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Integrating CRISPR knockout, interference, and activation to dissect the functional role and underlying signaling of microRNA-23a-27a-24-2 cluster in hepatocellular carcinoma

ABSTRACT: Integrating CRISPR knockout, interference, and activation to dissect the functional role and underlying signaling of microRNA-23a-27a-24-2 cluster in hepatocellular carcinoma

PROVIDER: PRJNA819411 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18463655.fastq.gz	Fastqsanger.gz
	SRR18463656.fastq.gz	Fastqsanger.gz
	SRR18463657.fastq.gz	Fastqsanger.gz
	SRR18463658.fastq.gz	Fastqsanger.gz
	SRR18463659.fastq.gz	Fastqsanger.gz

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Integrating CRISPR knockout, interference, and activation to dissect the functional role and underlying signaling of microRNA-23a-27a-24-2 cluster in hepatocellular carcinoma

Project description:The miR-23a-27a-24-2 cluster is overexpressed in human cancers, including hepatocellular carcinoma (HCC). However, the role of three mature miRNAs of the miR-23a-27a-24-2 cluster in HCC is still controversial. Also, the integrative role of the miR-23a-27a-24-2 cluster in HCC remains elusive. In the present study, using CRISPR knockout (KO), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) technologies, we established an endogenous miR-23a-27a-24-2 controllable and various knockout (KO) cell models, which were used to dissect the functional role and underlying signaling of the miR-23a-27a-24-2 cluster. Either miR-23a KO or miR-27a KO reduced cell growth in vitro and in vivo. Of particular note, endogenous miRNAs in the miR-23a-27a-24-2 cluster were effectively regulated by CRISPRi/a, identifying an integrated oncogenic role of the miR-23a-27a-24-2 cluster in HCC cells. Functional analysis showed that miR-23a KO and miR-27a KO led the cell cycle arrest, especially at the G2/M phase, by reducing CDK1/cyclin B activation in HCC cells. Furthermore, a high-throughput RNA-seq approach with miRNA target prediction identified the miR-23a/miR-27a-regulated gene network, further validated by various technologies. In addition, miR-23a and miR-27a have opposite roles in cell migration and mesenchymal-epithelial transition. However, an integrated analysis by CRISPRi/a suggested an oncogenic role of the miR-23a-27a-24-2 cluster in cell migration, which may require a miR-23a-BMPR2-Smad-Snail axis in HCC cells. Thus, our CRISPRi/a study offers a valuable research tool for dissecting the integrated role and underlying mechanism of an endogenous miRNA cluster. Also, this CRISPR approach may provide new routes of miRNA intervention for miRNA targeted therapy.

2024-09-30 | GSE199332 | GEO

Differential expression of genes after over expression of miR-23a~27a~24-2 cluster in human embryonic kidney cells

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Project description:The experiment was deigned to identify the genes which get altered after the over expression of miR-23a~27a~24-2 cluster in HEK293T cells. The HEK293T cells plated in 6-well plate were transfected with two different concentrations of cloned miRNA cluster i.e 2micrograms and 4micrograms per well. Biological duplicates of 3 samples were used viz. control HEK293T cells, HEK293T cells transfected with 2micrograms of the cloned cluster and HEK293T cells transfected with 4micrograms of the cloned cluster.

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2024-06-30 | GSE217629 | GEO

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Project description:Expression of the mirn23a cluster in murine hematopoietic progenitors blocks B cell development. Overexpressed individual cluster members miRs-23a, 24-2, and -27a in 70/3 mouse pre-B cells in order to examine changes in gene expression that would potentially reveal targets of the miRs relevant to hematopoiesis. Two independent 70Z/3 cell lines for each experimental group were generated (MSCV, miR-23a, miR-24-2, and miR-27a). RNA was generated and hybridized on Affymetrix microarrays.

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Project description:Gene expression profiling was carried out in Huh-7.5 cells in which miR-27a was over- or under-expressed. Transfection of cells with pre-miR-27a and pre-miR-control, or anti-miR-27a and anti-miR-control enabled down- and up-regulated genes to be determined, respectively. Replication and infectivity of the lipotrophic hepatitis C virus (HCV) is regulated by cellular lipid status. Among differentially expressed micro (mi)RNAs, we found that miR-27a was preferentially expressed in HCV-infected compared with hepatitis B virus (HBV)-infected liver. Gene expression profiling of Huh-7.5 cells showed that miR-27a regulates lipid metabolism by targeting the lipid synthetic transcriptional factor, RXRα, and the lipid transporter, ABCA1 Carrying out a Target Scan (Release 5.2) of miR-27a predicted 921 candidate target genes, and functional gene ontology enrichment analysis of these genes by MetaCore (Thomson Reuters, NY) showed that miR-27a could target the signaling pathways of cytoskeleton remodeling and lipid metabolism . To examine whether these signaling pathways were regulated by miR-27a, gene expression profiling was carried out in Huh-7.5 cells in which miR-27a was over- or under-expressed. Transfection of cells with pre-miR-27a and pre-miR-control, or anti-miR-27a and anti-miR-control enabled down- and up-regulated genes to be determined, respectively. Huh-7.5 cells with miR-27a over- or under-expressed

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