Mir-150-PTPMT1-Cardiolipin signaling in Pulmonary Arterial Hypertension
Pulmonary arterial hypertension (PAH) is a complex pulmonary vascular disease characterised by excessive vascular endothelial and smooth muscle cell proliferation, inflammation, and fibrosis, resulting in increased pulmonary vascular resistance and right ventricular hypertrophy. Circulating levels of endothelial miR-150 are reduced in PAH and act as an independent predictor of patient survival. Mechanistic links between endothelial miR-150 levels and vascular dysfunction in PAH are not well understood.In order to identify potential mediators of miR-150-induced effects, Human Pulmonary Artery Endothelial Cells (HPAECs) were transfected with control miRNA (non-targeting transfection control; Ambion Life Technologies, 4464076) or miR-150 mimic (Ambion Life Technologies, 4464066; Assay ID MC10070) at 20 nmol/L with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778150). RNA was extracted from trypsinised HPAECs using Monarch Total RNA Miniprep Kit (New England BioLabs), according to manufacturer's instructions. Next-generation RNA-sequencing of transfected HPAECs was performed in triplicate at the Imperial BRC Genomics Facility (Imperial College of London, UK). RNA libraries were prepared using TruSeq Stranded mRNA HT Sample Prep Kit (Illumina Inc.) according to the manufacturer's protocol. Briefly, 1 ug of high-quality total RNA (RNA Integrity Number Score >8.0) was used for polyadenylated RNA selection using poly-T oligo attached magnetic beads, followed by the fragmentation of poly-A containing mRNA. Cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase with random primers. The cDNA was further converted into double-stranded DNA that was end-repaired to incorporate the specific index adapters for multiplexing, followed by purification and amplification. The amplified libraries were examined using an Agilent 2100 Bioanalyzer and a Qubit. The samples for this study were pooled alongside 12 additional samples from other studies and together the 18 samples were sequenced across 4 lanes (2 x 100 bp) on a HiSeq 2500 using TruSeq SBS V3-HS kit (Illumina Inc.) in high output run mode (average of 34.4 million reads per sample).