RNA sequencing of (1) isolated retinal endothelial cells from wildtype and Zmiz1-KO endothelial cells and (2) control and Zmiz1 shRNA treated MS1 cells

Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 association with vascular system, its role in physiological and pathological angiogenesis is significantly unknown. Here, we use (1) MS1 cell line treated with control shRNA and Zmiz1 shRNA and (2) murine model to knockout Zmiz1 in endothelial cells using Cdh5CreERT2 and isolate retinal endothelial cells (iRECs) then performed RNA sequencing to profile transcriptional changes upon Zmiz1 deletion. Retinal ECs were isolated from P7 pups as previously described (Patel et al., 2022. JCI Insight). Briefly, isolation of ECs from the retinas was performed using the Miltenyi Neural tissue Dissociation Kit (P) (Miltenyi, 130-092-628). For each sample, 8-10 retinas were pooled and digested to obtain a single cell suspension. CD31+ cells were separated from the cell mixture via magnetic activated cell sorting. MS1 cells were transfected with 20μM of either control-shRNA or Zmiz1-shRNA using Lipofectamine3000 (Thermofisher, L3000015). Next, RNA was isolated from the cells and used for downstream RNA-sequencing. RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced on a Nextseq 500/550 platform. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub. Results: For iRECs , we found 2,364 differentially expressed genes of which 834 genes were upregulated while 1530 genes were downregulated. Downregulated genes were enriched in biological processes such as angiogenesis, vascular development, and cell growth. For MS1 cells, we found 1,711 differentially expressed genes of which 718 genes were upregulated while 993 genes were downregulated. Downregulated genes were enriched in biological processes such as regulation of angiogenesis. Conclusions: We assessed changes in transcriptional landscape in MS1 and iRECs following Zmiz1 deletion.