC cells. Here, we demonstrated that PF inhibited the proliferation of CRC as well as impeded CRC cell stem-like properties. Cancer stem-like cell possesses self-renewal and unlimited proliferation properties. Cancer stem cell is a key barrier for therapy resistance, metastasis, and relapse (35). In this study, PF-mediated inhibition of CRC stem-like properties suppressed tumor growth. Therefore, PF may be therapeutic strategy for CRC.

PF inhibits the progression of cancer through directly regulating gene expression or indirectly regulating non-coding RNAs (ncRNAs) (36,37). Here, we demonstrated that PF inhibited the expression of LINC01711, which is upregulated in CRC patients and cells. ncRNAs are involved in regulating biological processes through directly binding to functional proteins, such as RNA binding proteins, transcription factors, post-translational modification regulators (38-40). However, dysfunction of ncRNA (particularly lncRNA)-protein interaction contributes to the pathogenesis of cancer (41). In our study, LINC01711 bound to histone lysine methyltransferase KMT2D. KMT2D functions as an oncogenic role in various type of cancer, such as NSCLC, breast cancer, prostate cancer, as well as CRC (42-45). Previous studies have demonstrated that KMT2D could interact with lncRNA RNA-activated by DNA damage (NORAD) (46). In our study, we found that KMT2D could interacted with LINC01711, which promoted the histone methylation of KLF7. However, LINC01711 knockdown inhibited KMT2D-mediated histone methylation of KLF7, indicating that LINC01711 serves as a scaffold for the interaction between KMT2D and KLF7.

KLF7 is complicated in cancer cell stemness in numerous cancers. For example, KLF7 promotes oral cancer stemness via upregulating integrin subunit alpha 2 (47). KLF7 mediates pluripotency and self-renewal characteristics of cancer stem cells in high-grade serous ovarian cancer (48). Recent studies have also demonstrated that abnormally expressed KLF7 contributes to the progression of CRC. For example, KLF7 overexpression mediates the aggressiveness of CRC (49). KLF7 inhibits the apoptosis of CRC cells as well as promotes tumor growth (50). In the present study, KLF7 bound to LINC01711 promoter, driving transcription and upregulation of LINC01711. Therefore, LINC01711/KMT2C/KLF7 axis forms a positive feedback loop, amplifying the oncogenic role of LINC01711 in CRC. However, PF downregulated LINC01711, blocking the formation of this ternary in CRC.

Conclusions

In conclusion, PF inhibits proliferation and CRC cell stem-like properties via blocking LINC01711/KMT2D/KLF7 axis. However, clinical trials were lacking in this study. Moreover, although PF is shown to disrupt the LINC01711/KMT2D/KLF7 interaction, it is unclear whether PF directly targets any component of the complex or acts upstream. Our findings provide a novel theoretical basis for deeply exploring metastatic CRC treatment using PF.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-975/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-975/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-975/prf

Funding: This study was supported by Scientific Research Fund of Hunan University of Chinese Medicine (No. 2021XJJJ067) and the National Natural Science Foundation of China (No. 82374545).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-975/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. 2024-033-5) granted by The Second Affiliated Hospital of Hunan University of Chinese Medicine, in compliance with the national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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