Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of SMARCD1 knockdown in U937 leukemic cell line in untreated and vitamin D3 treated condition

ABSTRACT: SMARCD1, an auxiliary subunit of the SWI/SNF complex is essential for maintaining embryonic stem cell pluripotency and metabolic gene regulation. Here, we have tried to address the role of SMARCD1 in myeloid differentiation and leukemia. To address this, we transduced U937 cells with lentiviral particles expressing shcontrol and shSMARCD1. Next we subjected these cells to 30 nM vitamin D3 induction for 48 h. RNA-sequencing was conducted for shcontrol and shSMARCD1 in uninduced and vitamin D3 induced U937 cells. High quality RNA with RIN values above 9, was used to generate library using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) following manufacturer’s instructions. Finally barcoded libraries were pooled, and a final concentration of 1.2 pM was loaded using NextSeq 500/550 High Output Kit v2 150 Cycles (15057931, Illumia) and paired-end sequenced in a Nextseq 550 (Illumina).

INSTRUMENT(S): NextSeq 550

ORGANISM(S): Homo sapiens

SUBMITTER: Dr Punit Prasad

PROVIDER: E-MTAB-10983 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: In addition to its canonical functions, vitamin D has been proposed to be an important mediator of the immune system. Despite ample sunshine, vitamin D deficiency is prevalent (> 80%) in the Middle East, resulting in a high rate of supplementation. However, the underlying molecular mechanisms of the specific regimen and potential factors affecting an individual’s response to vitamin D are not well characterized. Objective: To characterize changes in blood transcriptomic and the potential mechanisms associated with vitamin D3 supplementation and response. Design: In this intervention study, one hundred vitamin D-deficient women were given a weekly oral dose (50,000 IU) of vitamin D3 for three months. A high-throughput targeted PCR, composed of 264 genes representing important blood transcriptomic fingerprints in health and disease states, was performed on pre- and post-supplementation blood samples to profile the molecular response to vitamin D3. Multivariate, network, gene ontology, and literature mining analyses were used for the interpretation of the transcriptomic profiling results. Results: We identified 54 differentially expressed genes that were strongly modulated by vitamin D3 supplementation. Network analyses showed significant changes in the immune-related pathways such as TLR4/CD14 and IFN receptors, and catabolic processes related to NFkB, which were subsequently confirmed by gene ontology enrichment analyses. We proposed a model for vitamin D3 response, using the reduced expression of the molecules involved and the receptor-mediated intra-cellular signaling leading to reduce cytokine production. Conclusions: Blood-transcriptomic profiles of vitamin D3 response were generated using a targeted blood gene panel. Vitamin D has a strong effect on the immune system, G-coupled protein receptor signaling, and the ubiquitin system. We highlighted the major molecular changes and biological processes induced by vitamin D3, which will help to further investigate the effectiveness of vitamin D supplementation among individuals in the Middle East.

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RNA-seq of SMARCD1 knockdown in U937 leukemic cell line in untreated and vitamin D3 treated condition

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