Integrative transcriptomics analysis for uterine leiomyosarcoma identifies aberrant activation of cell cycle-dependent kinases and their potential therapeutic significance.

Purpose: Uterine leiomyosarcoma is among the most aggressive gynecological malignancies. No effective treatment strategies have been established. This study aimed to identify novel therapeutic targets for uterine leiomyosarcoma based on transcriptome analysis and assess the preclinical efficacy of novel drug candidates. Experimental Design: Transcriptome analysis was carried out using fresh-frozen samples of six uterine leiomyosarcomas and three myomas. The Ingenuity Pathway Analysis was then used to identify potential therapeutic target genes for uterine leiomyosarcoma. Moreover, our results were validated using three independent datasets, including 40 uterine leiomyosarcomas. Then, the inhibitory effects of several selective inhibitors for the candidate genes were examined using the SK-UT-1, SK-LMS-1, and SKN cell lines. Results: We identified 512 considerably dysregulated genes in uterine leiomyosarcoma compared with myoma. The Ingenuity Pathway Analysis showed that the function of several genes, including CHEK1 and PLK1, were predicted to be activated in uterine leiomyosarcoma. Through an in vitro drug screening, PLK1 or CHEK1 inhibitors (BI 2536 or prexasertib) were found to exert a superior anti-cancer effect against cell lines at low nanomolar concentrations and induced cell cycle arrest. In SK-UT-1 tumor-bearing mice, BI 2536 monotherapy demonstrated a marked tumor regression. Moreover, the prexasertib and cisplatin combination therapy also reduced tumorigenicity and prolonged survival. Conclusion: We identified the upregulated expression of PLK1 and CHEK1; their kinase activity was considered to be activated in uterine leiomyosarcoma. BI 2536 and prexasertib demonstrate a significant anti-cancer effect; thus, cell cycle-related kinases may represent a promising therapeutic strategy for treating uterine leiomyosarcoma.

Age-related changes in microRNAs expression in cruciate ligaments of wild-stock house mice

Aim: Cruciate ligaments (CLs) of the knee joint are commonly injured following trauma or ageing. MicroRNAs (miRs) are potential therapeutic targets in musculoskeletal disorders. This study aimed to 1) identify if wild-stock house (WSH) mice are an appropriate model to study age-related changes of the knee joint and 2) investigate expression of miRs in ageing murine CLs. Methods: Knee joints were collected from 6 and 24 months old C57BL/6 and WSH mice (Mus musculus domesticus) for histological analysis. RNA extraction and qPCR gene expression were performed on CLs in 6, 12, 24, and 30 month WSH old mice. Expression of miR targets in CLs was determined, followed by analysis of predicted mRNA target genes and Ingenuity Pathway Analysis. Results: Higher CL and knee OARSI histological scores were found in 24 month old WSH mice compared to 6 and 12 month old C57BL/6 and 6 month old WSH mice (p< 0.05). miR-29a and miR-34a were upregulated in 30 month-old WSH mice in comparison to 6, 12 and 24-month-old WSH mice (p<0.05). Ingenuity Pathway Analysis on miR-29a and 34a targets was associated with inflammation through interleukins, TGFβ and Notch genes and p53 signalling. Collagen type I alpha 1 chain (COL1A1) correlated negatively with both miR-29a (r= -0.35) and miR-34a (r= -0.33). Conclusion: The findings of this study support WSH house mice as an accelerated ageing model of the murine knee joint. This study also indicated that miR-29a and 34a may be important regulators of COL1A1 gene expression in murine CLs.

Quantitative Glycoproteomics Analysis Identifies Novel FUT8 Targets and Signaling Networks Critical for Breast Cancer Cell Invasiveness

BackgroundWe recently showed that fucosyltransferase 8 (FUT8)-mediated core fucosylation of transforming growth factor-β receptor enhances its signaling and promotes breast cancer invasion and metastasis. However, the complete FUT8 target glycoproteins and their downstream signaling networks critical for breast cancer progression remain largely unknown.MethodWe performed quantitative glycoproteomics with two highly invasive breast cancer cell lines to unravel a comprehensive list of core-fucosylated glycoproteins by comparison to parental wild-type and FUT8-knockout counterpart cells. In addition, Ingenuity Pathway Analysis (IPA) was performed to highlight the most enriched biological functions and signaling pathways mediated by FUT8 targets. Novel FUT8 target glycoproteins with biological interest were functionally studied and validated by using LCA (Lens culinaris agglutinin) blotting and LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis.ResultsLoss-of-function studies demonstrated that FUT8 knockout suppressed the invasiveness of highly aggressive breast carcinoma cells. Quantitative glycoproteomics identified 140 common target glycoproteins. Ingenuity Pathway Analysis (IPA) of these target proteins gave a global and novel perspective on signaling networks essential for breast cancer cell migration and invasion. In addition, we showed that core fucosylation of integrin αvβ5 or IL6ST is crucial for breast cancer cell adhesion to vitronectin or enhanced cellular signaling to interleukin 6 and oncostatin M, two cytokines implicated in the breast cancer epithelial–mesenchymal transition and metastasis.ConclusionsOur report reveals a comprehensive list of core-fucosylated target proteins and provides novel insights into signaling networks crucial for breast cancer progression. These findings will assist in deciphering the complex molecular mechanisms and developing diagnostic or therapeutic approaches targeting these signaling pathways in breast cancer metastasis.

Gene Set Enrichment Analysis and Ingenuity Pathway Analysis to verify the impact on Wnt Signaling Pathway in Taodan Granules Treated Psoriasis

Background: Taodan granules (TDGs), traditional Chinese herbals, are effective in treating psoriasis. However, mechanisms of TDGs remain indistinct. The current study aims to indicate the molecular mechanisms of TDGs in treating psoriasis.Methods: Primarily, transcriptional profiling was applied to identify differentially expression genes (DEGs). The following was that we used Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) analysis for functional enrichment analysis. Eventually, RT-PCR was performed to validation. Results: The results revealed that TDGs could regulated the Wnt signaling pathway to ameliorate skin lesions of imiquimod (IMQ)-induced psoriatic mouse models. IPA core network associated with Wnt signaling pathways in TDGs for psoriasis was established. Thereinto zeste homolog (EZH) 2, CTNNB1, TP63, and WD repeat domain (WDR) 5 could be considered as upstream genes in the Wnt signaling pathway.Conclusions: The Wnt signaling pathway with these above upstream genes might be potential therapeutic targets of TDGs for psoriasis.

Separating the Wheat from the Chaff: The Use of Upstream Regulator Analysis to Identify True Differential Expression of Single Genes within Transcriptomic Datasets

The development of DNA microarray and RNA-sequencing technology has led to an explosion in the generation of transcriptomic differential expression data under a wide range of biologic systems including those recapitulating the monogenic muscular dystrophies. Data generation has increased exponentially due in large part to new platforms, improved cost-effectiveness, and processing speed. However, reproducibility and thus reliability of data remain a central issue, particularly when resource constraints limit experiments to single replicates. This was observed firsthand in a recent rare disease drug repurposing project involving RNA-seq-based transcriptomic profiling of primary cerebrocortical cultures incubated with clinic-ready blood–brain penetrant drugs. Given the low validation rates obtained for single differential expression genes, alternative approaches to identify with greater confidence genes that were truly differentially expressed in our dataset were explored. Here we outline a method for differential expression data analysis in the context of drug repurposing for rare diseases that incorporates the statistical rigour of the multigene analysis to bring greater predictive power in assessing individual gene modulation. Ingenuity Pathway Analysis upstream regulator analysis was applied to the differentially expressed genes from the Care4Rare Neuron Drug Screen transcriptomic database to identify three distinct signaling networks each perturbed by a different drug and involving a central upstream modulating protein: levothyroxine (DIO3), hydroxyurea (FOXM1), dexamethasone (PPARD). Differential expression of upstream regulator network related genes was next assessed in in vitro and in vivo systems by qPCR, revealing 5× and 10× increases in validation rates, respectively, when compared with our previous experience with individual genes in the dataset not associated with a network. The Ingenuity Pathway Analysis based gene prioritization may increase the predictive value of drug–gene interactions, especially in the context of assessing single-gene modulation in single-replicate experiments.

Transkriptomik Veri Yaklaşımı ile Kutanöz Leyşmanyazisteki Moleküler Sinyal Yolakların ve Regülatör Moleküllerin Tanımlanması

Leishmaniasis is a disease caused by the genus Leishmania spp., which are intracellular parasites. Depending on parasite species and host immune response, there are three basic clinical forms of the disease: cutaneous, mucocutaneous, and visceral leishmaniasis. Cutaneous leishmaniasis is a chronic disease and characterized by the presence of ulcerated skin lesions. The type of skin pathology seen during disease is determined in part by the infecting Leishmania spp., but also by a combination of inflammatory and antiinflammatory host immune response factors resulting in diverse clinical outcomes. In this study, it was aimed to determine the genes, molecular signaling mechanisms and biological functions of the molecules that play a role in the pathogenesis of the disease and immune response and determine host-parasite interactions in mice that are naturally resistant and susceptible to Leishmania major and Leishmania braziliensis. For this, transcriptomic series GSE56029 was downloaded from “Gene Expression Omnibus” (GEO) data base, including expression profiling of twenty-four tissue samples that were recovered from both naive mice and mice (BALB/c, C57BL/6) infected with L.major and L.braziliensis. Then, “Differentially Expressed Genes” (DEGs) were identified by limma package in R script. FDR q< 0.05 and absolute log2FC> 2 as threshold values were accepted in the analysis. Subsequently, functional and pathway enrichment analyses were performed for the DEGs by “Ingenuity Pathway Analysis” (IPA). For each of DEGs, p< 0.01, FDR q< 0.01, and absolute log2FC> 1 were used and analyzed with the software program IPA 8.0. Ingenuity Pathway Analysis revealed the most enrichment pathways to be the inflammation, dendritic cell maturation and “Triggering Receptor Expressed on Myeloid Cells 1” (TREM-1) signal mechanisms and that the DEGs related to the regulation of immune system process were closely associated with the progress of cutaneous leishmaniasis. The upstream regulator analysis predicted that TNF-α, IFNy, IL-1 β, IL-10RA and “Signal Transducer and Activator of Transcription-1” (STAT-1) are the regulators that explained gene expression changes causing biological activities in the tissues. Chemical compounds that may have anti-leishmanial effects were also identified in the study. In this study, the mechanisms belonging to the parasite species and host that determine the resistance/susceptibility phenotype were attempted to elucidate. Assessment of gene expression patterns, cytokine/chemokines, and signaling pathways in BALB/c and C57BL/6 mice infected with L.major and L.braziliensis will provide a better understanding of the potential mechanisms underlying infection from a genetic perspective. These results may guide for the future studies in terms of developing potential biomarkers for the diagnosis and prognosis prediction of cutaneous leishmaniasis and providing information about new treatment targets.