Integrated multiple microarray studies by robust rank aggregation to identify immune-associated biomarkers in Crohn's disease based on three machine learning methods.

Crohn's disease (CD) is a complex autoimmune disorder presumed to be driven by complex interactions of genetic, immune, microbial and even environmental factors. Intrinsic molecular mechanisms in CD, however, remain poorly understood. The identification of novel biomarkers in CD cases based on larger samples through machine learning approaches may inform the diagnosis and treatment of diseases. A comprehensive analysis was conducted on all CD datasets of Gene Expression Omnibus (GEO); our team then used the robust rank aggregation (RRA) method to identify differentially expressed genes (DEGs) between controls and CD patients. PPI (protein‒protein interaction) network and functional enrichment analyses were performed to investigate the potential functions of the DEGs, with molecular complex detection (MCODE) identifying some important functional modules from the PPI network. Three machine learning algorithms, support vector machine-recursive feature elimination (SVM-RFE), random forest (RF), and least absolute shrinkage and selection operator (LASSO), were applied to determine characteristic genes, which were verified by ROC curve analysis and immunohistochemistry (IHC) using clinical samples. Univariable and multivariable logistic regression were used to establish a machine learning score for diagnosis. Single-sample GSEA (ssGSEA) was performed to examine the correlation between immune infiltration and biomarkers. In total, 5 datasets met the inclusion criteria: GSE75214, GSE95095, GSE126124, GSE179285, and GSE186582. Based on RRA integrated analysis, 203 significant DEGs were identified (120 upregulated genes and 83 downregulated genes), and MCODE revealed some important functional modules in the PPI network. Machine learning identified LCN2, REG1A, AQP9, CCL2, GIP, PROK2, DEFA5, CXCL9, and NAMPT; AQP9, PROK2, LCN2, and NAMPT were further verified by ROC curves and IHC in the external cohort. The final machine learning score was defined as [Expression level of AQP9 × (2.644)] + [Expression level of LCN2 × (0.958)] + [Expression level of NAMPT × (1.115)]. ssGSEA showed markedly elevated levels of dendritic cells and innate immune cells, such as macrophages and NK cells, in CD, consistent with the gene enrichment results that the DEGs are mainly involved in the IL-17 signaling pathway and humoral immune response. The selected biomarkers analyzed by the RRA method and machine learning are highly reliable. These findings improve our understanding of the molecular mechanisms of CD pathogenesis.

Identification of a Ferroptosis-Related Signature Model Including mRNAs and lncRNAs for Predicting Prognosis and Immune Activity in Hepatocellular Carcinoma.

BACKGROUND: Ferroptosis is a novel form of regulated cell death involved in tumor progression. The role of ferroptosis-related lncRNAs in hepatocellular carcinoma (HCC) remains unclear. METHODS: RNA-seq and clinical data for HCC patients were downloaded from The Cancer Genome Atlas (TCGA) Genomic Data Commons (GDC) portal. Bioinformatics methods, including weighted gene coexpression network analysis (WGCNA), Cox regression, and least absolute shrinkage and selection operator (LASSO) analysis, were used to identify signature markers for diagnosis/prognosis. The tumor microenvironment, immune infiltration and functional enrichment were compared between the low-risk and high-risk groups. Subsequently, small molecule drugs targeting ferroptosis-related signature components were predicted via the L1000FWD and PubChem databases. RESULTS: The prognostic model consisted of 2 ferroptosis-related mRNAs (SLC1A5 and SLC7A11) and 8 ferroptosis-related lncRNAs (AC245297.3, MYLK-AS1, NRAV, SREBF2-AS1, AL031985.3, ZFPM2-AS1, AC015908.3, MSC-AS1). The areas under the curves (AUCs) were 0.830 and 0.806 in the training and test groups, respectively. Decision curve analysis (DCA) revealed that the ferroptosis-related signature performed better than all pathological characteristics. Multivariate Cox regression analysis showed that the risk score was an independent prognostic factor. The survival probability of low- and high-risk patients could be clearly distinguished by the principal component analysis (PCA) plot. The risk score divided HCC patients into two distinct groups in terms of immune status, especially checkpoint gene expression, which was further supported by the Gene Ontology (GO) biological process, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Finally, several small molecule drugs (SIB-1893, geldanamycin and PD-184352, etc) targeting ferroptosis-related signature components were identified for future reference. CONCLUSION: We constructed a new ferroptosis-related mRNA/lncRNA signature for HCC patients. The model can be used for prognostic prediction and immune evaluation, providing a reference for immunotherapies and targeted therapies.

Integrated Analysis of Multiple Microarray Studies to Identify Novel Gene Signatures in Ulcerative Colitis.

Background: Ulcerative colitis (UC) is a chronic, complicated, inflammatory disease with an increasing incidence and prevalence worldwide. However, the intrinsic molecular mechanisms underlying the pathogenesis of UC have not yet been fully elucidated. Methods: All UC datasets published in the GEO database were analyzed and summarized. Subsequently, the robust rank aggregation (RRA) method was used to identify differentially expressed genes (DEGs) between UC patients and controls. Gene functional annotation and PPI network analysis were performed to illustrate the potential functions of the DEGs. Some important functional modules from the protein-protein interaction (PPI) network were identified by molecular complex detection (MCODE), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), and analyses were performed. The results of CytoHubba, a plug for integrated algorithm for biomolecular interaction networks combined with RRA analysis, were used to identify the hub genes. Finally, a mouse model of UC was established by dextran sulfate sodium salt (DSS) solution to verify the expression of hub genes. Results: A total of 6 datasets met the inclusion criteria (GSE38713, GSE59071, GSE73661, GSE75214, GSE87466, GSE92415). The RRA integrated analysis revealed 208 significant DEGs (132 upregulated genes and 76 downregulated genes). After constructing the PPI network by MCODE plug, modules with the top three scores were listed. The CytoHubba app and RRA identified six hub genes: LCN2, CXCL1, MMP3, IDO1, MMP1, and S100A8. We found through enrichment analysis that these functional modules and hub genes were mainly related to cytokine secretion, immune response, and cancer progression. With the mouse model, we found that the expression of all six hub genes in the UC group was higher than that in the control group (P < 0.05). Conclusion: The hub genes analyzed by the RRA method are highly reliable. These findings improve the understanding of the molecular mechanisms in UC pathogenesis.

Overexpression of the immediate early response 5 gene increases the radiosensitivity of HeLa cells.

The effects of the immediate early response 5 (IER5) gene on the sensitivity of HeLa cells to radiation remain unclear. In the present study, stably transfected HeLa cells resulting in the knockdown or overexpression of IER5 were investigated. In addition, xenografts of normal, IER5-silenced and -overexpressed HeLa cells were injected into nude mice and examined. The results demonstrated that the radiosensitivity of the IER5-overexpressed HeLa cells was significantly increased compared with that of the normal and IER5-silenced cells. The upregulation of IER5 effectively decreased cell proliferation and IER5 silencing promoted cell proliferation compared with that in the normal HeLa cells. Following irradiation of the cells with IER5 knockdown, cell cycle was arrested at the G2/M phase and an increase in the proportion of S phase cells was observed. By contrast, the overexpression of IER5 led to an increase in the proportion of G1 phase cells. Furthermore, the upregulation of IER5 inhibited tumor growth in vivo. The present findings demonstrate that the IER5 gene affects the radiosensitivity of HeLa cells and serves an important role in cell proliferation, suggesting that this gene may be a potential radiotherapeutic target in cervical cancer.

[Effects of illumination and seed-soaking reagent on seed germination of Solanum nigrum].

To explore a rapid seed germination method for hyperaccumulator Solanum nigrum, a germination experiment with different illumination and seed-soaking treatments was conducted in constant temperature box and greenhouse, with filter as burgeon base. Under illumination, the germination rate was about 5 times high of that without illumination (P < 0.05), indicating that illumination was one of the prerequisites for the seed germination of S. nigrum. All test seed-soaking reagents could significantly improve the germination rate of S. nigrum (P < 0.05), with the best effect of H2O2. The seeds treated with H2O2 had the shortest germination time. The germination rate of seeds soaked but without cleaning was 2-3 times as high as that of seeds soaked and cleaned with water.

Induced expression of the IER5 gene by gamma-ray irradiation and its involvement in cell cycle checkpoint control and survival.

The immediate-early response gene 5 (IER5) was previously shown, using microarray analysis, to be upregulated by ionizing radiation. Here we further characterized the dose- and time-dependency of radiation-induced expression of IER5 at doses from 0.5 to 15 Gy by quantitative real-time PCR analyses in HeLa cells and human lymphoblastoid AHH-1 cells. A radiation-induced increase in the IER5 mRNA level was evident 2 h after irradiation with 2 Gy in both cell lines. In AHH-1 cells the expression reached a peak at 4 h and then quickly returned to the control level, while in HeLa cells the expression only remained increased for a short period of time at around 2 h after irradiation before returning to the control. After high-dose irradiation (10 Gy), the induction of the IER5 expression was lower and delayed in AHH-1 cells as compared with 2-Gy irradiated cells. In HeLa cells, at this dose, two peaks of increased expression were observed 2 h and 12-24 h post-irradiation, respectively. RNA interference technology was employed to silence the IER5 gene in HeLa cells. siRNA-mediated suppression of IER5 resulted in an increased proliferation of HeLa cells. Cell growth and survival analyses demonstrated that suppression of IER5 significantly increased the radioresistance of HeLa cells to radiation doses of up to 6 Gy, but barely affected the sensitivity of cells at 8 Gy. Moreover, suppression of IER5 potentiated radiation-induced arrest at the G2-M transition and led to an increase in the fraction of S phase cells. Taken together, we propose that the early radiation-induced expression of IER5 affects the radiosensitivity via disturbing radiation-induced cell cycle checkpoints.

[Hyperaccumulative characteristics of 7 widely distributing weed species in composite family especially Bidens pilosa to heavy metals].

Hyperaccumulator is the main point of phytoremediating contaminated soils by heavy metals, and the identification of hyperaccumulator is still the difficult and key step of phytoremediation. The outdoor pot-culture experiment was used to study the hyperaccumulative characteristics of 7 widely distributing weed species in Northeast of China to heavy metals. The results in screening experiment showed that Taraxacum mongolicum and Bidens pilosa indicated strong tolerance to Cd single and Cd-Pb-Cu-Zn combined pollution, their Cd concentration in shoot were higher than that in roots, and the Cd enhancement factors (ratio of heavy metal concentration in shoot to that in soil) in shoots were greater than 1 too, which displayed that the two plants were with Cd hyperaccumulative characteristics. In concentration gradient experiment, Cd concentration in leaves of B. pilosa were all greater than 100 mg x kg(-1) the minimum of Cd-hyperaccumulator should have under the conditions of 25, 50, 100 mg x kg(-1) Cd added. Meanwhile, the shoot biomass of B. pilosa did not reduce significantly (p <0.05), Cd concentration in its shoots were higher than those in roots. But for T. mongolicum, Cd concentration in its shoots were not greater than 100 mg x kg(-1) in any treatment. Thus, only B. pilosa can be regarded as Cd-hyperaccumulator.