Regulação dos genes IKBKB e BCL2 por microRNA e expressão de genes apoptóticos em tecidos com trissomia do cromossomo 21

Abstract

Introduction: Trisomy of chromosome 21 results in various dysfunctions, including cognitive impairment and immune system deficiency. MicroRNAs (miRNAs) influence biological processes such as apoptosis and cell proliferation, playing a significant role in disorders associated with Down syndrome (DS). Objectives: To assess the gene and protein expression of IKBKB and BCL2 in disomic and trisomic fibroblasts for chromosome 21, after transfection with miR-942-5p and miR-378a-3p, and to analyze, through bioinformatics, the expression of apoptotic genes (BCL2, BAX, BBC3, and CASP7) in post-mortem brains of individuals with DS. Materials and Methods: Fibroblasts were transfected with miRNA mimics and inhibitors to evaluate their interactions with the BCL2 and IKBKB genes. RNAs and proteins were extracted and analyzed by quantitative real-time PCR and Western Blotting, respectively. Microarray data of apoptotic genes expressed in brains of 58 DS individuals and 58 controls, from the public GEO database, were analyzed using the bioinformatics tool GEO2R; protein interactions were investigated using STRING-DB and Metascape. Results: The expression of IKBKB decreased after transfection with miR-942-5p mimic in disomic (p<0.0001) and trisomic (p<0.0001) fibroblasts, while it increased in disomic (p<0.0001) and trisomic (p=0.02) fibroblasts with the inhibitor. The expression of BCL2 in disomic fibroblasts decreased after transfection with miR-942-5p mimic (p=0.0016) and increased with the inhibitor (p=0.01). In trisomic fibroblasts, BCL2 expression decreased with the mimic (p<0.0001) and inhibitor (p<0.0001). For miR-378a-3p, BCL2 expression in disomic fibroblasts transfected with the mimic did not show significant difference (p=0.09), but increased with the inhibitor (p<0.0001). In trisomic fibroblasts, BCL2 expression decreased with mimic (p<0.0001) and inhibitor (p=0.0001). Western Blotting analysis did not show significant differences in the protein expression of IKBKB and BCL2 between transfected fibroblasts and negative controls. Bioinformatics analysis revealed that BCL2 expression increased during the prenatal period (logFC=1,16) and in the age ranges of 13-22 years (logFC=0,71) and 30-39 years (logFC=0,93). Increased expression was also observed for BBC3 during the prenatal period (logFC=0,65) and between 40-42 years (logFC=0,67) and for CASP7 between 13-22 years (logFC=0,66) and 40-42 years (logFC=0,77). BAX expression decreased between 30-39 years (logFC=-1,10). In the ventrolateral frontal cortex of DS, increased expression of BCL2 (logFC=0,74) was observed. STRING-DB analysis showed a highconfidence (0,700) and significant (p=2,2e-06) interaction network between BCL2, BAX, BBC3, and CASP7, indicating their importance in regulating apoptosis in DS. Conclusion: It is suggested that miR-942-5p regulates IKBKB independently of trisomy 21 and regulates BCL2 only in disomic fibroblasts. The findings from the others transfection assays are not consistent with the expected function of the miRNAs. The discrepancy between mRNA and protein levels of IKBKB and BCL2 indicates the complexity of post-transcriptional regulation. In brain samples, the differential expression of the genes BCL2, BBC3, CASP7, and BAX in DS across different age groups suggests an imbalance in apoptotic pathway regulation in DS. The increased expression of BCL2 in the ventrolateral frontal cortex suggests a potential importance of this gene expression in specific brain areas of DS individuals, possibly contributing to cellular survival and protection against neurodegeneration.