FUS/CircZEB1/miR-128-3p/LBH Feedback Loop Promotes Malignant Phenotype of GSCs via TNF-α Mediated NF-κB Signaling Pathway

Neurology Oncology Life Sciences Immunology Medicine Biochemistry Genetics
Glioblastoma CircZEB1 Glioma Stem Cells Limb-Bud and Heart NF-κB Signaling Pathway

The Role of the Novel Circular RNA circZEB1 in the Malignant Phenotype of Glioblastoma Stem Cells and Its Molecular Mechanism

Research Background

Glioblastoma (GBM) is one of the most aggressive and fatal primary tumors in the central nervous system, with a very poor prognosis for patients. Even with surgery, radiotherapy, and chemotherapy, the average survival period is still less than 15 months. The characteristics of this malignant tumor, such as growth, invasion, self-renewal, and recurrence, are closely related to glioma stem cells (GSCs). Due to their crucial role in tumor proliferation, invasion, self-renewal, and resistance to radiotherapy and chemotherapy, GSCs have become key targets in GBM treatment research. In recent years, the role of circular RNAs (circRNAs) in different cancers has gradually been unveiled. They regulate gene expression by acting as “sponges” for microRNAs (miRNAs) and are involved in numerous physiological and pathological processes. Therefore, discovering new circRNAs related to GSCs may provide new therapeutic avenues for GBM treatment.

Research Source

This study was completed by research teams from the First Affiliated Hospital of China Medical University, the Affiliated People’s Hospital of China Medical University, and Shanghai Tenth People’s Hospital. The primary researchers include Guoqing Zhang, Yang Jiang, Zhichao Wang, among others. The study was published in the journal “Cancer Cell International” in 2024, with the article DOI number being 10.1186/s12935-024-03526-8.

Research Objective

This study aims to reveal the mechanistic role of a novel circular RNA, circZEB1, in GBM, specifically exploring whether it affects the malignant phenotype of GBM through the regulation of the tumor necrosis factor alpha (TNF-α)-mediated NF-κB signaling pathway in glioma stem cells. The study also investigates whether circZEB1 can influence tumor growth and invasion capabilities through a positive feedback loop with miR-128-3p, LBH, and FUS proteins.

Research Methods

Experimental Procedures and Samples

The study collected tumor tissue samples from 70 glioma patients who underwent surgery at the First Affiliated Hospital of China Medical University between 2015 and 2019. These samples were graded according to WHO standards (20 cases of grade II, 25 cases of grade III, and 25 cases of grade IV). For the control group, the study collected 10 normal brain tissue samples. Additionally, primary glioma stem cells (GSC) were isolated from grade IV specimens of 6 GBM patients for subsequent experiments.

Experimental Steps

  1. CircRNA Screening: The study employed the circRNA gene chip GSE109569 from the GEO database to screen circRNAs in GBM and used the differential gene analysis tool LIMMA to identify 3377 upregulated circRNAs. Among the screened circRNAs, the study further validated the high expression of circZEB1.

  2. RNA Immunoprecipitation Experiments: Through RNA immunoprecipitation (RIP) experiments, the study demonstrated direct binding between circZEB1 and miR-128-3p and FUS proteins. This experiment confirmed the function of circZEB1 as a “sponge” for miR-128-3p and revealed the regulatory effect of the FUS protein on the stability of circZEB1.

  3. Functional Experiments: By knocking down and overexpressing circZEB1 and LBH, the study conducted a series of cell proliferation, invasion, sphere formation, and ELDA experiments to analyze the effect of circZEB1 and LBH on the malignant phenotype of GSCs. The results showed that high expression of circZEB1 significantly enhanced the proliferation and invasion capability of GSCs, whereas its knockdown reduced these capabilities.

  4. Signal Pathway Analysis: Through GSEA analysis and Western blot detection, the study revealed that high expression of LBH could upregulate the transcription level of TNF-α, thus activating the NF-κB signaling pathway. The activation of this signaling pathway further promoted the malignant phenotype of GBM.

  5. Animal Experiments: The study also validated the in vivo tumor-promoting effect of circZEB1 in mouse models. Through subcutaneous injection of GSC cells overexpressing or knocking down circZEB1, it was observed that overexpression of circZEB1 significantly increased tumor growth and invasion capability, further proving its crucial role in the malignant phenotype of GBM.

Research Results

  1. High Expression of circZEB1 Closely Related to Malignant Progression and Poor Prognosis of GBM: circZEB1 was significantly overexpressed in GBM patients and associated with poor survival prognosis. Kaplan-Meier survival analysis showed that patients with high circZEB1 expression had significantly shorter survival times.

  2. circZEB1 Promotes the Malignant Phenotype of GSCs: Functional experiments demonstrated that high expression of circZEB1 promotes the proliferation, invasion, and self-renewal capability of GSCs, while its knockdown significantly inhibits these malignant traits. Moreover, the overexpression of circZEB1 could upregulate the expression of stem cell markers such as Nanog, Nestin, Oct4, Sox2, and CD133, further enhancing the stemness of GSCs.

  3. LBH Enhances the Malignant Phenotype of GSCs Through the NF-κB Signaling Pathway: The study found that high expression of LBH could significantly increase the expression of TNF-α, thus activating the NF-κB signaling pathway. Inhibiting the expression of TNF-α effectively reverses the proliferative and invasive abilities of GSCs induced by LBH, demonstrating the activating effect of LBH on the NF-κB signaling pathway.

  4. circZEB1 Upregulates LBH by “Sponging” miR-128-3p: The study showed that circZEB1 can upregulate LBH expression by “sponging” miR-128-3p. A series of functional experiments and Luciferase reporter experiments confirmed the interaction between circZEB1 and miR-128-3p and demonstrated the crucial role of miR-128-3p in the malignant phenotype of GSCs.

  5. FUS Protein Has a Significant Impact on the Stability of circZEB1: The study further found that the RNA-binding protein FUS can bind with circZEB1 and maintain its stability by extending its half-life. At the same time, LBH can form a positive feedback loop of FUS/circZEB1/miR-128-3p/LBH by upregulating the expression of FUS, thus promoting tumor growth and the malignant phenotype.

Research Conclusion

This study uncovers a novel role of circZEB1 in GSCs and, through its positive feedback loop with miR-128-3p, LBH, and FUS proteins, activates the TNF-α-mediated NF-κB signaling pathway, ultimately leading to the enhancement of the malignant phenotype of GBM. The research suggests that circZEB1 may serve as a potential biomarker and therapeutic target for GBM, providing a new direction for future GBM treatment.

Research Significance and Prospects

This study provides new insights into the mechanism of circRNAs in GBM, especially in the regulation of the malignant phenotype of GSCs. The high expression of circZEB1 in GSCs and its feedback loop with miR-128-3p, LBH, and FUS not only reveal the molecular basis of malignant progression in GSCs but also offer potential therapeutic targets for GBM treatment. Additionally, the study’s findings on the role of the TNF-α/NF-κB signaling pathway in tumors offer new ideas for research into other types of malignant tumors.