Elucidating the TSC2-mediated transcriptional regulation of miR-514b-3p

Abstract

The PI3K-AKT-MTOR signalling axis is pivotal in regulating cell survival, proliferation, and growth. Tuberous sclerosis complex (TSC) is a well-established negative regulator of this pathway, which primarily acts by suppressing the MTORC1 activity. This complex is composed of the following three proteins: TSC1, TSC2, and TBC1D7. While the cytoplasmic role of TSC2 (tuberin) as a component of TSC assembly in regulating PI3K-AKT-MTOR signalling is well characterised, emerging evidence points toward its nuclear functions. Previous work from our laboratory has identified TSC2 as a transcription factor, where it binds to the promoter of the EREG (Epiregulin) gene and represses its expression. Building on this foundation, the present study has investigated the transcriptional role of TSC2 in the context of microRNA (miRNA) gene regulation.

Using the miRNA microarray profiling of TSC2 knockdown cells from an oral squamous cell carcinoma (OSCC) cell line SCC131, we have identified 19 upregulated and 24 downregulated miRNAs. Of these, miR-514b-3p emerged as one of the most significantly upregulated miRNAs. We have validated the upregulation of miR-514b-3p following TSC2 knockdown. Further, we have observed the downregulation of miR-514b-3p following TSC2 overexpression, suggesting the TSC2-mediated negative regulation of miR-514b-3p expression. Moreover, we have observed that TSC2 negatively regulates the MIR514B promoter activity in an NLS-dependent manner. Furthermore, using chromatin immunoprecipitation (ChIP), we have demonstrated that TSC2 acts as a transcription factor by binding to the promoter of the MIR514B gene. Interestingly, we identified TSPAN9, a known tumour suppressor gene, as a direct downstream target of miR-514b-3p. We have demonstrated that miR-514b-3p binds to the 3’UTR of TSPAN9 and negatively regulates its expression in a dose-dependent manner. In contrast, TSC2 positively regulates TSPAN9 levels by repressing miR-514b-3p, thereby establishing a novel TSC2–miR-514b-3p–TSPAN9 regulatory axis. Furthermore, we have demonstrated that miR-514b-3p regulates cell proliferation, apoptosis, and anchorage-independent growth of OSCC cells, in part, by targeting the 3’UTR of TSPAN9.

In addition, we have also explored the crosstalk between TSC2–miR-514b-3p–TSPAN9 axis and the canonical PI3K-AKT-MTOR signalling. We have found that miR-514b-3p positively regulates, whereas TSPAN9 negatively regulates the PI3K-AKT-MTOR pathway. Further, we have demonstrated that AKT acts as an upstream modulator of the TSC2–miR-514b-3p–TSPAN9 axis via TSC2 nuclear localisation. Importantly, we have also observed that the miR-514b-3p and TSPAN9 levels remain unchanged following rapamycin treatment, suggesting that the TSC2-mediated regulation of miR-514b-3p/TSPAN9 is a function of nuclear TSC2 and is independent of the MTORC1 activity. Taken together, this study has provided 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.