Differential expression of oncogenic lncRNAs NEAT1 and MALAT1 in 2D monolayer vs. 3D tumoroid culture and its implications in cancer progression

Abstract Long non-coding RNAs (lncRNAs) have emerged as crucial regulators of cellular processes, overturning their previous classification as “junk” DNA. Their involvement in various cellular functions makes them potential therapeutic targets in a range of diseases, particularly in tumorigenesis and cancer. Among these lncRNAs, MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) and NEAT1 (Nuclear-Enriched Abundant Transcript 1) are prominent oncogenic lncRNAs with complex regulatory roles across multiple cancer types. Although the roles of these lncRNAs in cancer have been extensively studied, the majority of these investigations have been conducted in 2D culture systems. While 2D cell cultures are well-established and widely accepted models in cell biology, they lack the physiological complexity of 3D tumor architecture. In contrast, 3D cultures, where cells grow as three-dimensional clusters, better mimic in vivo conditions, making them essential for understanding tumor microenvironment (TME) dynamics. Despite the importance of 3D models, there is a lack of literature exploring lncRNA expression in 2D monolayers versus 3D cultures across different cancer types. This study examines the expression and function of the lncRNAs NEAT1 and MALAT1 in 3D tumoroids compared to 2D monolayer cultures, aiming to bridge the gap between in vitro models and the complex in vivo tumor microenvironment. We addressed this gap by quantifying the expression differences of NEAT1 and MALAT1 using qRT-PCR in breast cancer, liver cancer, cervical cancer, and glioblastoma (GBM) cells grown in both 2D and 3D cultures. Significant variations in NEAT1 and MALAT1 expression were observed between 2D and 3D cultures across these cancer types, signifying the need to study lncRNAs in 3D micro-environments. Furthermore, we established correlations between NEAT1 expression and cancer traits such as stemness, invasion, glucose transporter expression, and epithelial-mesenchymal transition (EMT) in GBM 3D tumoroids compared to 2D monolayers. Using siRNA to downregulate NEAT1 in GBM tumoroids, we demonstrated that reducing NEAT1 expression to the levels comparable to 2D cultures led to a decrease in the expression of mRNA markers associated with stemness, invasion, glucose transporters, and EMT. Additionally, siRNA-mediated downregulation of NEAT1 in 3D tumoroids directly impacted cancer properties, as validated by phenotypic assays, where reduced proliferation, migration, and invasion were observed in GBM (comparing 3D to 2D models). Therefore, our work provides new insights into the expression of two key oncogenic lncRNAs in 3D microenvironments of various cancers and lays the foundation for integrating lncRNAs as important molecular players within 3D culture systems, offering a better understanding of the in vivo complexity of the TME.