ABSTRACT
Huntington's disease (HD) is caused by an expansion of CAG trinucleotide repeat in the HTT gene; the exact pathogenesis of HD currently remains unclear. One of the promising directions in the study of HDs is to determine the molecular mechanism underlying the development and role of microRNAs (miRNAs). This study aimed to identify the profile of miRNAs in an HD human cell line model as diagnostic biomarkers for HD. To study HD, the human SH-SY5Y HD cell model is based on the expression of two different forms: pEGFP-Q23 and pEGFP-Q74 of HTT. The expression of Htt protein was confirmed using aggregation assays combined with immunofluorescence and Western blotting methods. miRNA levels were measured in SH-SY5Y neuronal cell model samples stably expressing Q23 and Q74 using the extraction-free HTG EdgeSeq protocol. A total of 2083 miRNAs were detected, and 354 (top 18 miRNAs) miRNAs were significantly differentially expressed (DE) (p < 0.05) in Q23 and Q74 cell lines. A majority of the miRNAs were downregulated in the HD cell model. Moreover, we revealed that six DE miRNAs target seven genes (ATN1, GEMIN4, EFNA5, CSMD2, CREBBP, ATXN1, and B3GNT) that play important roles in neurodegenerative disorders and showed significant expression differences in mutant Htt (Q74) when compared to wild-type Htt (Q23) using RT-qPCR (p < 0.05 and 0.01). We demonstrated the most important DE miRNA-mRNA profiles, interaction binding sites, and their related pathways in HD using experimental and bioinformatics methods. This will allow the development of novel diagnostic strategies and provide alternative therapeutic routes for treating HD.
ABSTRACT
Sortilin is an important regulator with potential tumour-suppressor function by limiting EGFR signalling. In this study, we undertook a comprehensive expression analysis of sortilin transcript variants and the DNA methylation status of their corresponding promoters in human non-small cell carcinomas (NSCLCs). RNA/DNA was extracted from 81 NSCLC samples and paired normal tissue. mRNA expression was measured by qPCR and DNA methylation determined by pyrosequencing. BigDye-terminator sequencing was used to confirm exon-8 alternative splicing. Results demonstrated that both SORT1A and SORT1B variants were downregulated in lung tumours. The SORT1A/SORT1B expression ratio was higher in tumours compared to normal tissue. SORT1B promoter hypermethylation was detected in lung tumours compared to normal lung (median difference 14%, Mann-Whitney test p = 10-6). Interestingly, SORT1B is hypermethylated in white blood cells, but a small and very consistent drop in methylation (6%, p = 10-15) was observed in the lung cancer cases compared to control subjects. We demonstrate that the SORT1B exon-8 splice variation, reported in sequence databases, is also a feature of SORT1A. The significantly altered quantitative and qualitative characteristics of sortilin mRNA in NSCLC indicate a significant involvement in tumour pathogenesis and may have significant impact for its utility as a predictive marker in lung cancer management.