Integrated machine learning-driven disulfidptosis profiling: CYFIP1 and EMILIN1 as therapeutic nodes in neuroblastoma.

BACKGROUND: Neuroblastoma (NB), a prevalent pediatric solid tumor, presents formidable challenges due to its high malignancy and intricate pathogenesis. The role of disulfidptosis, a novel form of programmed cell death, remains poorly understood in the context of NB. METHODS: Gaussian mixture model (GMM)-identified disulfidptosis-related molecular subtypes in NB, differential gene analysis, survival analysis, and gene set variation analysis were conducted subsequently. Weighted gene co-expression network analysis (WGCNA) selected modular genes most relevant to the disulfidptosis core pathways. Integration of machine learning approaches revealed the combination of the Least absolute shrinkage and selection operator (LASSO) and Random Survival Forest (RSF) provided optimal dimensionality reduction of the modular genes. The resulting model was validated, and a nomogram assessed disulfidptosis characteristics in NB. Core genes were filtered and subjected to tumor phenotype and disulfidptosis-related experiments. RESULTS: GMM clustering revealed three distinct subtypes with diverse prognoses, showing significant variations in glucose metabolism, cytoskeletal structure, and tumor-related pathways. WGCNA highlighted the red module of genes highly correlated with disulfide isomerase activity, cytoskeleton formation, and glucose metabolism. The LASSO and RSF combination yielded the most accurate and stable prognostic model, with a significantly worse prognosis for high-scoring patients. Cytological experiments targeting core genes (CYFIP1, EMILIN1) revealed decreased cell proliferation, migration, invasion abilities, and evident cytoskeletal deformation upon core gene knockdown. CONCLUSIONS: This study showcases the utility of disulfidptosis-related gene scores for predicting prognosis and molecular subtypes of NB. The identified core genes, CYFIP1 and EMILIN1, hold promise as potential therapeutic targets and diagnostic markers for NB.

miR-375-3p targets YWHAB to attenuate intestine injury in neonatal necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) is a significant contributor to neonatal mortality. This study aimed to investigate the role of high levels of miR-375-3p in breast milk in the development of NEC and elucidate its mechanism. METHODS: Differential expression of miR-375-3p in the intestines of breast-fed and formula-fed mice was confirmed using real-time polymerase chain reaction (RT-PCR). NEC mice models were established, and intestinal injury was assessed using HE staining. RT-PCR and Western blot were conducted to examine the expression of miR-375-3p, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein ß (YWHAB), as well as the inflammatory in IEC-6 cells, and intestinal tissues obtained from NEC mice and patients. Flow cytometry and cell counting kit-8 (CCK-8) were employed to elucidate the impact of miR-375-3p and YWHAB on cell apoptosis and proliferation. RESULTS: Breastfeeding increases miR-375-3p expression in the intestines. The expression of miR-375-3p in NEC intestinal tissues exhibited a significant decrease compared to the healthy group. Additionally, the expression of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) was higher in the NEC group compared to the control group. Down-regulation of miR-375-3p inhibited IEC-6 cell proliferation, increased apoptosis, and elevated secretion of inflammatory factors. Bioinformatics revealed that YWHAB may be a target of miR-375-3p. RT-PCR and Western blot indicated a down-regulation of YWHAB expression in intestines of NEC patients and mice. Furthermore, YWHAB was found to be positively connected with miR-375-3p. Knockdown miR-375-3p down-regulated YWHAB expression in cells. Inhibition of YWHAB exhibited similar effects to miR-375-3p in IEC-6 cells. YWHAB plasmid partially reverse cellular functional impairment induced by miR-375-3p knockdown. CONCLUSIONS: Breastfeeding elevated miR-375-3p expression in intestines in neonatal mice. MiR-375-3p leads to a decrease in apoptosis of intestinal epithelial cells, an increase in cell proliferation, and a concomitant reduction in the expression of inflammatory factors partly through targeting YWHAB.

Circular RNA MTCL1 targets SMAD3 by sponging miR-145-5p for regulation of cell proliferation and migration in Hirschsprung's disease.

BACKGROUND: Hirschsprung's disease (HSCR) is a congenital disorder resulting from abnormal development of the enteric nervous system (ENS). Given the complexity of its pathogenesis, it is important to investigate the role of epigenetic inheritance in its development. As Circ-MTCL1 is abundant in brain tissue and colon tissue, whether it has a significant part in the development of ENS is worth exploring. This study clarifies its role in HSCR and identifies the specific molecular mechanisms involved. METHODS: Diseased and dilated segment colon tissues diagnosed as HSCR were collected for the assessment of gene expression levels using RT-PCR. EdU and CCK-8 assays were adopted to evaluate cell proliferation, and Transwell assay was adopted to assess cell migration. The interaction between Circ-MTCL1, miR-145-5p and SMAD3 was confirmed by dual luciferase reporter gene analysis, RT-PCR and Western blotting. RESULTS: Circ-MTCL1 was down-regulated in the aganglionic colon tissues. The decreased expression of Circ-MTCL1 associated with a reduction in cell migration and proliferation. Bioinformatics analysis and cellular experiments confirmed its role might have been associated with the inhibition of miR-145-5p. MiR-145-5p was up-regulated in HSCR diseased segment colon tissues, exhibiting a negative correlation with Circ-MTCL1. Overexpression of miR-145-5p reversed the inhibition of cell migration and proliferation associated with Circ-MTCL1 down-regulation. The expression of SMAD3 was inhibited by miR-145-5p. The overexpression of SMAD3 eliminated the miR-145-5p-associated inhibition of cell migration and proliferation. Overexpression of miR-145-5p reversed the inhibitory effects of Circ-MTCL1 down-regulation-associated inhibition of cell migration and proliferation, while suppressing SMAD3 expression. Conversely, overexpression of SMAD3 counteracted the miR-145-5p-associated inhibition of cell migration and proliferation. CONCLUSIONS: Circ-MTCL1 may function as a miR-145-5p sponge, regulating the expression of SMAD3 and influencing cell migration and proliferation, thus participating in the development of HSCR.

Plasma exosomal miR-199a-3p downregulates cell proliferation and migration in Hirschsprung's disease by targeting mTOR.

BACKGROUND: Plasma exosomal microRNAs have been suggested to be potential biomarkers of disease. However, the exosomal microRNAs in Hirschsprung's disease (HSCR) are still unclear. In this study, we analyzed the miRNA profiles of HSCR and elucidated the mechanism of the selected miR-199a-3p in the development of HSCR. METHODS: Plasma exosomes were isolated, and exosomal miRNA high-throughput sequencing was performed to obtain differentially expressed miRNAs. CCK-8 and Transwell assay were used to determine the function of the most differentially expressed miRNA, which was confirmed in tissue specimen. Thereafter, target genes of the selected miRNAs were predicted by the databases. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes Genomes (KEGG) analysis, and protein-protein interaction network (PPI) construction of possible target genes were used to perform enrichment analysis and interaction. Finally, the PCR, Western blot and recovery experiment were used to confirm the function of target gene, mammalian target of rapamycin (mTOR), in vitro. RESULTS: The expression of miR-199a-3p was upregulated in plasma exosomes and diseased colonic tissues of patients with HSCR. In vitro, miR-199a-3p can inhibit cell proliferation and migration. Bioinformatic analysis suggested that mTOR might be a potential target of miR-199a-3p in HSCR. mTOR was discovered to be downregulated by miR-199a-3p in vitro. The negative connection between mTOR and miR-199a-3p was confirmed in tissue samples. mTOR can partially reverse the effect of miR-199a-3p on cell proliferation and migration function in vitro. CONCLUSIONS: miR-199a-3p suppresses cell growth and motility, partially by targeting mTOR. Plasma exosomal miR-199a-3p, a diagnostic marker, is crucial for the development of HSCR.

microRNAs expression profiling analysis in Hirschsprung disease by microarray / 中华实用儿科临床杂志

Objective To investigate the microRNAs(miRNAs)expression profiles in the bowels of patients with Hirschsprung disease(HSCR),and to explore the role of differentially expressed miRNAs in the pathogenesis of HSCR. Methods Twenty - seven HSCR tissues,including spastic segments and distending segments,were obtained from patients with HSCR during operation. Then miRNA microarrays were used to investigate the miRNA expression profiles in 6 HSCR specimens. Bioinformatics software was used to predict target genes of miRNA. Three miRNAs (miR - 145 - 3p,miR - 4505 and miR - 1260a)were chosen and quantificational real - time(qRT)- PCR was per-formed to verify the different expression of those three miRNAs in 27 HSCR tissues. Results The expression of 26 miRNAs in an aganglionic colon segment was found to be more than two fold greater than that in ganglionic segment tis-sues,including 19 up - regulated miRNAs and 7 down - regulated miRNAs in patients with HSCR(all P ﹤ 0. 05). Tar-get genes of miRNAs were found,such as SOX10,RET,L1CAM. qRT - PCR showed the expression of miR - 145 - 3p (1. 42 ± 0. 42,aganglionic segment vs 0. 90 ± 0. 31,ganglionic segment)and miR - 4505(1. 30 ± 0. 30,aganglionic segment vs 0. 76 ± 0. 22,ganglionic segment)displayed a statistical difference between groups(all P ﹤ 0. 001). Be-sides,the expressions of miR - 145 - 3p(1. 53 ± 0. 46,long - segment type vs 1. 16 ± 0. 12,short - segment type)and miR - 4505(1. 42 ± 0. 26,long - segment type vs 1. 00 ± 0. 16,short - segment type)showed a statistical difference be-tween different types(all P ﹤ 0. 001),but miR - 1260a(1. 11 ± 0. 25,aganglionic segment vs 0. 99 ± 0. 21,ganglionic segment)did not show differential expression between different groups(P = 0. 064). Conclusions Abnormal expression of miRNAs was found in HSCR spastic segments,suggesting that miRNAs may be involved in the pathogenesis of HSCR.