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RNA-sequencing data of T-AP1-labeled NCI-N87 xenograft tumor cells sorted according to the fluorescence

ABSTRACT: RNA-sequencing data of T-AP1-labeled NCI-N87 xenograft tumor cells sorted according to the fluorescence

PROVIDER: PRJNA752281 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15348352_1.fastq.gz	Fastqsanger.gz
	SRR15348352_2.fastq.gz	Fastqsanger.gz
	SRR15348354_1.fastq.gz	Fastqsanger.gz
	SRR15348354_2.fastq.gz	Fastqsanger.gz
	SRR15348356_1.fastq.gz	Fastqsanger.gz

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RNA-sequencing data of T-AP1-labeled NCI-N87 xenograft tumor cells sorted according to the fluorescence

Project description:Oxidative stress is recognazed as a hallmark of cancer. However, little is known about intratumor heterogeneity of reactive oxygen species (ROS) and its influence on tumor behavior, largely due to technical challenges inherent in detecting intratumor ROS at single-cell resolution. We have developed a tumor-targeted H2O2 probe (T-AP1) by conjugating trastuzumab with both Alexa Fluor 647 (AF647), an H2O2-insensitive fluorophore, and peroxy green 1 (PG1), an irreversible H2O2-sensitive chemical probe to provide a “ratiometric” readout, allowing H2O2 measurements independent of accumulated T-AP1 levels. Given the ability of T-AP1 to persistently mark tumor cells that experience H2O2 exposure, we employed cell sorting to isolate tumor cells with high (PG1highAF647+) and low (PG1lowAF647+) H2O2 exposure levels from T-AP1–labeled NCI-N87 xenograft tumors. PG1highAF647+ and PG1lowAF647+ cells obtained via cell sorting from three biologically independent N87 tumors were subjected to RNA sequencing.

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Project description:We evaluate the effects of treatment with the product Poliprotect (F) on human NCI-N87 cells in order to measure the overall activity of the product on biological functions of interest We used microarrays to detail the transcriptional response obtained by treating NCI-N87 cells with HBSS pH1 (acid-induced damage), HBSS pH7.4 (control pH) , Poliprotect (F)(apical and basolateral treatment)

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Project description:MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are often dysregulated in disease. The recent development of the CRISPR-Cas9 gene-editing system, composed of the Cas9 nuclease in complex with a single guide RNA (sgRNA), allows researchers to direct DNA cleavage at a predetermined site and to conduct genome-scale knockout screens. To determine the functional role of miRNAs in cancer, we designed and constructed a library of 7,382 sgRNAs to target 85% of the 1,881 annotated human miRNA stem-loops. We then examined the role of miRNAs in HeLa cell fitness by monitoring the change in frequency of each sgRNA over time. We identified 44 pro-proliferative miRNAs from two replicate experiments, including miR-31, a known cervical cancer overexpressing miRNA that enhances HeLa cell proliferation. We also examined the role of miRNAs in NCI-N87 gastric cancer cells and identified 10 pro-fitness and 10 anti-fitness miRNAs. In both screens, many of the pro-fitness miRNAs identified are overexpressed in tumors cervical tumors for HeLa or gastric tumors for NCI-N87. In summary, we present a CRISPR miRNA-targeted screen which was able to identify both known and novel fitness-associated miRNAs in the HeLa and NCI-N87 cell lines.

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