Aug 2026· Cellular Signalling· Vol 148, pp.
112831
· 0 citations· 39 references
Medicine
TL;DR
The identification of the circTLL1-90aa/NT5C2/Ras/PI3K axis not only expands the functional repertoire of the non-coding genome but also provides new insights into the complexity of drug resistance.
Abstract
Background
Acquired resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor, remains a major clinical challenge in the treatment of non-small cell lung cancer (NSCLC). Although circular RNAs (circRNAs) have been increasingly implicated in drug resistance, most studies have focused on their canonical role as microRNA sponges, while their capacity to encode functional micropeptides remains largely unexplored. This study aimed to identify novel circRNAs involved in osimertinib resistance and to characterize their regulatory functions at the protein level.
Methods
Osimertinib-resistant (OR) NSCLC cell lines were established and validated. High-throughput RNA sequencing was performed to compare the circRNA expression profiles between parental and OR cells. The function of the candidate circRNA was assessed through a series of in vitro and in vivo experiments, including cell viability assays, apoptosis analysis, and xenograft mouse models. Mechanistic investigations involved mass spectrometry, co-immunoprecipitation and western blotting to explore its protein-coding potential and downstream signaling pathways.
Results
We identified a novel circRNA, termed circTLL1, that was stably and significantly upregulated in OR-NSCLC cells. Functionally, overexpression of circTLL1 promoted osimertinib resistance, whereas its knockdown restored drug sensitivity both in vitro and in vivo. Mechanistically, we discovered that circTLL1 harbors an open reading frame (ORF) that is translated into a novel 90-amino-acid protein, which we designated circTLL1-90aa. Further investigation revealed that circTLL1-90aa directly interacts with and promotes the degradation of 5'-nucleotidase, cytosolic II (NT5C2), thereby uncoupling nucleotide metabolism from its normal regulatory constraints. The consequent downregulation of NT5C2 leads to elevated GTP levels and leading to the sustained activation of the downstream Ras/PI3K/AKT signaling pathway.
Conclusion
Our findings unveil a previously unrecognized circRNA/micropeptide/metabolism cascade underlying osimertinib resistance. The identification of the circTLL1-90aa/NT5C2/Ras/PI3K axis not only expands the functional repertoire of the non-coding genome but also provides new insights into the complexity of drug resistance. Given its selective upregulation in resistant cells, circTLL1-90aa holds promise both as a predictive biomarker for treatment stratification and as an actionable therapeutic target, offering a novel strategy to overcome osimertinib resistance in NSCLC patients.
BACKGROUND
Osimertinib resistance is a major challenge in the treatment of EGFR-mutated lung adenocarcinoma (LUAD), and the role of LINC00511 in this process remains unclear.
METHODS
We analyzed LINC00511 expression, patient prognosis, and its correlation with EIF2AK3 (encoding PERK) in LUAD using the GEPIA2 database, and further validated its circulating levels in patient peripheral blood by RT-qPCR. In vitro experiments, we constructed osimertinib-resistant H1650-OS and H1975-OS cell lines and regulated the expression of LINC00511 using genetic means. We evaluated the effects on resistance through functional experiments and used Western blot to detect PERK/Nrf2 and ER stress (ERS)-related proteins. Finally, we established a xenograft model to validate the in vitro findings in vivo.
RESULTS
The results showed that elevated LINC00511 levels in patients with osimertinib resistance, and positively correlated with PERK. Knockdown of LINC00511 could block the activation of the PERK/Nrf2 axis, inducing ERS. In vivo experiments confirmed that silencing LINC00511 enhances the inhibitory effect of osimertinib on osimertinib-resistant LUAD. Mechanistically, LINC00511 inhibits ERS through the PERK-Nrf2 pathway, thereby driving resistance.
CONCLUSION
These findings provide clinical evidence for the early diagnosis and risk stratification of Osimertinib resistance, and suggest that targeting the LINC00511-PERK-Nrf2 axis may become a novel therapeutic strategy for restoring drug sensitivity, offering potential targets for the development of intelligent diagnostic and therapeutic nanomaterials.
Qingyang Lei, Jing Peng, Na Li et al.· Cellular Signalling· 0 citations
Elucidating the complex molecular drivers of non-small cell lung cancer (NSCLC) and identifying novel therapeutic targets are urgently needed. Although circPRKCA is implicated in tumorigenesis, its role in NSCLC remains poorly characterized. This study investigates the biological functions and regulatory mechanisms of circPRKCA in NSCLC. CircPRKCA expression and downstream target expression were assessed via qRT-PCR and Western blotting. In vitro functional assays evaluated circPRKCA's role in NSCLC cells. Regulatory mechanisms were further examined using database analysis, RNA immunoprecipitation (RIP), and dual-luciferase reporter assays. In vivo tumor growth was evaluated in mouse xenograft models. CircPRKCA was significantly upregulated in NSCLC and promoted tumor growth and metastasis. Our data suggest that circPRKCA may act as a scaffold for RNA-binding proteins (RBPs), potentially facilitating the recruitment of SRSF1 to HIF-1A mRNA, which appeared to correlate with increased HIF-1A stability. HIF-1A overexpression enhanced NSCLC cell proliferation via PKM2-mediated glycolysis. High circPRKCA expression correlated with poor survival in NSCLC patients. CircPRKCA-SRSF1 axis enhanced proliferation and suppressed apoptosis by stabilizing HIF-1A and modulating PKM2-driven glycolysis. These findings reveal novel diagnostic biomarkers and therapeutic strategies for NSCLC.
Hai-Ting Chen, Xing-Xing Jin, Leilei Tao et al.· Experimental Cell Research· 0 citations
: Background: Circular RNAs have emerged as important regulators of non-small cell lung cancer progression through competing endogenous RNA networks, but the specific role and mechanism of CircATRNL1 in NSCLC remain unclear. This study investigates the mechanistic role of CircATRNL1 in the malignant progression of non-small cell lung cancer (NSCLC). Methods: Gene expression levels in NSCLC cell lines were quantified using reverse transcription quantitative PCR (RT-qPCR). Functional assays, including Western blot analysis of epithelial– mesenchymal transition (EMT)-related proteins, wound-healing assays, and Transwell migration and invasion assays, were performed to assess the effects of CircATRNL1 on cellular motility and invasive potential. Potential RNA–RNA interactions were predicted using the ENCORI and CircBank databases, followed by luciferase reporter assays to validate these interactions experimentally. To determine whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway mediated the biological effects of insulin-like growth factor-binding protein 3 (IGFBP3), rescue experiments were performed using the PI3K inhibitor LY294002. Results: CircATRNL1 was significantly upregulated in NSCLC cell lines and showed typical circular RNA characteristics, including RNase R resistance and enhanced transcript stability (*** p < 0.001). CircATRNL1 knockdown inhibited NSCLC cell migration and invasion, whereas CircATRNL1 overexpression exerted the opposite effects. Mechanistically, CircATRNL1 functioned as a sponge for miR-103a-3p and relieved miR-103a-3p mediated repression of IGFBP3, as validated by luciferase reporter and rescue assays (** p < 0.01). IGFBP3 further activated PI3K/AKT signaling by increasing p-PI3K/PI3K and p-AKT/AKT ratios, while LY294002 attenuated IGFBP3-induced migration and invasion. Conclusions: CircATRNL1 promotes NSCLC cell migration and invasion by acting as a ceRNA for miR-103a-3p, thereby regulating IGFBP3 and activating PI3K/AKT signalling. These findings highlight the CircATRNL1/miR-103a-3p/IGFBP3/PI3K-AKT axis as a potential regulatory mechanism contributing to in vitro migration and invasion in NSCLC.
Jiawei Liu, Tao Huang, Qi You et al.· Biocell (Mendoza)· 0 citations
BACKGROUND
Bladder cancer (BCa) is a common urological malignancy with high recurrence rates and limited treatment options for advanced disease. Circular RNAs (circRNAs) are emerging as key regulators of cancer progression, but the roles of most circRNAs in BCa remain poorly understood.
OBJECTIVE
To investigate the expression, function and mechanism of circPCMTD1 (hsa_circ_0001801) in BCa, focusing on its m6A methylation and its role as a ceRNA regulating the miR-3681-5p/HPSE/PI3K/AKT axis.
METHODS
circPCMTD1 expression was analyzed in GEO datasets, 30 paired BCa tissues and cell lines by qRT-PCR. Circular structure and stability were confirmed by RNase R and actinomycin D assays. Subcellular localisation was determined by fractionation and FISH. Gain- and loss-of-function experiments assessed circPCMTD1 effects on BCa cell malignancy. A subcutaneous xenograft model was used for in vivo validation. m6A sites were predicted by SRAMP, and circPCMTD1-EIF4A3 interaction validated by RIP. Protein-protein docking predicted EIF4A3-METTL3 binding. miRNA targets were screened by ENCORI and confirmed by qRT-PCR and RNA-FISH. miR-3681-5p targets were identified by transcriptome sequencing and database analysis. Western blotting assessed HPSE, PI3K, AKT and p-AKT. Rescue experiments verified the circPCMTD1/miR-3681-5p/HPSE axis.
RESULTS
circPCMTD1 was upregulated in BCa tissues and cells, stable and cytoplasmic. Knockdown suppressed BCa proliferation, migration, invasion and Tumor growth in vivo, while overexpression promoted these phenotypes. circPCMTD1 contained nine predicted m6A sites, interacted with EIF4A3, and EIF4A3 docked with METTL3. circPCMTD1 overexpression increased METTL3, METTL14, FTO and YTHDF2. circPCMTD1 directly sponged miR-3681-5p. miR-3681-5p acted as a tumor-suppressive miRNA. HPSE was a direct target of miR-3681-5p and overexpressed in BCa. circPCMTD1 positively regulated HPSE, PI3K and p-AKT, whereas miR-3681-5p had opposite effects. Rescue experiments confirmed that circPCMTD1 promoted BCa through sponging miR-3681-5p and activating HPSE/PI3K/AKT.
CONCLUSIONS
EIF4A3 facilitates METTL3-mediated m6A methylation of circPCMTD1. Methylated circPCMTD1 acts as a ceRNA sponging miR-3681-5p, derepressing HPSE and activating PI3K/AKT to drive BCa progression. This study identifies a novel EIF4A3/m6A-circPCMTD1/miR-3681-5p/HPSE/PI3K/AKT axis and suggests potential therapeutic targets for BCa.
Lingbao Xiang, Yutong Li, Xuan Dong et al.· Cellular Signalling· 0 citations
INTRODUCTION
Ovarian cancer (OC) is the third most aggressive gynecological malignancy worldwide, and platinum resistance is a common malignant feature of OC associated with poor clinical prognosis. Circular RNA (circRNA) has been reported in many tumor progressions, including drug resistance. In this study, a new circRNA that mediates OC progression by acting as a ceRNA was identified.
METHODS
Through high-throughput transcriptome sequencing and bioinformatic analyses, the CircSERPINE2/miR-513a-5p/miR-526b-3p/SEMA7A axis was identified as a potential key factor in the development of platinum resistance in OC. CCK-8, colony formation assay, and flow cytometry confirmed the role of CircSERPINE2 in regulating platinum resistance in OC. Relevant patents were cited by the China National Intellectual Property Administration (CNIPA).
RESULTS
In OC cells and subcutaneous xenograft tumors, downregulation of CircSERPINE2 was associated with elevated expression of miR-513a-5p/miR-526b-3p, inactivation of the SEMA7A/ IL-6 pathway, and reduced tumor size following platinum treatment. Furthermore, the CircSERPINE2/SEMA7A axis mediated IL-6 autocrine in OC cells and significantly decreased platinum-induced apoptosis. Moreover, the CircSERPINE2/SEMA7A axis promoted M2 polarization of macrophages in the tumor microenvironment, which in turn impaired platinum sensitivity in ovarian cancer cells.
DISCUSSION
Given that circRNAs can act as promising clinical diagnostic biomarkers and therapeutic targets, the development of circRNA vaccine nanocomposites represents a novel and promising therapeutic strategy to potentiate tumor-killing efficacy when combined with chemotherapy regimens. On this basis, our subsequent research will focus on the rational design and development of a tailored nano-drug delivery system for this application.
CONCLUSION
CircSERPINE2 promotes platinum resistance in OC cells and induces an immunosuppressive environment through the miR-513a-5p/miR-526b-3p/SEMA7A axis, highlighting its potential as a target for overcoming platinum resistance in OC.
Zhi-Xing Niu, Yuqing Mo, Bikang Yang et al.· Recent Patents on Anti-Cance...· 0 citations
Introduction Renal cell carcinoma (RCC) is a fatal urological malignancy with limited therapeutic options and poor prognosis. Emerging evidence has shown that a number of circular RNAs (circRNAs) exert crucial effects on carcinogenesis. Nevertheless, the biological role of circEVI5 in RCC remains poorly clarified. This study aimed to elucidate the clinical significance, biological functions, and molecular mechanisms of circEVI5 in RCC pathogenesis. Methods The expression profile of circEVI5 was first screened based on GEO and TCGA-KIRC datasets, and further verified in RCC cell lines (A498, 786-O, ACHN, CAKI-1, OSRC-2) and normal renal tubular epithelial HK-2 cells using qRT-PCR. Functional assays (CCK-8, Transwell) and a subcutaneous xenograft model were employed to assess circEVI5’s role in proliferation, migration, and invasion. RNA pull-down, RIP, and dual-luciferase reporter assays were conducted to validate interactions among circEVI5, miR-433, and GBP2. Clinical correlations were further analyzed based on TCGA datasets and immunohistochemical staining results. Results CircEVI5 was significantly upregulated in RCC tissues (median FC=6.619; P<0.05) and cell lines, correlating with advanced tumor stage (median FC=7.691; P<0.05) and poor survival (P=0.035). Knockdown of circEVI5 via small interfering RNA inhibited cell viability, migration, and invasion in vitro and suppressed tumor growth in vivo. Mechanistically, circEVI5 acted as a cytoplasmic molecular sponge for miR-433, which was downregulated in RCC and inversely correlated with GBP2 expression. RNA pull-down and RIP assays confirmed direct binding between circEVI5 and miR-433, while luciferase assays identified GBP2 as a miR-433 target. Rescue experiments demonstrated that miR-433 inhibition or recombinant GBP2 reversed the anti-tumor effects of circEVI5 knockdown. Clinically, GBP2 overexpression in RCC tissues predicted reduced survival, and miR-433 levels were inversely correlated with both circEVI5 (P<0.0001) and GBP2 (P=0.0003). Discussion Our findings establish circEVI5 as a novel oncogenic driver that facilitates RCC progression through the miR-433/GBP2 regulatory axis. The circEVI5/miR-433/GBP2 network represents a potential therapeutic target, and circEVI5 may serve as a prognostic biomarker for RCC patients. This study highlights the critical role of circRNA-mediated miRNA sponging in RCC pathogenesis and provides a framework for developing RNA-based therapies.
Yang Xiong, Jin Li, Zhi-Peng Tang et al.· Frontiers in Oncology· 0 citations
