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EXPRESSION OF MICRORNAS MIR21, MIR146A, AND MIR155 IN TSC
Introduction
Tuberous sclerosis complex (TSC) is a multisystem genetic disorder resulting from a mutation in the TSC1 or TSC2 genes 1-3. The TSC1 and TSC2 products, hamartin and tuberin, form a protein complex that suppresses the mammalian target of rapamycin (mTOR) pathway 3, 4. The mTOR pathway is a key regulator of cell metabolism, growth, proliferation and survival 5. mTOR has also been shown to be involved in the regulation of both the innate and adaptive immune responses 6-9.
The cerebral involvement in TSC gives rise to a large spectrum of neurological manifestations including epilepsy and cognitive disabilities 10. Epilepsy, in particular, is often associated with significant morbidity and mortality in TSC patients 10 and its early and adequate treatment represents a major challenge.
Cortical tubers, subependymal nodules, and subependymal giant cell tumors (SGCT) represent typical brain lesions of TSC underlying the neurological manifesta- tions. 11-14. Both cortical tubers and subependymal giant cell astrocytomas (SEGA) are characterized by prominent activation of pro-inflammatory signaling pathways, includ- ing in particular the IL-1β signaling pathway 15, 16. Activation of the IL-1 receptor (R)/ Toll-like receptor (TLR) pathway has also been observed in fetal TSC brain lesions 17, 18. Interestingly, abnormal activation of IL-1β signaling pathway has been recently reported in astrocytes before epilepsy onset in a mouse model of TSC 19.
MicroRNAs (miRNAs) have recently been identified as key post-transcriptional regulators of gene expression during brain development and have been also shown to critically contribute to different neurological disorders 20-23, including epilepsy 24, 25. Increasing evidence has also implicated miRNAs in the regulation of both adaptive and innate immune responses 26-28, including specific inflammatory networks activated in epi- lepsy (for reviews see 29, 30.
Several studies have recently supported the role of microRNAs in inflammatory processes in epilepsy-associated pathologies 31-35, with prominent up-regulation of miR- NAs, such as miR146a, miR21 and miR155, specifically involved in the modulation of the IL-1R/TLR pathway 26-28, 36-38.
In the present study, we investigated the expression and cellular distribution of miR21, miR146 and miR155, three miRNAs involved in the regulation of the IL-1R/TLR path- way, in TSC brain tissue (cortical tuber and perituberal epileptogenic cortex). We also evaluated possible correlations between the miRNA levels and the expression of putative targets in glial cells, as well as their relationship with IL-1β expression levels and the clin- ical course of epilepsy. To provide better insights into the functional consequences of the deregulation of these mRNAs in TSC we investigated their regulation and release in response to IL-1β stimulation in both human fetal astrocytes and TSC-SEGA-derived cell cultures and further evaluated their role in regulating the TLR/IL-1R-interleukin-1 recep- tor-associated kinase (IRAK)- nuclear factor kappa-B (NF-κB) signaling.
Materials and Methods
Subjects
The cases included in this study were obtained from the archives of the departments of neuropathology of the Academic Medical Center (AMC, University of Amsterdam, the Netherlands), the University Medical Center Utrecht (UMCU, the Netherlands),
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