Dynamic feedback between IL-6 and TWIST1 drives pancreatic cancer progression

Introduction

Pancreatic cancer, primarily manifested as pancreatic ductal adenocarcinoma (PDAC), is known for its aggressive nature and poor prognosis (1). Recent advances in the study of PDAC at the genomic, epigenetic, and metabolic levels have shed light on its complex activation pathways and intricate signaling crosstalk, offering valuable insights into the mechanisms underlying tumor progression, as well as potential therapeutic targets (2-4). Among these mechanisms, cytokines play a critical role as central regulators of cell signaling pathways. They are pivotal in shaping tumor biology, influencing critical processes including cell proliferation, invasion, and metastasis. Additionally, cytokines contribute to the development of chemoresistance through autocrine and paracrine signaling (5,6). Notably, interleukin-6 (IL-6) has emerged as one of the most extensively studied cytokines, with strong associations with PDAC progression, metastasis, and resistance to therapy (7,8).

IL-6, known for its multifaceted role, is critical in the development of chronic inflammation and tumor-associated inflammation, primarily through the regulation of complex cytokine networks. It has been strongly implicated in the progression and metastasis of several cancers, including breast, prostate and cervical cancers (9-11). In PDAC, IL-6 has emerged as a critical factor driving tumor progression, with elevated serum IL-6 levels closely associated with disease severity and poor prognosis (12). Findings from genetically engineered mouse models have further confirmed its essential role in pancreatic tumorigenesis (13). Moreover, IL-6 has been demonstrated to trigger epithelial-to-mesenchymal transition (EMT) in cancer cells, as evidenced in breast cancer models. During this process, there is a downregulation of epithelial markers such as E-cadherin, while mesenchymal markers like Vimentin and N-cadherin are upregulated. Additionally, transcription factors that regulate EMT, including Snail and TWIST1, are activated. These findings underscore the dual role of IL-6 as a pro-inflammatory mediator and a key driver of tumor progression and metastasis (14). By facilitating EMT and reshaping the tumor microenvironment, IL-6 represents a promising therapeutic target, with its signaling pathways offering new opportunities for the development of innovative treatment strategies.

TWIST1, a key transcription factor within the basic helix-loop-helix (bHLH) family, facilitates EMT by specifically binding to E-box DNA sequences. As a critical regulator of EMT, TWIST1 has been strongly linked to enhanced tumor invasiveness and poor prognosis across multiple malignancies, including breast, gastric, and pancreatic cancers (15-17). Its oncogenic role extends beyond promoting tumor cell migration and invasion to include modulation of the tumor microenvironment, further contributing to cancer progression. Recent studies have highlighted the emerging interplay between IL-6 and TWIST1, particularly in the context of cancer-associated fibroblasts (CAFs) in gastric cancer, where IL-6 has been shown to upregulate TWIST1 expression via activation of the STAT3 signaling pathway, thereby accelerating EMT and tumor development (18). However, the precise relationship between IL-6 and TWIST1 in PDAC remains poorly understood. Notably, the involvement of microRNAs (miRNAs) in post-transcriptional regulation of IL-6 and TWIST1 interactions presents an intriguing but largely unexplored mechanism, underscoring the need for further investigation into their molecular crosstalk in PDAC.

In this study, we investigate the miRNA-mediated regulatory mechanisms between IL-6 and TWIST1 to explore their functional interplay in PDAC. Previous research has established the critical roles of IL-6 and TWIST1 in various malignancies; however, their interaction in PDAC remains inadequately understood. We hypothesize that TWIST1 regulates IL-6 expression, thereby initiating a positive feedback loop with IL-6 and miR-543, which further enhances TWIST1 expression. This loop may drive the progression of PDAC by influencing critical cellular behaviors. Our study aims to provide direct evidence for the existence of this regulatory loop and clarify its role in the pathological processes of PDAC. By uncovering these mechanisms, we hope to establish a conceptual framework that positions the IL-6/miR-543/TWIST1 feedback loop as a potential therapeutic target, offering new insights into precision treatment strategies for PDAC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tgh.amegroups.com/article/view/10.21037/tgh-2026-0021/rc).

Methods

Cell culture

In this study, we utilized human pancreatic ductal epithelial cells (hTERT-HPNE) and various PDAC cell lines, including BxPC-3, Capan-2, MIA PaCa-2, and PANC-1, all sourced from the American Type Culture Collection (ATCC). Cells were grown in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 ℃ with 5% CO₂ in a humidified environment.

miR-543 mimic/inhibitor and viral transduction

The miR-543 mimic and inhibitor were purchased from GenePharma (Suzhou, China, Table S1). When the cell density reached 50–70%, they were transfected using the Lipofectamine 3000 reagent (Invitrogen, USA) at the concentrations specified by the manufacturer. Lentiviruses for the overexpression of TWIST1, IL-6, and miR-543 (LV17-TWIST1, LV17-IL6 and LV12-miR543) were also obtained from GenePharma (Suzhou, China). These lentiviruses were employed to infect Capan-2 and PANC-1 cell lines, resulting in the establishment of stable clones that overexpress the corresponding genes for subsequent experiments.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of IL-6 in the culture supernatant were assessed with a commercially available ELISA kit (J&L Biological, China). Briefly, 100 µL of sample or standard was pipetted into each well and incubated at 37 ℃ for 60 minutes, followed by the addition of 100 µL of biotin-antibody and a subsequent 60-minute incubation. After discarding the liquid, wells were washed three times with 300 µL wash buffer. Subsequently, 100 µL of Streptavidin-HRP was added, followed by a 30-minute incubation of the plate. After another wash, TMB substrate was then added, the plate was left to incubate in the dark for 15 minutes. The reaction was terminated by adding 50 µL of stop solution, and absorbance was measured at 450 nm using a microplate reader.

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

RNA was extracted, and mRNA was reverse transcribed as previously reported (19). For miRNA analysis, reverse transcription and subsequent qRT-PCR were conducted using the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Japan). Gene expression was normalized using GAPDH and U6 as internal controls for mRNA and miRNA, respectively, with data processed using the 2^−ΔΔCT method. The sequences of the primers are provided in Table S2.

Western blot

Cell extracts were prepared with RIPA buffer (Servicebio, China). Proteins were separated by SDS-PAGE, transferred to PVDF (Millipore, USA), incubated with antibodies and detected with ECL, following the manufacturer’s protocols. Antibody details are in Table S3.

Sample collection and patient information

Serum samples from healthy individuals and PDAC patients, as well as pancreatic tissue and tumor specimens, were collected at Sun Yat-sen Memorial Hospital, Sun Yat-sen University, between 2013 and 2021. Serum samples from PDAC patients were obtained at the time of diagnosis, while healthy controls had no known history of pancreatic disease or malignancy. Pancreatic tissue samples were discarded specimens obtained during surgical resection. Clinical and follow-up data were also collected for disease analysis. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of Sun Yat-sen Memorial Hospital (SYSKY-2024-1056-01) and informed consent was obtained from all participants prior to sample collection.

Immunohistochemistry

The sections were deparaffinized and rehydrated, followed by antigen retrieval. To block endogenous peroxidase activity, the sections were incubated with hydrogen peroxide, and non-specific binding was minimized by applying a blocking solution. Following the primary antibody incubation, the sections were washed and exposed to the appropriate secondary antibody. After staining with diaminobenzidine, the antigen’s localization and expression were evaluated under a microscope. Immunohistochemical scores were assigned based on both the staining intensity and the proportion of positive cells.

Dual-Luciferase reporter assay

Plasmids were introduced into PDAC cells via Lipofectamine 3000 (Invitrogen, USA). Following transfection, dual-luciferase reporter assays (Yeasen, China) were performed to measure Firefly and Renilla luciferase activities. The luciferase signal ratio reflects the expression and regulation of the target gene. For miRNA-target validation, the TWIST1 3’-UTR containing the predicted miR-543 binding sites was cloned into the pmirGLO vector. For transcriptional regulation analysis, the IL-6 promoter region (wild-type and mutant constructs) was cloned into the same vector. Luciferase activity was assessed with a dual-luciferase reporter system, measuring Firefly luminescence at 560 nm and Renilla luminescence at 450 nm.

Bioinformatics analysis

GEPIA was used to analyze the expression of IL-6 and TWIST1 and their correlation in pancreatic cancer and normal pancreatic tissues (http://gepia.cancer-pku.cn/) (20). Potential miRNA targets that could bind to TWIST1 were predicted using the TargetScan, miRDB, and miRTarBase algorithms. Additionally, TWIST1 binding sites on the IL-6 promoter were identified through prediction analysis with the JASPAR database (https://jaspar.genereg.net/) (21).

Chromatin immunoprecipitation (ChIP) assay

The BeyoChIP™ Enzymatic ChIP Assay Kit (Beyotime, China) was used to perform ChIP assays. Cells were crosslinked using formaldehyde to stabilize protein-DNA interactions and then collected after quenching with glycine. The chromatin was fragmented, and nuclear pellets were prepared before treatment with MNase. Specific antibodies were employed to immunoprecipitate DNA fragments associated with the target protein, incubating overnight at 4 ℃. The samples were then incubated with Protein A/G Magnetic Beads/Salmon Sperm DNA for 1 hour, and the protein-DNA complexes were isolated using a magnetic stand and washed thoroughly. After reverse crosslinking and DNA purification, qRT-PCR was performed to detect the enrichment of IL-6 promoter DNA fragments immunoprecipitated by the TWIST1 antibody. Primers for the IL-6 promoter are provided in Table S2. PCR products were subjected to separation and visualization using a 2% agarose gel.

Cell proliferation assays

CCK-8 as well as colony formation assays were performed referring to our previous study (19).

Migration and invasion assays

Following the procedures outlined in our previous research (22), we conducted the wound healing and transwell migration/invasion tests.

Experimental animals

All animals (BALB/c-Nude) used in this study were obtained from the Experimental Animal Center of Sun Yat-sen University and were maintained under standard laboratory conditions. The mouse orthotopic pancreatic cancer xenograft model was used to evaluate the IL-6/miR-543/TWIST1 signaling in pancreatic cancer growth and the therapeutic effect of tocilizumab, an anti-IL-6 receptor monoclonal antibody. Consistent with our previous studies, pancreatic cancer cell suspensions were injected into the pancreas of mice via small animal surgery. Post-surgery, tumor progression was monitored through regular in vivo fluorescence imaging. Mice in the immunoglobulin G (IgG) and tocilizumab (5 mg/kg) treatment groups were administered the respective treatments every 2 days for a total of 20 days. At the end of the experiment, mice were anaesthetised with isoflurane (2–3% in oxygen) and euthanised by cervical dislocation while fully unconscious. Tumor tissue samples were collected for analysis to assess histological characteristics and the expression of relevant molecular markers. All animal experiments were performed under a project license (SYSU-IACUC-2024-002317) granted by the Ethics Committee of Sun Yat-sen University, in compliance with institutional guidelines for the care and use of animals.

Statistical analysis

GraphPad Prism 9.0 (GraphPad Software, USA) was used for statistical analysis, with data expressed as the mean ± standard deviation (SD). Normality of data distribution was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using the Student’s t-test or Mann-Whitney U test as appropriate. One-way analysis of variance (ANOVA) was used for multiple group comparisons. Pearson correlation analysis was employed to explore the relationship between two variables. Kaplan-Meier curves were used for survival analysis. Experiments were carried out in triplicate, with significance determined at P<0.05.

Results

IL-6, IL-6R and TWIST1 were highly expressed and associated with poor prognosis in human PDAC

Using GEPIA analysis of GTEx and TCGA datasets, we found that IL-6 and TWIST1 were significantly upregulated in PDAC tissues compared with normal pancreatic tissues, and their expression levels were positively correlated (Figure 1A). Serum levels of IL-6 were measured in 10 healthy individuals and 16 pancreatic cancer patients, showing significantly higher levels in the latter group (Figure 1B). Additionally, qRT-PCR analysis of samples from nine PDAC patients, including both tumor and adjacent normal tissues, revealed higher expression of IL-6 and TWIST1 in tumor tissues, with a positive correlation between the two (Figure 1C). To investigate the connection between IL-6, IL-6R, and TWIST1, we performed immunohistochemical staining on PDAC tissue microarrays. This revealed stronger positive staining for IL-6, IL-6R, and TWIST1 in tumor tissues compared to adjacent normal tissues (Figure 1D). Clinical data analysis indicates that lymph node metastasis is associated with the expression of IL-6, TWIST1, and IL-6R, and that cholesterol levels correlate with the expression of IL-6R (Tables S4-S6). Survival analysis of follow-up patients indicated that those with low TWIST1 expression had significantly longer overall survival than those with high expression (P=0.03, Figure 1E). Similar findings were observed for IL-6 and IL-6R (P=0.01, P=0.02, Figure 1E).

Figure 1 IL-6, IL-6R and TWIST1 are highly expressed and associated with poor prognosis in human patients with PDAC. (A) IL-6 and TWIST1 are highly expressed in PDAC in GTEx and TCGA datasets and positively correlate with each other. (B) Serum levels of IL-6 were detected by ELISA in normal and pancreatic cancer patients. (C) qRT-PCR detection of IL-6 and TWIST1 expression in pancreatic cancer and paracancerous normal tissues. (D) Representative IHC images of IL-6, TWIST1 and IL-6R in PDAC microarrays. (E) Kaplan-Meier survival analysis of IL-6, TWIST1, and IL-6R expression. *, P<0.05; **, P<0.01. ELISA, enzyme-linked immunosorbent assay; GTEx, Genotype-Tissue Expression; IHC, immunohistochemistry; IL-6, interleukin-6; IL-6R, interleukin-6 receptor; N, normal; PAAD, pancreatic adenocarcinoma; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction; T, tumor; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TWIST1, Twist family bHLH transcription factor 1.

miR-543 binds and regulates TWIST1 expression

To identify potential miRNAs regulating TWIST1 expression, we performed predictive analyses using miRTarBase, TargetScan, and miRDB databases. A Venn diagram was used to illustrate the overlap among the predicted results, identifying four common candidate miRNAs: hsa-miR-543, hsa-miR-2467-3p, hsa-miR-580-3p, and hsa-miR-2115-3p. Based on predictive scores, we selected miR-543 as the primary candidate regulator (Figure 2A). Analysis of tumor tissues from nine PDAC patients revealed that miR-543 expression was significantly lower than in adjacent normal tissues and showed a negative correlation with TWIST1 expression (Figure 2B). Additionally, we assessed miR-543 expression in the normal pancreatic ductal epithelial cell line hTERT-HPNE and several pancreatic cancer cell lines (BxPC-3, Capan-2, MIA PaCa-2, and PANC-1), observing lower miR-543 levels in the cancer cell lines (Figure 2C). Consequently, Capan-2 and PANC-1 cells were chosen for further study. qRT-PCR confirmed the transfection efficiency of miR-543 mimics and inhibitors (Figure 2D). At both mRNA and protein levels, TWIST1 expression was significantly reduced following miR-543 mimic transfection, whereas it was upregulated by miR-543 inhibitor transfection (Figure 2E,2F, Figure S1A). To verify whether miR-543 binds to the predicted site within the TWIST1 3’UTR (Figure 2G), we conducted a luciferase reporter assay. As shown in Figure 2H, the miR-543 mimic markedly decreased luciferase activity in cells transfected with the wild-type TWIST1 3’UTR, while the mutant 3’UTR was unaffected. Conversely, the miR-543 inhibitor increased luciferase activity in the wild-type 3’UTR.

Figure 2 miR-543 binds and regulates TWIST1 expression. (A) Venn diagram of potential miRNAs predicted to bind TWIST1 by miRTarBase, TargetScan, and miRDB. (B) qRT-PCR to detect miR-543 expression in pancreatic cancer and paracancerous normal tissues. (C) Expression of miR-543 in normal pancreatic ductal epithelial cells (hTERT-HPNE) and pancreatic cancer cells (BxPC-3, Capan-2, MIA PaCa-2, PANC-1). (D) qRT-PCR detection of miR-543 mimic and inhibitor efficiency. (E) qRT-PCR to detect the expression of TWIST1 with miR-543 mimic and inhibitor treatment of Capan-2 and PANC-1 cells. (F) Western blot to detect the expression of TWIST1 with miR-543 mimic and inhibitor treatment of Capan-2 and PANC-1 cells. (G) Schematic representation of the predicted binding regions of miR-543 and TWIST1 and its wild-type and mutant sequences. (H) Luciferase activity of Capan-2 and PANC-1 with miR-543 mimic or inhibitor effect. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. MT, mutant type; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TWIST1, Twist family bHLH transcription factor 1; WT, wild-type.

IL-6 regulates TWIST1 expression through miR-543 and promotes EMT

To investigate whether IL-6 regulates TWIST1 via miR-543, PDAC cells were exposed to varying concentrations of recombinant IL-6 (5, 10, 20, 50, 100 ng/mL). qRT-PCR analysis revealed that TWIST1 expression increased while miR-543 expression decreased in response to IL-6 treatment (Figure 3A). Consistently, Western blot analysis showed elevated TWIST1 protein levels, with a particularly significant increase at 50 ng/mL (Figure 3B, Figure S1B); hence, this concentration was selected for subsequent experiments. These results suggest a dose-dependent effect of IL-6 on the miR-543/TWIST1 axis within the tested range. To examine the relationship between IL-6/TWIST1 and EMT activity, we assessed the expression of EMT-related genes at both mRNA and protein levels. In Capan-2 and PANC-1 cells treated with IL-6, N-cadherin and Vimentin expression were upregulated, while E-cadherin was downregulated (Figure 3C,3D). To validate that IL-6 modulates TWIST1 expression and promotes EMT through miR-543, we treated the cells with IL-6 and miR-543 mimic. Results showed that the miR-543 mimic reversed IL-6-induced changes in TWIST1 expression and EMT markers (Figure 3E,3F, Figure S1C).

Figure 3 IL-6 regulates the expression of TWIST1 and promotes EMT through miR-543. (A) qRT-PCR was performed to detect the expression of miR-543 and TWIST1 in Capan-2 and PANC-1 cells at different concentrations of IL-6. (B) Western blot of TWIST1 expression in Capan-2 and PANC-1 at different concentrations of IL-6. (C) mRNA levels of E-cadherin, N-cadherin and Vimentin in PDAC cells with IL-6 (50 ng/mL). (D) Protein expression of TWIST1, E-cadherin, N-cadherin and Vimentin. (E,F) qRT-PCR and Western blot for detection of EMT-related genes in Capan-2 and PANC-1 cells by IL-6 in combination with or without miR-543 mimic. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. EMT, epithelial-to-mesenchymal transition; IL-6, interleukin-6; NC, negative control; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TWIST1, Twist family bHLH transcription factor 1.

TWIST1 binds to IL-6 promoter and regulates the secretion of IL-6

A complex regulatory interplay exists between IL-6 and TWIST1, where IL-6 can upregulate TWIST1 expression via miR-543, thereby promoting tumor EMT. However, whether TWIST1, as a key transcription factor, can in turn regulate IL-6 expression, forming a bidirectional regulatory network, remains unclear. To investigate this, we examined IL-6 expression in parental PDAC cells and in cells overexpressing TWIST1. Elevated IL-6 expression was observed at both mRNA and protein levels in TWIST1-overexpressing cells (Figure 4A,4B, Figure S1D). Additionally, ELISA analysis of cell supernatants confirmed higher IL-6 secretion in TWIST1-overexpressing cells (Figure 4C). Based on these findings, we hypothesized that TWIST1 may regulate IL-6 transcription by binding directly to the IL-6 promoter region. Using the JASPAR database, we identified three potential TWIST1 binding sites within the IL-6 promoter (Figure 4D,4E). To validate these sites, ChIP analysis was performed, revealing that TWIST1 binds to site 2 and site 3 but not site 1 (Figure 4F). The PCR-amplified ChIP products were further analyzed by electrophoresis, which showed distinct bands for TWIST1 immunoprecipitation at sites S2 and S3 (Figure 4G). Luciferase reporter assays further showed that TWIST1 significantly enhanced IL-6 promoter activity, whereas disruption of the predicted binding region markedly attenuated this effect, further supporting direct transcriptional regulation of IL-6 by TWIST1 (Figure 4H).

Figure 4 TWIST1 binds to the IL-6 promoter and regulates IL-6 secretion. (A,B) RNA and protein expression levels of TWIST1 and IL-6 in Capan-2 and PANC-1 cells overexpressing TWIST1. (C) ELISA detection of IL-6 in supernatants of Capan-2 and PANC-1 overexpressing TWIST1. (D) Prediction of possible binding sites of TWIST1 to the IL-6 promoter region in JASPAR. (E) Schematic diagram of TWIST1 binding IL-6 promoter region. (F) ChIP detection of TWIST1 binding to the IL-6 promoter in Capan-2 and PANC-1 cells using control IgG or anti-TWIST1 antibody. (G) Agarose gel electrophoresis of ChIP-qPCR amplification products. (H) Luciferase reporter assays evaluating IL-6 promoter activity regulated by TWIST1. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. ChIP, chromatin immunoprecipitation; IgG, immunoglobulin G; IL-6, interleukin-6; NC, negative control; OE, overexpression; PDAC, pancreatic ductal adenocarcinoma; qPCR, quantitative polymerase chain reaction; TWIST1, Twist family bHLH transcription factor 1.

IL-6/miR-543/TWIST1 pathway promotes PDAC progression

Both in vitro and in vivo experiments were conducted to assess the functional impact of the IL-6/miR-543/TWIST1 pathway in PDAC. CCK-8 and colony formation assays showed that IL-6 promoted the proliferation of Capan-2 and PANC-1 cells, whereas miR-543 inhibited cell proliferation and reversed the effects of IL-6 (Figure 5A, Figure S2A). Western blot analysis revealed that miR-543 promoted apoptosis in PDAC cells and counteracted the anti-apoptotic effects of IL-6 (Figure 5B, Figure S2B). Wound healing and transwell assays further demonstrated that IL-6 enhanced the migration and invasion of Capan-2 and PANC-1 cells, whereas miR-543 inhibited these effects and attenuated IL-6-induced cell migration and invasion (Figure 5C,5D, Figure S2C,S2D).

Figure 5 IL-6/miR-543/TWIST1 pathway promotes PDAC progression. (A) CCK-8 assay for proliferation of PDAC cells upon overexpression of IL-6 and/or overexpression of miR-543. (B) Western blot to detect the expression of BAX and Bcl-2 in different groups of PDAC cells. (C) Wound healing assay to detect the migration of different groups of PDAC cells. Magnification: ×40. (D) Transwell migration and invasion assay of cells overexpressing IL-6 and/or overexpressing miR-543. Crystal violet staining; magnification: ×100. (E) Flowchart of the mouse experiment. (F) Images of mice in the overexpression of IL-6 and/or overexpression of miR-543 groups and tumors. (G) Weight and volume of tumors in different groups of mice. (H) Immunohistochemistry of TWIST1, IL-6, Ki-67, E-cadherin, N-cadherin and vimentin in different groups of mouse tumors. *, P<0.05; **, P<0.01; ns, not significant. CCK-8, Cell Counting Kit-8; IL-6, interleukin-6; NC, negative control; OE, overexpression; PDAC, pancreatic ductal adenocarcinoma; TWIST1, Twist family bHLH transcription factor 1.

To validate the role of the IL-6/miR-543/TWIST1 pathway in vivo, stable IL-6 or miR-543 overexpressing PANC-1 cells were implanted into the pancreases of nude mice, establishing an orthotopic xenograft model (Figure 5E). Tumor growth was significantly enhanced by IL-6 overexpression, leading to increased tumor volume and weight compared to the control group. Conversely, forced expression of miR-543 inhibited tumorigenesis, significantly reducing tumor volume and weight when compared to the negative control. In line with the in vitro findings, miR-543 reduced the tumor-enhancing effects of IL-6 (Figure 5F,5G). In IL-6 overexpressing xenograft tumors, TWIST1, N-cadherin, Vimentin, Bcl-2, and Ki67 were upregulated, whereas Bax and E-cadherin were downregulated. Conversely, miR-543 overexpression produced the opposite effects on these markers in xenograft tumors (Figure 5H, Figure S2E). In summary, the IL-6/miR-543/TWIST1 pathway regulates tumorigenesis in PDAC.

IL-6R antibody blocks IL-6/miR-543/TWIST1 loop to inhibit PDAC progression

In this study, we uncovered the IL-6/miR-543/TWIST1 loop as a key driver of pancreatic cancer progression. To further validate these findings, we used the anti-IL-6R antibody tocilizumab to inhibit IL-6 signaling and block the loop. qRT-PCR, Western blot, and ELISA analyses showed that TWIST1 overexpression increased IL-6 and TWIST1 expression while decreasing miR-543 levels, whereas tocilizumab exerted the opposite effects, with no significant changes observed in the IgG group (Figure 6A, Figure S3A,S3B). Assays of colony formation further demonstrated that TWIST1 promoted the proliferation of Capan-2 and PANC-1 cells, whereas tocilizumab treatment inhibited cell proliferation (Figure 6B, Figure S3C). Western blot analysis showed that overexpression of TWIST1 inhibited apoptosis in PDAC cells, while tocilizumab promoted apoptosis (Figure 6C, Figure S3D).

Figure 6 IL-6R antibody blocks the IL-6/miR-543/TWIST1 loop to inhibit PDAC progression. (A) qRT-PCR for IL-6, IL-6R, TWIST1 and miR-543 levels in Capan-2 and PANC-1 cells overexpressing or not overexpressing TWIST1 treated with Tocilizumab or IgG. (B) Colony formation ability of Capan-2 and PANC-1 cells in different treatments. Crystal violet staining. (C) Western blot detection of BAX and Bcl-2 expression in PDAC cells of different treatment groups. (D) Flowchart of the animal experiments. (E) Representative graphs of in vivo imaging of different groups of mice. (F) Survival curves of different groups of pancreatic cancer mice. (G) Immunohistochemistry of tumors in different groups of mice with pancreatic cancer. (H) The mechanism diagram of Tocilizumab blocking the IL-6/miR-543/TWIST1 loop to inhibit PDAC progression. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. IgG, immunoglobulin G; IHC, immunohistochemistry; IL-6, interleukin-6; NC, negative control; OE, overexpression; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TWIST1, Twist family bHLH transcription factor 1.

Next, we validated the in vivo effect of tocilizumab using a nude mouse orthotopic xenograft model (Figure 6D). In vivo fluorescence imaging showed that TWIST1 overexpression promoted tumor growth, whereas tocilizumab treatment suppressed tumor progression, consistent with tumor weight and volume measurements, while IgG had no significant effect (Figure 6E, Figure S4A,S4B). Kaplan-Meier analysis revealed that mice overexpressing TWIST1 had a shorter survival time compared to controls. Furthermore, neither IgG treatment in the negative control nor TWIST1 overexpression groups improved survival, while tocilizumab treatment significantly prolonged survival (Figure 6F). Immunohistochemical analysis showed increased expression of TWIST1, IL-6, IL-6R, Ki-67, and Bcl-2 and reduced Bax expression in the TWIST1 overexpression group, whereas tocilizumab treatment produced the opposite effects, with no significant changes observed in the IgG group (Figure 6G, Figure S4C). Together, these findings highlight the essential role of the IL-6/miR-543/TWIST1 loop in the progression of pancreatic cancer, with Tocilizumab effectively disrupting this loop, thereby inhibiting tumor growth and extending survival in vivo (Figure 6H).

Discussion

In this study, we observed elevated levels of IL-6 and TWIST1, along with a marked downregulation of miR-543 in PDAC tissues, which correlated with poor patient prognosis. Mechanistically, miR-543 directly binds to the 3’-UTR of TWIST1, suppressing its expression. Conversely, IL-6 promotes the upregulation of TWIST1 by downregulating miR-543, highlighting the pivotal role of the IL-6/miR-543/TWIST1 axis in PDAC progression. This signaling pathway demonstrates oncogenic properties, regulating cell proliferation, migration, invasion, and apoptosis. Additionally, TWIST1 binds to the IL-6 promoter and enhances IL-6 transcription, potentially contributing to a positive regulatory loop involving the IL-6/miR-543/TWIST1 axis. In vivo, miR-543 overexpression significantly reduced the tumor growth of nude mice caused by overexpression of IL-6. Moreover, treatment with Tocilizumab, an IL-6 receptor antagonist, disrupted this feedback loop and effectively inhibited tumor growth.

PDAC, the predominant and most aggressive form of pancreatic cancer, is marked by rapid progression, early metastasis, and a poor prognosis (23,24). Despite significant research efforts, the underlying molecular mechanisms driving PDAC remain complex and not fully understood. Studies have revealed a range of genetic, epigenetic, and metabolic alterations that converge to disrupt critical cellular processes (25). In particular, cytokines such as IL-6 have emerged as key mediators of these processes, influencing both tumor cells and stromal components (26). IL-6, a key cytokine in the IL-6 family, plays a role in regulating immune responses during inflammation and also promotes oncogenic activities (27). Our findings demonstrate elevated IL-6 levels in the serum and increased IL-6 mRNA expression in primary tumor tissues of pancreatic cancer patients, both of which are positively correlated with poor prognosis. Moreover, the IL-6R is upregulated in pancreatic cancer tissues. Based on these observations, IL-6 stimulation was performed in vitro to evaluate IL-6R-mediated downstream signaling activation. IL-6R activation engages the JAK family of tyrosine kinases, which subsequently stimulate downstream pathways, including STAT, MAPK, and PI3K (28-30). Through these pathways, IL-6 promotes the transcription of key molecules involved in cell cycle progression, apoptosis, and angiogenesis, such as cyclin D1, ZIP4, and VEGF (30-32). Additionally, IL-6 enhances the invasive and metastatic potential of pancreatic cancer cells by inducing EMT transcription factors, including Snail1 and TWIST1 (33,34).

TWIST1, a well-established EMT regulator, plays a pivotal role in cancer progression by enhancing the migratory and invasive capacities of tumor cells (35,36). Our findings reveal that TWIST1 expression is significantly upregulated in PDAC tissues compared to adjacent normal tissues, underscoring its involvement in disease pathogenesis. Functional assays further demonstrate that TWIST1 overexpression not only accelerates cell proliferation but also reduces apoptosis in PDAC cell lines, highlighting its dual role in promoting tumor growth and survival. Importantly, we identified a positive correlation between IL-6 and TWIST1 expression levels in PDAC tissues, suggesting a potential regulatory relationship. Consistent with this, IL-6 overexpression in PDAC cells markedly increased TWIST1 expression, providing mechanistic insights into how IL-6 signaling contributes to the aggressive behavior of PDAC through TWIST1-mediated pathways. These findings reinforce the notion that the IL-6/TWIST1 axis is a critical driver of PDAC progression and a potential target for therapeutic intervention.

IL-6 is known to regulate gene expression through transcriptional, post-transcriptional, and epigenetic mechanisms, playing a significant role in cancer progression (37-39). miRNAs are short non-coding RNAs, participating in the post-transcriptional regulation of the targeted gene expression and influencing cancer activities (40,41). In this study, we identified miR-543 as a target of IL-6, revealing that the IL-6/miR-543/TWIST1 axis may contribute to PDAC pathogenesis (42-44). Although the association between IL-6 signaling and TWIST1 has been reported previously, our findings provide additional insight by revealing a miR-543-mediated regulatory pathway linking IL-6 to TWIST1 activation. While miR-543 has been implicated in tumorigenesis in other cancers, its role in PDAC is not well understood. Our findings show that miR-543 is significantly downregulated in PDAC tissues and cell lines, suggesting it acts as a tumor suppressor in this context. Moreover, IL-6 suppresses miR-543 expression, which in turn upregulates TWIST1, a key regulator of EMT and metastasis. These findings suggest that IL-6 may promote PDAC progression not only through previously reported EMT-related pathways, but also through miR-543-mediated post-transcriptional regulation of TWIST1.

This study highlights a positive feedback loop between IL-6 and TWIST1 in PDAC, a mechanism that may drive tumor progression. Previous studies have demonstrated TWIST1 binding to IL-6 promoters, enhancing its transcription in various contexts (45,46). Our findings expand on this by showing that TWIST1 overexpression induces IL-6 expression and secretion in PDAC cells, with ChIP assays confirming IL-6 as a direct target of TWIST1. This loop amplifies oncogenic signaling, promoting EMT and tumor growth. From a therapeutic perspective, disrupting this pathway offers potential benefits. Tocilizumab, a monoclonal antibody targeting the IL-6 receptor, has been extensively investigated in both preclinical and clinical settings for its immunomodulatory effects. Several studies have demonstrated its ability to suppress IL-6-driven inflammation in cancers and inflammatory diseases (47,48). In cancer models, IL-6 blockade using tocilizumab has been shown to inhibit tumor growth and reshape the tumor microenvironment by reducing the infiltration of immunosuppressive myeloid cells (49,50). Our findings further support the therapeutic potential of targeting the IL-6/miR-543/TWIST1 axis in PDAC. These findings suggest that targeting this feedback loop could be a promising approach for PDAC treatment. Further studies are warranted to validate its clinical applicability.

Several limitations of this study should be noted. The functional experiments were mainly performed in established PDAC cell lines and rely in part on overexpression approaches, which may not fully reflect the endogenous tumor context. Although an orthotopic mouse model was used to support the in vivo relevance, it cannot completely recapitulate the complexity of the tumor microenvironment. In addition, the clinical cohort size was relatively limited. Future studies should further validate these findings in more physiologically relevant models, such as patient-derived organoids or patient-derived xenografts, and in larger independent clinical cohorts to strengthen the translational implications of these findings.

Conclusions

In conclusion, our findings provide evidence that IL-6 upregulates TWIST1 expression while downregulating miR-543 in pancreatic cancer, thereby suggesting the critical role of the IL-6/miR-543/TWIST1 axis in tumor progression. Importantly, we show that TWIST1 transcriptionally activates IL-6, which in turn influences miR-543 to regulate TWIST1, forming a positive feedback loop. This mutually reinforcing loop may amplify the oncogenic effects of both IL-6 and TWIST1, contributing to the progression of PDAC. Notably, our study highlights the therapeutic potential of targeting this feedback loop using Tocilizumab, an IL-6R monoclonal antibody. Tocilizumab effectively suppresses tumor cell proliferation, downregulates EMT-related markers, and disrupts the IL-6/miR-543/TWIST1 axis. Collectively, these findings reveal a mutually reinforcing loop between IL-6 and TWIST1, suggesting that disrupting this loop could represent a novel therapeutic strategy for pancreatic cancer.

Acknowledgments

The abstract of this study was previously presented at The 5th International Symposium on Frontiers in Molecular Science (2025).

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tgh.amegroups.com/article/view/10.21037/tgh-2026-0021/rc

Data Sharing Statement: Available at https://tgh.amegroups.com/article/view/10.21037/tgh-2026-0021/dss

Peer Review File: Available at https://tgh.amegroups.com/article/view/10.21037/tgh-2026-0021/prf

Funding: This work was supported by grants from the National Natural Science Foundation of China (grant No. 82273409), Guangdong Basic and Applied Basic Research Foundation (grant Nos. 2023A1515010300 and 2024A1515010673), and Guangzhou Municipal Science and Technology Project (grant No. 2024B03J1246).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tgh.amegroups.com/article/view/10.21037/tgh-2026-0021/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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of Sun Yat-sen Memorial Hospital (SYSKY-2024-1056-01) and informed consent was obtained from all participants prior to sample collection. All animal experiments were performed under a project license (SYSU-IACUC-2024-002317) granted by the Ethics Committee of Sun Yat-sen University, in compliance with institutional 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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