[Non-small cell lung cancer-derived exosomal circular RNA circEZH2 activates fibroblasts by regulating the miR-495-3p...

Objective: To study the effects and mechanisms of activation of human lung fibroblasts (MRC-5) by exosomal RNA hsa _ circ _ 0006357 (circEZH2) derived from non-small cell lung cancer. Methods: Western blot was used to detect exosome molecular markers, reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and cell invasion assays to detect the effect of non-small cell lung cancer-derived exosomes on MRC-5 activation. A circRNA microarray analysis was performed in serum exosomes from patients with non-small cell lung cancer (collected at Ningbo University People's Hospital, September 2023), and levels of circEZH2 were measured in serum exosomes from non-small cell lung cancer by RT-qPCR analysis. The effects of circEZH2 on MRC-5 activation were explored using wound healing assays, Transwell assays, RT-qPCR, cellular immunofluorescence, and western blot. The regulatory effect of circEZH2 on miR-495-3p/TPD52 axis and NF-κB pathway through dual-luciferase assay, immunofluorescence, and western blot. Results: Exosomes derived from non-small cell lung cancer cells were shown to promote MRC-5 cell invasiveness, the number of cells invaded in co-culture with exosomes derived from normal human bronchial epithelial cells was (42±5), and the number of cells invaded in co-culture with exosomes derived from non-small cell lung cancer cells (SPC-A1, H1299, A549 cells) was (246±7), (89±4), (69±14), expression of markers of fibroblast activation (α-SMA, FAP), and cytokines (IL-6, IL-8, P＜0.05). CircEZH2 expression was significantly upregulated in the serum exosomes of non-small cell lung cancer (P＜0.01). Alternatively, co-culture of exosomes derived from non-small cell lung cancer cells with MRC-5 cells promoted circEZH2 expression (P＜0.05). Functionally, overexpression of circEZH2 promoted MRC-5 cell migration and invasion [the cell migration rates were (30.81±2.54)% and (60.29±8.34)%, respectively, and the cell invasion numbers were (48.00±13.58) and (115.00±9.50), respectively, P＜0.05]. RT-qPCR, western blot, and immunofluorescence assays demonstrated a significant increase in the expression of the pro-inflammatory genes IL-6 and IL-8 in MRC-5 cells as well as the activation markers α-SMA and FAP in fibroblasts (P＜0.05) following the expression of circEZH2. Mechanistically, circEZH2 may function as ceRNA to regulate miR-495-3p, promote the expression of TPD52, and activate the NF-κB pathway to promote the activation of MRC-5. Conclusion: Exosomal circEZH2, derived from non-small cell lung cancer, may promote the activation of fibroblasts by activating the NF-κB pathway through the miR-495-3p/TPD52 axis.