mRNA and lncRNA profiling of hypoxic human pulmonary artery endothelial cells treated with siRNA-mediated knockdown of KMT2E-AS1 or KMT2E
Purpose: In lung tissue of mice with experimental PH, we identified an upregulated lncRNA 5031425E22 which mapped to a human ortholog KMT2E-AS1. Across mammals, this lncRNA gene sits adjacent (head-to-head) to the chromosomal location of the histone lysine N-methyltransferase 2E gene (KMT2E), a member of a family of regulators controlling histone 3 lysine 4 trimethylation (H3K4Me3) and chromatin remodeling. We found that both mouse lncRNA E22 and human KMT2E-AS1 were increased in multiple in vivo rodent and human instances of PH. This study was designed to compare the RNA profile of human pulmonary arterial endothelial cells (HPAECs) treated with siRNA targeting KMT2E-AS1, KMT2E and negative control respectively. Method: Primary HPAECs were grown in EBM-2 basal medium supplemented with EGM-2 MV BulletKit (Lonza). Experiments were performed at passages 5 to 8. Cultured HPAECs were transfected with siRNAs targeting either negative control, KMT2E-AS1 or KMT2E, then exposed to hypoxic stress (0.2% oxygen) for 24 hours. Cells were harvested for total RNA extraction, and total RNA samples received ribosomal depletion and directional (stranded) RNA library prep. mRNA and lncRNA profiles were generated by deep sequencing, using NextSeq P3 flow cell (2 x 101bp, 40M reads/sample). RNA-Seq reads were mapped against the human genome build hg19 using STAR aligner and the read counts for each gene were calculated using featureCounts. Gene expression was normalized by DESeq2 with variance-stabilizing transformation (VST) and the low expression genes with total counts across all samples less than 10 were excluded. The differential gene expression analysis was performed using DESeq2 with the adjusted p-value cutoff of 0.05. The Benjamini-Hochberg method was used for multiple test correction. GSEA was conducted on differentially expressed genes with the enrichR R package [4] using the "GO_Biological_Process_2021", "Reactome_2022", "KEGG_2021_Human" databases. Overall design: Hypoxic HPAEC RNA profile with siRNA knockdown of either KMT2E-AS1 or KMT2E