Nuclear TSC2 drives miR-514b-3p transcription to regulate PI3K-AKT-MTOR signalling: Implications for OSCC pathogenesis

Abstract The PI3K-AKT-MTOR signalling pathway is pivotal in regulating cell survival, proliferation, and growth. TSC2 or tuberin acts as a key negative regulator of this pathway by forming a cytoplasmic complex with TSC1 and TBC1D7. While the cytoplasmic role of TSC2 is well established, emerging evidence suggests its nuclear functions. Previously, we have identified TSC2 as a transcriptional repressor of the EREG gene. Building on this foundation, the present study has investigated the transcriptional role of TSC2 in miRNA gene regulation. A genome-wide miRNA microarray profiling of TSC2-depleted SCC131 cells identified 19 upregulated and 24 downregulated miRNAs. Of these, miR-514b-3p emerged as one of the most upregulated miRNAs. The RT-qPCR analysis showed that TSC2 knockdown results in a robust miR-514b-3p upregulation, whereas TSC2 overexpression suppresses its expression in SCC131 cells. Moreover, TSC2 negatively regulates the MIR514B promoter activity in an NLS-dependent manner. Our ChIP analysis confirmed the direct binding of TSC2 to the MIR514B promoter, establishing miR-514b-3p as a transcriptional target of TSC2. We further identified TSPAN9 as a direct downstream target of miR-514b-3p. TSC2 positively regulates TSPAN9 levels by repressing miR-514b-3p, thereby establishing a novel TSC2/miR-514b-3p/TSPAN9 regulatory axis. We further uncovered a crosstalk between TSC2/miR-514b-3p/TSPAN9 axis and the canonical PI3K-AKT-MTOR signalling pathway, where miR-514b-3p positively and TSPAN9 negatively regulates this pathway. Interestingly, AKT functions as an upstream regulator of this axis by modulating the nuclear localization of TSC2. Collectively, this study provides new insights into the non-canonical, nucleus-dependent transcriptional functions of TSC2, thus expanding its role beyond cytoplasmic signalling regulation and underscoring its significance in the cellular signalling networks. Graphical abstract The diagrammatic representation of the TSC2/miR-514b-3p/TSPAN9 axis and its interaction with the canonical PI3K-AKT-MTOR pathway. Abbreviations: PI3K, Phosphatidylinositol-3 kinase; TSC1, Tuberous sclerosis complex subunit 1; TSC2, Tuberous sclerosis complex subunit 2; TBC1D7, Tre2-Bub2-CDC16 domain family member 7; RHEB, Ras homolog enriched in brain; GTP, Guanosine tri phosphate; GDP, Guanosine di phosphate; MTORC1, Mechanistic target of rapamycin kinase complex 1; S6K1, p70 ribosomal protein S6 kinase; 4E-BP-1, eIF4E-binding protein 1; and TSPAN9, Tetraspanin 9. This figure was created in BioRender (https://app.biorender.com/). Competing Interest Statement The authors have declared no competing interest. Footnotes The paper's title has been updated. The article's language has improved, resulting in better flow and readability. No changes in findings of the study.