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A combined strategy of global gene expression analysis and multiplex PCR established a 13-member gene signature for the diagnosis of human hepatocellular carcinoma

ABSTRACT: Aberrant gene expression between HCC tumor tissues, histologically normal adjacent liver tissues and peripheral blood mononuclear cell (PBMC) samples from healthy individuals and patients with pancreatic carcinoma, gastric carcinoma and HCC were identified using Affymetrix gene arrays. A 13-member HCC-specific gene signature was identified and validated via custom-designed quantitative multiplexed gene expression analysis using the GenomeLab GeXP Genetic Analysis System (GeXP). Peripheral blood mononuclear cell (PBMC) from healthy individuals, patients with pancreatic carcinoma, gastric carcinoma, and HCC were isolated and total RNA was extracted for Affymetrix gene microarray analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: K. Man Hui

PROVIDER: S-ECPF-GEOD-36076 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundThe oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest.ResultsIn this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized.ConclusionTaken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.

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A combined strategy of global gene expression analysis and multiplex PCR established a 13-member gene signature for the diagnosis of human hepatocellular carcinoma

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