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Next Generation Sequencing Facilitates Quantitative Analysis of HGF treated (HGF+) and matched control (HGF-) HepG2 cells

ABSTRACT: Next Generation Sequencing Facilitates Quantitative Analysis of HGF treated (HGF+) and matched control (HGF-) HepG2 cells

PROVIDER: PRJNA551437 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9608240_1.fastq.gz	Fastqsanger.gz
	SRR9608240_2.fastq.gz	Fastqsanger.gz
	SRR9608237_1.fastq.gz	Fastqsanger.gz
	SRR9608237_2.fastq.gz	Fastqsanger.gz
	SRR9608238_1.fastq.gz	Fastqsanger.gz

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Next Generation Sequencing Facilitates Quantitative Analysis of HGF treated (HGF+) and matched control (HGF-) HepG2 cells

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Project description:The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristics that permit infection. We developed an novel analytical approach to test this hypothesis viz. the HC-04 response to hepatocyte growth factor (HGF). We used online two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) to quantify protein expression profiles in HC-04 pre-/post-HGF treatment and validated these results by RT-qPCR and microscopy. We successfully increased protein identification efficiency over offline-2D methods by 12-fold using less sample material, allowing robust protein quantification. We observed expected up-regulation and down-regulation of EMT protein markers in response to HGF, but also unexpected cellular responses. HC-04 is thus susceptible to HGF-mediated signaling but to a lesser degree than what we observed for HepG2, a widely used, but poor malaria HCC model. Our analytical approach to understanding the basic biology of HC-04 helps us understand the factors that may influence its utility as a model for malaria liver-stage development. We observed that HC-04 treatment with HGF prior to the addition of Plasmodium falciparum sporozoites did not facilitate cell invasion, arguing for unlinking the effect of HGF on malaria liver stage development from hepatocyte invasion. Finally, our 2D-LC-MS/MS approach and broadly applicable experimental strategy should prove useful in the analysis of various hepatocyte-pathogen interactions, tumor progression and early disease events.

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