Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq of human hematopoietic stem and progenitor cells to study membrane repair following electroporation (EP) or filtroporation (FP) disruption based strategies for intracellular delivery

ABSTRACT: This experiment investigates how different intracellular delivery methods, specifically filtroporation (FP) and electroporation (EP) affect gene editing efficiency and cellular responses in human hematopoietic stem and progenitor cells. By comparing these methods, results show how mechanical versus electrical membrane disruption influences gene expression, cell fate, and membrane repair mechanisms. This study also explores the molecular processes involved in rapid membrane repair, highlighting the roles of calcium signaling and key repair proteins such as GRAF1, SNAP23, and CHMP4B.

INSTRUMENT(S): Qiagen RNeasy Mini Kit, R, Illumina NovaSeq 6000, NEBNext Ultra II Directional RNA Library Prep, Linux Bash Scripting, With L-glutamine supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (10,000 units/mL penicillin and 10 mg/mL streptomycin) (Gibco).

ORGANISM(S): Homo sapiens

SUBMITTER: Isaura Frost

PROVIDER: E-MTAB-15741 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Chronic pulmonary inflammation in the form of chronic obstructive pulmonary disease (COPD) has been consistently shown to increase the risk of lung cancer. Therefore, identification of chemopreventive agents with anti-inflammatory effects, in addition to antiproliferative and apoptotic activities, is indispensable. Recently, we found that combinations of silibinin (Sil) and indole-3-carbinol (I3C) significantly inhibited lung tumorigenesis induced by 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) and enhanced by chronic treatment with the inflammatory agent lipopolysaccharide (LPS). In this study, we described gene expression profiling of lung tissues using RNA-seq to determine the gene expression signature in inflammation-driven lung tumors and modulation of this signature by the chemopreventive agents Sil and I3C. Methods: Total RNA extracted from lung tissues of control and treated mice were processed for mRNA sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed for transcript abundance at the gene level using CLC Bio Genomics Workbench and differential gene expression analysis was performed using the built-in Empirical Analysis of Differential Gene Expression (DGE) based on 'Exact Test' method. qRTâPCR validation was performed using SYBR Green assays. Results and conclusions: We found that 330, 2,957, and 1,143 genes were differentially regulated in mice treated with NNK, LPS, and NNK + LPS, respectively. The expression of inflammation-and immunity-related genes was significantly more deregulated in lung tissues of mice treated with LPS alone compared to mice treated with NNK + LPS. Among 1,143 genes differentially regulated in the NNK + LPS group, the expression of 162 genes and associated signaling pathways were significantly modulated by I3C and/or Sil + I3C. These genes include cytokines, chemokines, and genes with a well-established role in inflammation and/or tumorigenesis such as c-ros oncogene 1 (Ros1), the EGFR ligands amphiregulin and epiregulin, Cyp1a1, and the circadian rhythm genes Arntl, and Npas2. To our knowledge, this is the first report that provides insight into genes that are differentially expressed during inflammation-driven lung tumorigenesis and the modulation of these genes by chemopreventive agents. Lung tissue mRNA profiles of mice treated with control vehicle, NNK, LPS, NNK+LPS, or NNK+LPS supplemented with chemopreventive agent(s) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

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RNA-Seq of human hematopoietic stem and progenitor cells to study membrane repair following electroporation (EP) or filtroporation (FP) disruption based strategies for intracellular delivery

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