A potential pathogenic hypoxia-related gene HK2 in necrotizing enterocolitis (NEC) of newborns.

BACKGROUND: Necrotizing enterocolitis (NEC) is a disastrous gastrointestinal disease of newborns, and the mortality rate of infants with NEC is approximately 20%-30%. The exploration of pathogenic targets of NEC will be conducive to timely diagnosis of NEC. METHODS: The whole transcriptome RNA sequencing was performed on NEC samples to reveal the expression of lncRNAs, circRNAs, miRNAs and mRNAs. Using differential expression analysis, cross analysis, target prediction, enrichment analysis, the pathogenic ceRNA network and target was found. RESULTS: Preliminarily, 281 DEmRNAs, 21 DEmiRNAs, 253 DElncRNAs and 207 DEcircRNAs were identified in NEC samples compared with controls. After target prediction and cross analyses, a key ceRNA regulatory network was built including 2 lncRNAs, 4 circRNAs, 2 miRNAs and 20 mRNAs. These 20 mRNAs were significantly enriched in many carbohydrate metabolism related pathways. After cross analysis of hypoxia-, carbohydrate metabolism-related genes, and 20 core genes, one gene HK2 was finally obtained. Dendritic cells activated were significantly differentially infiltrated and negatively correlated with HK2 expression in NEC samples. CONCLUSIONS: The promising pathogenic hypoxia-related gene HK2 has been firstly identified in NEC, which might also involve in the carbohydrate metabolism in NEC.

The potential regulatory role of the non-coding RNAs in regulating the exogenous estrogen-induced feminization in Takifugu rubripes gonad.

Estrogen plays a pivotal role in the early stage of sex differentiation in teleost. However, the underlying mechanisms of estrogen-induced feminization process are still needed for further clarification. Here, the comparative analysis of whole-transcriptome RNA sequencing was conducted between 17beta-Estradiol induced feminized XY (E-XY) gonads and control gonads (C) in Takifugu rubripes. A total of 57 miRNAs, 65 lncRNAs, and 4 circRNAs were found to be expressed at lower levels in control-XY (C-XY) than that in control-XX (C-XX), and were up-regulated in XY during E2-induced feminization process. The expression levels of 24 miRNAs, and 55 lncRNAs were higher in C-XY than that in C-XX, and were down-regulated in E2-treated XY. Furthermore, a correlation analysis was performed between miRNA-seq and mRNA-seq data. In C-XX/C-XY, 114 differential expression (DE) miRNAs were predicted to target to 904 differential expression genes (DEGs), while in C-XY/E-XY, 226 DEmiRNAs were predicted to target to 2,048 DEGs. In C-XX/C-XY, and C-XY/E-XY, KEGG pathway enrichment analysis showed that those targeted genes were mainly enriched in MAPK signaling, calcium signaling, steroid hormone biosynthesis and ovarian steroidogenesis pathway. Additionally, the competitive endogenous RNA (ceRNA) regulatory network was constructed by 24 miRNAs, 21 lncRNAs, 4 circRNAs and 5 key sex-related genes. These findings suggested that the expression of critical genes in sex differentiation were altered in E2-treated XY T. rubripes may via the lncRNA-miRNA-mRNA regulation network to facilitate the differentiation and maintenance of ovaries. Our results provide a new insight into the comprehensive understanding of the effects of estrogen signaling pathways on sex differentiation in teleost gonads.

Integrated whole transcriptome analysis for the crucial regulators and functional pathways related to cardiac fibrosis in rats.

BACKGROUND: Cardiac fibrosis has gradually gained significance in the field of cardiovascular disease; however, its specific pathogenesis remains unclear. This study aims to establish the regulatory networks based on whole-transcriptome RNA sequencing analyses and reveal the underlying mechanisms of cardiac fibrosis. METHODS: An experimental model of myocardial fibrosis was induced using the chronic intermittent hypoxia (CIH) method. Expression profiles of long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) were acquired from right atrial tissue samples of rats. Differentially expressed RNAs (DERs) were identified, and functional enrichment analysis was performed. Moreover, a protein-protein interaction (PPI) network and competitive endogenous RNA (ceRNA) regulatory network that are related to cardiac fibrosis were constructed, and the relevant regulatory factors and functional pathways were identified. Finally, the crucial regulators were validated using qRT-PCR. RESULTS: DERs, including 268 lncRNAs, 20 miRNAs, and 436 mRNAs, were screened. Further, 18 relevant biological processes, such as "chromosome segregation, " and 6 KEGG signaling pathways, such as "cell cycle, " were significantly enriched. The regulatory relationship of miRNA-mRNA-KEGG pathways showed eight overlapping disease pathways, including "pathways in cancer." In addition, crucial regulatory factors, such as Arnt2, WNT2B, GNG7, LOC100909750, Cyp1a1, E2F1, BIRC5, and LPAR4, were identified and verified to be closely related to cardiac fibrosis. CONCLUSION: This study identified the crucial regulators and related functional pathways in cardiac fibrosis by integrating the whole transcriptome analysis in rats, which might provide novel insights into the pathogenesis of cardiac fibrosis.

Integrated Analysis of Coding and Non-coding RNAs Reveals the Molecular Mechanism Underlying Salt Stress Response in Medicago truncatula.

Salt stress is among the most severe abiotic stresses in plants worldwide. Medicago truncatula is a model plant for legumes and analysis of its response to salt stress is helpful for providing valuable insights into breeding. However, few studies have focused on illustrating the whole-transcriptome molecular mechanism underlying salt stress response in Medicago truncatula. Herein, we sampled the leaves of Medicago truncatula treated with water or NaCl and analyzed the characteristics of its coding and non-coding RNAs. We identified a total of 4,693 differentially expressed mRNAs (DEmRNAs), 505 DElncRNAs, 21 DEcircRNAs, and 55 DEmiRNAs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that their functions were mostly associated with metabolic processes. We classified the lncRNAs and circRNAs into different types and analyzed their genomic distributions. Furthermore, we predicted the interactions between different RNAs based on the competing endogenous RNA (ceRNA) theory and identified multiple correlation networks, including 27 DEmiRNAs, 43 DEmRNAs, 19 lncRNAs, and 5 DEcircRNAs. In addition, we comprehensively analyzed the candidate DEmRNAs and ceRNAs and found that they were involved in Ca+ signaling, starch and sucrose biosynthesis, phenylpropanoid and lignin metabolism, auxin and jasmonate biosynthesis, and transduction pathways. Our integrated analyses in salt stress response in Medicago truncatula revealed multiple differentially expressed coding and non-coding RNAs, including mRNAs, lncRNAs, circRNAs, and miRNAs, and identified multiple DEmRNA and ceRNA interaction pairs that function in many pathways, providing insights into salt stress response in leguminous plants.

Construction of a focal adhesion signaling pathway-related ceRNA network in pelvic organ prolapse by transcriptome analysis.

Objective: Pelvic organ prolapse (POP) affects a large proportion of adult women, but the pathogenesis of POP remains unclear. The increase in global population aging will impose a substantial medical burden. Herein, we aimed to explore the related RNAs regulating the occurrence of POP and provide potential therapeutic targets. Method: Tissue biopsies were collected from the anterior vaginal wall of six women with POP and six matched subjects without POP. The profiles of mRNAs, circRNAs, lncRNAs, and miRNAs were obtained by whole transcriptome RNA sequencing. Result: The findings revealed that 71 circRNAs, 76 known lncRNAs, 84 miRNAs, and 931 mRNAs were significantly altered (p < 0.05 and |log2FC| > 1). GO and KEGG enrichment analyses indicated that the differentially expressed genes (DEGs) were mainly enriched in the focal adhesion signaling pathway. FLT, ITGA9, VEGFD, PPP1R12B, and ROCK2 were identified as focal adhesion signaling pathway-related hub genes by protein-protein interaction network analysis. Based on the relationships between the DEGs and miRNA, lncRNA and circRNA targets, we constructed a focal adhesion signaling pathway-related ceRNA network. The ceRNA network includes hsa_circ_0002190/hsa_circ_0046843/lnc-CARMN -miR-23a-3p - ROCK2 and hsa_circ_0001326/hsa_circ_0007733/lnc-AC107959/lnc-TPM1-AS - miR-205-5p - ROCK2/PPP1R12B/VEGFD. Moreover, abnormalities in the cytoskeleton in fibroblasts from individuals with POP were observed. Conclusion: In this study, a focal adhesion signaling pathway-related ceRNA network was constructed, and this network may serve as a target for finding suitable drugs for the treatment of POP.

Improving lung cancer risk stratification leveraging whole transcriptome RNA sequencing and machine learning across multiple cohorts.

BACKGROUND: Bronchoscopy for suspected lung cancer has low diagnostic sensitivity, rendering many inconclusive results. The Bronchial Genomic Classifier (BGC) was developed to help with patient management by identifying those with low risk of lung cancer when bronchoscopy is inconclusive. The BGC was trained and validated on patients in the Airway Epithelial Gene Expression in the Diagnosis of Lung Cancer (AEGIS) trials. A modern patient cohort, the BGC Registry, showed differences in key clinical factors from the AEGIS cohorts, with less smoking history, smaller nodules and older age. Additionally, we discovered interfering factors (inhaled medication and sample collection timing) that impacted gene expressions and potentially disguised genomic cancer signals. METHODS: In this study, we leveraged multiple cohorts and next generation sequencing technology to develop a robust Genomic Sequencing Classifier (GSC). To address demographic composition shift and interfering factors, we synergized three algorithmic strategies: 1) ensemble of clinical dominant and genomic dominant models; 2) development of hierarchical regression models where the main effects from clinical variables were regressed out prior to the genomic impact being fitted in the model; and 3) targeted placement of genomic and clinical interaction terms to stabilize the effect of interfering factors. The final GSC model uses 1232 genes and four clinical covariates - age, pack-years, inhaled medication use, and specimen collection timing. RESULTS: In the validation set (N = 412), the GSC down-classified low and intermediate pre-test risk subjects to very low and low post-test risk with a specificity of 45% (95% CI 37-53%) and a sensitivity of 91% (95%CI 81-97%), resulting in a negative predictive value of 95% (95% CI 89-98%). Twelve percent of intermediate pre-test risk subjects were up-classified to high post-test risk with a positive predictive value of 65% (95%CI 44-82%), and 27% of high pre-test risk subjects were up-classified to very high post-test risk with a positive predictive value of 91% (95% CI 78-97%). CONCLUSIONS: The GSC overcame the impact of interfering factors and achieved consistent performance across multiple cohorts. It demonstrated diagnostic accuracy in both down- and up-classification of cancer risk, providing physicians actionable information for many patients with inconclusive bronchoscopy.