Dataset Information

Dietary genistein supplementation alters mRNA expression profile and alternative splicing signature in the thymus of chicks with lipopolysaccharide challenge.

ABSTRACT: Genistein is abundant in the soybean products, which exerts prominent effects on immune function. Little information is available about the effect of dietary genistein on thymic transcriptome, especially when suffering from lipopolysaccharide challenge. In this study, 180 one-day-old male broilers were randomly allocated to 3 groups: nonchallenged chicks given a basal diet (CON), and lipopolysaccharide-challenged chicks fed a basal diet (LPS), or lipopolysaccharide-challenged chicks fed a basal diet supplemented with 40 mg/kg genistein (GEN). Lipopolysaccharide injection induced thymocyte apoptosis and inflammatory reactions in the chicks. The results showed dietary genistein significantly reduced the percentage of CD3+ T lymphocytes by 10.04% and CD4+/CD8+ T lymphocyte ratio by 21.88% in the peripheral blood induced by lipopolysaccharide injection (P < 0.05). In addition, genistein significantly reduced the thymus index by 50% and apoptotic index by 12.34% induced by LPS challenge (P < 0.05). Transcriptomic analysis identified 1,926 DEGs (1,014 upregulated and 912 downregulated, P < 0.05) between GEN and LPS groups, which altered the mRNA expression profile and signaling pathways (Toll-like receptor, and NOD-like receptor signaling pathway) in the thymus. Furthermore, 5 splicing (AS) isoforms of the Drosophila Disabled-2 (DAB2) gene were detected, which were significantly upregulated in the GEN group compared with that in the LPS group. In summary, dietary genistein supplementation altered the RNA expression profile and AS signatures in the thymus, and alleviated immune response against lipopolysaccharide challenge.

SUBMITTER: Huang Z

PROVIDER: S-EPMC8666715 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of biological process. The goals of this study are to characterize the effects of genistein on the thymic transcriptome of chicks, when suffering from lipopolysaccharide challenge using RNA-Seq technology Methods: Thymic mRNA profiles of 21-day-old chicks with basic diet and lipopolysaccharide-treated, or lipopolysaccharide+genistein treated chick mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 70 million sequence reads per sample to the chicken genome and identified 25,016 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 6 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. We identified 712 DEGs (367 upregulated and 345 downregulated) between the LPS vs CON group with a |fold change| ＞1 (P ≤0.05, Figure 4 a). There are 1926 DEGs (1014 upregulated and 912 downregulated) in the GEN group compared with the LPS groupAltered expression of 6 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Conclusions: Our research has evidenced a novel finding in chicks that dietary genistein could inhibit the increased percentage of CD3+ T lymphocytes, and CD4+/CD8+ T lymphocyte ratio in the peripheral blood induced by lipopolysaccharide injection. Furthermore, dietary genistein exposure altered the mRNA expression profile and AS signatures in the thymus, and regulated the immune response, along with the improvement of thymus index and apoptotic index after lipopolysaccharide challenge. It would help to understand the role of dietary genistein to the regulation of immune function and provide theoretical support for improving healthy production of poultry.

Project description:BackgroundAlternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.MethodsWe recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.ResultsMass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.ConclusionProteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

Project description:BackgroundIncreasing evidence indicated a close relationship between aberrant splicing variants and carcinoma, whereas comprehensive analysis of prognostic alternative splicing (AS) profiling in breast cancer (BRCA) is lacking and largely unknown.MethodsRNA-seq data and corresponding clinical information of BRCA patients were obtained and integrated from The Cancer Genome Atlas (TCGA). Then SpliceSeq software was used to assess seven AS types and calculate the Percent Spliced In (PSI) value. Univariate followed by stepwise multivariate Cox regression analyses identified survival associated AS events and constructed the AS signature, which were further sent for enrichment analysis, respectively. Besides, the splicing correlation network was constructed. Additionally, nomogram incorporating AS signature and clinicopathological characteristics was developed and its efficacy was evaluated with respect to discrimination, calibration and clinical utility.ResultsA total of 45,421 AS events were detected, among which 3071 events were found associated with overall survival (OS) after strict filtering. Parent genes of these prognostic events were involved in BRCA-related processes including NF-kappaB and HIF-1 signaling pathway. Besides, the final prognostic signature built with 20 AS events performed well with an area under the curve (AUC) of receiver operating characteristic (ROC) curve up to 0.957 for 5 years. And gene set enrichment analysis (GSEA) also confirmed the candidate 20 AS events contributed to progression of BRCA. Moreover, the nomogram that incorporated 20-AS-event-based classifier, age, pathological stage and Her-2 status showed good calibration and moderate discrimination, with C-index of 0.883 (95% CI, 0.844-0.921). Decision curve analysis (DCA) confirmed more benefit was added to survival prediction with our nomogram, especially in 5 or 8 years with threshold probability up to 80%. Finally, splicing correlation network revealed an obvious regulatory pattern of prognostic splicing factors (SF) in BRCA.ConclusionThis study provided a systematic portrait of survival-associated AS events involved in BRCA and further presented a AS-clinicopathological nomogram, which could be conveniently used to assist the individualized prediction of long-term survival probability for BRCA patients. And a series of bioinformatic analysis provided a promising perspective for further uncovering the underlying mechanisms of AS events and validating therapeutic targets for BRCA.

Dataset Information

Dietary genistein supplementation alters mRNA expression profile and alternative splicing signature in the thymus of chicks with lipopolysaccharide challenge.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets