Search

Long non-coding RNAs (lncRNAs) have been proven to be critical gene regulators of development and disease. The main aim of the present study was to elucidate the lncRNA-mRNA regulation network in ischemic stroke induced by middle cerebral artery occlusion (MCAO) using RNA sequencing (RNA-seq) in rats. lncRNA expression profiles were screened in brain tissues to identify a number of differentially expressed lncRNAs (DELs) and genes (DEGs) by RNA-seq. Reverse transcription-quantitative polymerase chain reaction was performed to further confirm the lncRNA expression data. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to mine mRNA functions, and a lncRNA-mRNA network was constructed. Additionally, cis- and trans-regulatory gene analyses of DELs were predicted. A total of 134 DELs (fold change >2, false discovery rate <0.05) and 1,006 DEGs (fold change >2 and P<0.05) were identified. Eighteen lncRNAs were predicted to regulate heme oxygenase 1, mitotic checkpoint serine/threonine kinase B, chemokine ligand 2 and DNA Topoisomerase IIα, amongst other genes. These genes are all associated with a cellular response to inorganic substances, alkaloids, estradiol, reactive oxygen species, metal ions, oxidative stress, and are associated with metabolic pathways, chemokine signaling pathways, malaria, Parkinson's disease, the cell cycle and other GO and KEGG pathway enrichments. The present study identifies novel DELs and an lncRNA-mRNA regulatory network that may allow for an improved understanding of the molecular mechanism of ischemic stroke induced by MCAO. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 813716. Background RNA modifications affect gene expression through the regulation of RNA metabolism. N6-methyladenosine (m6A) is the most abundant post-transcriptional modification that occurs in RNAs. Its dynamic expression is regulated by the "writer complex" (methyltransferases) and "erasers" (demethylases) and affects numerous biological functions, including mammalian embryonic stem cell (ESC) fate specification. However, the role of m6A in human cardiomyocyte (CM) lineage specification remains unclear. Purpose In this study, we aim to investigate the impact of m6A modification on early human cardiomyocyte differentiation, following the dynamic expression of m6A modification of human induced pluripotent stem cells (hiPSC) into cardiomyocytes (hiPSC-CMs). Methods hiPSCs were differentiated into hiPSC-CMs by mesodermal induction, followed by inhibition of WNT-signaling.  We collected hiPSC derivates at different stages of the differentiation protocol: hiPSCs, hiPSC-derived cardiac mesoderm cells, hiPSC-derived cardiomyocyte progenitors (hiPSC-CPCs), and mature hiPSC-CMs. Protein levels of m6A key regulators were analyzed. To systematically profile the expression of m6A modification, we subjected hiPSC derivates to m6A immunoprecipitation combined with deep sequencing (MeRIP-seq) and RNA-seq. Enrichment analyses of gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were conducted to elucidate the biological significance of differentially expressed and methylated genes. Results m6A distribution analysis on hiPSC derivates revealed a conserved pattern on a transcriptome-wide level: methylation sites are mainly located nearby the stop coding regions. However, we observed upregulated levels of writer proteins during the transition of hiPSC-derived cardiac mesoderm cells into hiPSC-CPCs. Interestingly, the dynamic changes in writer protein levels toward hiPSC-CPC transition were accompanied by a higher number of significantly upregulated and hyper-methylated mRNA transcripts. GO and KEGG analyses indicated hyper-methylated upregulated transcripts are enriched in muscle cell differentiation, cardiac physiology and calcium and MAPK signaling pathways regulating heart contraction. Conclusion For the first time, our study provides evidence that m6A modification is a mediator of early human cardiomyocyte differentiation. The role of specific writer regulators and individual m6A transcripts will be further investigated. [ABSTRACT FROM AUTHOR]