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Expression of profilin 2 isoforms was associated with lung metastasis of colorectal cancer as revealed by a comprehensive gene expression study using mice model

ABSTRACT: Expression of profilin 2 isoforms was associated with lung metastasis of colorectal cancer as revealed by a comprehensive gene expression study using mice model

PROVIDER: PRJNA1061500 | ENA |

REPOSITORIES: ENA

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Project description:Transcriptome analysis via high-throughput sequencing (RNA-Seq) captures significant changes in gene expression. The purpose of this study is to elucidate the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type control (LdPfn+/+). This research will enable us to gain insight into the role of profilin in Leishmania cells. The data has been validated by qRT-PCR. Data analysis results are further supported by flow cytometry, immunofluorescence experiments and by western blot demonstrating a depletion in the protein expression of a eukaryotic translation initiation factor in the cells with depleted levels of profilin protein. Methods: Total RNA from three independent biological replicates from each of wild-type (LdPfn+/+) and profilin knockout (LdPfn+/-) Leishmania promastigotes was extracted and after library preparation, RNA sequencing was carried out on Illumina HiSeq 2500. The sequence reads that passed quality filters were aligned to the Leishmania donovani (BPK282A1) genomic data obtained from TriTrypDB version 51 using Bowtie2 (-x option). All analyses were carried out using the Tophat pipeline with the following versions: Tophat v2.1.1, Bowtie2 v2.3.5.1. The HTSeq version 0.12.4 (htseq-count -f option) was used to count the number of reads aligned to protein-coding genes. DeSeq tool was used for differential gene expression analysis between samples in protein-coding genes. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq datasets generated in this study with each sample control (LdPfn+/+) and profilin knockout (LdPfn+/-) have at least 30 million read pairs. RNA-seq data were aligned to the L.donovani genome (Leishmania donovani BPK282A1, NCBI taxon ID: 981087) and 8135 transcripts were identified in our data set. Data analysis revealed that a total of 254 genes were differentially expressed (DE) having a p-value <0.05. The data obtained from RNA-Seq have been validated by real-time PCR of 18 genes, 8 UP regulated and 10 down-regulated. Results of qRT-PCR analysis showed a strong correlation with the RNA-seq data. Inferences drawn from transcriptomic analysis were further validated by flow cytometry, immunofluorescence experiments and western blotting, revealing a critical role during the early phases of Leishmania cell division. Conclusions: Present study represents a comprehensive report elucidating the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type (LdPfn+/+). This RNA-Seq data along with validation by qPCR, flow cytometry experiments, and immunofluorescent microscopy experiments indicate that profilin might play a critical role during the early phases of Leishmania cell division.

Project description:Calcium oxalate raphide crystals are found in bundles in intravacuolar membrane chambers of specialized idioblasts cells of most plant families including many crop plants and are found in most tissues. Raphides are needle-shaped, often with barbed ends, and their morphology and biomineralization pattern have been a focus of much research. In the family Araceae, raphides have been proposed to cause acridity in crops such as taro (Colocasia esculenta (L.) Schott). Acridity is irritation that causes swelling of lips, mouth and throat, as well as itchiness and pain when raw or insufficiently cooked tissues are eaten; this response varies among taro consumers. Since raphides in some other foods do not cause acridity, and since acridity can be inactivated by cooking and/or protease treatment, it is possible that a toxin or allergen-like compound is associated with the crystals. Using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) peptide sequencing of selected peptides from purified raphides and the results from taro apex transcriptome sequencing, we have shown that profilins, known allergens, are present on raphides. One of the five raphide profilins genes was highly expressed in the apex and had a 17-amino-acid insert that significantly increased that profilin’s epitope peak. This insert was predicted to be coiled and exposed on the surface of the folded peptide. A second profilin had a 2-amino-acid insert that also had a greater B-cell epitope prediction. The taro profilins showed 83 to 92% similarity to known characterized profilins while other raphide peptides showed greater than 90% similarity to other higher plants. The presence on the raphides of peptides normally associated with mitochrondria (ATP synthase), chloroplasts (chaperonin cpn 60 kDa), cytoplasm (actin, profilin) and vacuole (V-type ATPase) indicates a multistage biocrystallation process ending with possible invagination of the tonoplast and addition of mucilage that may be derived from the Golgi. Actin might play a crucial role in the generation of the needle-like raphides. We also showed that commercial allergen test strips for hazelnuts, where profilin is a secondary allergen, have potential for screening in a plant breeding program to reduce acridity and during food processing to avoid over-cooking.

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Expression of profilin 2 isoforms was associated with lung metastasis of colorectal cancer as revealed by a comprehensive gene expression study using mice model

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