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Enhanced CLIP-seq analysis of PTBP1 and IGF2BP2 binding targets

ABSTRACT: To investigate the binding targets of PTBP1 (PTB) and IGF2BP2 (IMP2) in glioma cells, we carried out an unbiased analysis of transcripts bound by PTBP1 and IGF2BP2 using enhanced crosslinking immunoprecipitation followed by high-throughput sequencing (eCLIP) in A172 glioma cells. We repeated the eCLIP method twice in each experiment, and found that the ratio of PTBP1 and IGF2BP2 binding mRNAs overlap was very high.

ORGANISM(S): Homo sapiens

PROVIDER: GSE192792 | GEO | 2022/01/02

REPOSITORIES: GEO

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Project description:High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. Tumor cells release extracellular vesicles (EVs) that can polarize macrophages towards tumor-associated macrophages (TAMs), which play a role in tumor progression. However, there is a lack of understanding of IMP2’s effect on TAMs. EVs were isolated from colorectal cancer HCT116 parental (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells via ultracentrifugation and tangential flow filtration. EVs were characterized according to MISEV guidelines and microRNA cargo was assessed by microRNA-Seq. Primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype by EVs and the expression of genes and surface markers was assessed by qPCR and flow cytometry, respectively. Morphological changes of macrophages as well as the migratory potential of cancer cells were assessed by the IncuCyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified in live cells in a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish embryos, and macrophages were isolated by fluorescence-activated cell sorting. EV samples from both WT and KO EVs had a similar size and concentration and were positive or CD9, CD81, CD63, and ALIX. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs, while the expression of tumor-suppressing TNF and IL6 was reduced. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. They exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate. microRNA-seq revealed a significant difference in the microRNA composition of WT and KO EVs. Macrophages transfected with miR-181a-5p mimic showed a lower level of the phosphatase DUSP6. Zebrafish macrophages upon polarization with WT EVs showed lower expression of IL6 and TNF. Our results show that IMP2 can determine the cargo of EVs released by cancer cells, thereby modulating the EVs’ actions on macrophages. Expression of IMP2 is responsible for the secretion of EVs that polarize macrophages toward a tumor-promoting phenotype.

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Enhanced CLIP-seq analysis of PTBP1 and IGF2BP2 binding targets

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