Regulação da expressão dos genes TSC1 e TSC2 na via de mTORC1

Abstract

Introduction: Tuberous sclerosis complex (TSC) is a multisystemic disease characterized by benign tumors in various organs and caused by pathogenic variants in the tumor suppressor genes TSC1 or TSC2. The TSC1 and TSC2 proteins inhibit the mTORC1 pathway, which is central to the control of cell growth and proliferation. mTORC1 activity has been linked to the differential expression of specific genes. However, it is unknown whether the pathway's activity has any effect on the mRNA and protein levels expressed by the TSC complex (TSC1, TSC2, and TBC1D7). The unkempt protein (UNK), encoded by the UNK gene, is an RNA-binding protein that regulates mRNA expression in neuronal differentiation processes, serving as a constituent of mTORC1. Previous results from our lab identified co-expression of TSC1 and UNK mRNA in tumors without pathogenic alterations in TSC1 and TSC2. There is limited information on the regulation of TSC1 and TSC2 gene expression in modulating mTORC1 pathway activity. Moreover, it is unclear whether UNK regulates mRNA expression of certain genes in this pathway. Aims: The specific aims of this study were to assess (i) the effect(s) of the mTOR pathway on the RNA and protein levels expressed by TSC1, TSC2, TBC1D7, and UNK genes; and (ii) whether shRNA-mediated silencing of UNK alters the protein levels of TSC1 and TSC2 in HEK293T cells. Methods: Differential gene expression analyses were performed on publicly available transcriptome data obtained from HEK293T cells treated with rapamycin, SH-SY5Y cell line submitted to UNK knocking-down, or soleus muscle from mice that had been treated with rapamycin or caloric restriction for 15 months. In the laboratory, HEK293T cells were treated with rapamycin for 24 hours. A cell line was established by transduction of lentiviral particles modified to express short-hairpin RNA (shRNA) targeting UNK. Western blotting was conducted for quantitative comparison of target proteins. Results: HEK293T cells subjected to mTORC1 pathway repression by rapamycin treatment for 72 hours had an increase in TSC2 mRNA levels. Transcriptome analysis of soleus muscle from mice treated in vivo with rapamycin for 15 months revealed an increase in Tsc2 mRNA and a reduction in Tsc1 and Tbc1d7 mRNA. UNK knock-down in human SH-SY5Y cells resulted in a reduction in TSC2 mRNA. Analysis of the TSC1-TSC2-TBC1D7 complex proteins in unmodified HEK293T cells showed an increase in UNK levels 24 hours after starvation, which was maintained during consecutive rapamycin treatment for an additional 24 hours. A HEK293T cell line with significant reduction of UNK protein was generated, and no change was observed in the levels of any of the three TSC1-TSC2-TBC1D7 complex proteins. Conclusions: Our data indicate that the mTORC1 pathway has a negative effect on TSC2 mRNA levels in HEK293T cells and mouse soleus muscle. In HEK293T cells, mTORC1 pathway inactivity increases UNK protein levels. Reduction of UNK protein in this cell line does not alter the levels of the TSC1-TSC2-TBC1D7 complex proteins. In...