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Title: RNA-seq analysis of b0-HUDEP2 and HUDEP2 cells following erythroid differentiation

ABSTRACT: Purpose: The goal of this study is to compare the altered transcriptional program (RNA-seq) and cellular adaptation to stress caused by excess a-globin synthesis. Results: 100bp paired-end polyA-enriched RNA sequencing were performed with a depth of minimum of 20 million read counts per sample. The sequencing library was sequenced on the Illumina HiSeq2500 at Azenta. Raw data then went through read quality assessment by FastQC (version 0.11.5), mapping to the human genome (GRch38) by the STAR aligner and gene-level reads quantification using the Rsubread package. RNA-seq data analysis identified 1870 upregulated and 1549 down regulated genes on Day 0 (FDR <=0.05) and 1924 upregulated and 1960 down regulated genes on Day 8 (FDR <=0.05). MA plot analysis of differentially expressed genes uncovered an altered transcriptional program favouring γ-globin expression not solely dependent upon the HRI ATF4-BCL11A axis. Instead, we noted attenuated expression of several key γ-globin co-repressors, including SOX6, ZBTB7A, KLF1 and GATA1, which coincided with increased expression of co-activators such as CBFB/NY-FB and GATA2 following erythroid differentiation. Gene Set Enrichment Analysis (GSEA) software was used to determine significantly enriched gene sets. Downregulated DEGs in β0 HUDEP 2 cells in pathways related to haeme metabolism, hypoxia, MTORC1, unfolded protein response and TNFA/NFkB signalling were enriched. Meanwhile, upregulated DEGs in β0 HUDEP-2 cells related to G-protein-coupled receptors (GPCR) signalling, IL-2/STAT5 and hypoxia signalling pathway were also retrieved. Pathways shown to be significantly altered in both β0 HUDEP-2 cells and patient group include apoptosis, MAPK signalling and NFKB pathway thus provided complementary insights in transcriptome profiling. Conclusions: Our study represents analysis of HUDEP2 transcriptomes, with biologic replicates, generated by RNA-seq technology. The data analysis reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of the cellular adaptation to intrinsic stress caused by excess a-globin synthesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200504 | GEO | 2024/04/04

REPOSITORIES: GEO

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Project description:MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) crucial for post-transcriptional and translational regulation of cellular and developmental pathways. Study of miRNAs in erythropoiesis thus elucidates the process and facilitates diagnosis and therapy development for related disorders. Here, we used high-throughput DNA Nanoball small RNA sequencing to characterise sncRNAs, and in particular miRNAs, comprehensively in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and on two influential erythroid cell lines with adult and fetal expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential expression (DE) to erythroid differentiation, cell type, and fetal and adult hemoglobin expression patterns. Sequencing results were validated by reverse-transcription quantitative PCR (RT-qPCR) of selected DE miRNAs (miR-451a, miR-182-5p, miR-146a-5p, miR-221-3p, miR-222-3p) and indicated overall common erythroid differentiation signatures in primary cells and cell lines, in terminal stages of erythropoiesis characterized by decreased complexity of global miRNA expression and reciprocally increased expression of individual lineage-specific miRNAs. Interestingly, against a backdrop of evident clustering and thus similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted readily interpretable discrepancies between both cell types. Moreover, differential expression analysis between the closely related HUDEP-2 and HUDEP-1 cells provided pointers to miRNAs, transcription factors (TFs), target genes, and pathways mechanistically linked to globin switching. In resulting TF-miRNA co-regulatory networks, TFs known to be implicated in hemoglobin switching, including the major γ-globin repressors BCL11A and ZBTB7A, were targeted by many co-regulated miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

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Title: RNA-seq analysis of b0-HUDEP2 and HUDEP2 cells following erythroid differentiation

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