Comparative Analysis of Transcriptomic Changes including mRNA and microRNA Expression Induced by the Xenoestrogens Zearalenone and Bisphenol A in Human Ovarian Cells.

Xenoestrogens are natural or synthetic compounds that mimic the effect of endogenous estrogens and might cause cancer. We aimed to compare the global transcriptomic response to zearalenone (ZEA; mycotoxin) and bisphenol A (BPA; plastic additive) with the effect of physiological estradiol (E2) in the PEO1 human ovarian cell line by mRNA and microRNA sequencing. Estrogen exposure induced remarkable transcriptomic changes: 308, 288 and 63 genes were upregulated (log2FC > 1); 292, 260 and 45 genes were downregulated (log2FC < -1) in response to E2 (10 nM), ZEA (10 nM) and BPA (100 nM), respectively. Furthermore, the expression of 13, 11 and 10 miRNAs changed significantly (log2FC > 1, or log2FC < -1) after exposure to E2, ZEA and BPA, respectively. Functional enrichment analysis of the significantly differentially expressed genes and miRNAs revealed several pathways related to the regulation of cell proliferation and migration. The effect of E2 and ZEA was highly comparable: 407 genes were coregulated by these molecules. We could identify 83 genes that were regulated by all three treatments that might have a significant role in the estrogen response of ovarian cells. Furthermore, the downregulation of several miRNAs (miR-501-5p, let-7a-2-3p, miR-26a-2-3p, miR-197-5p and miR-582-3p) was confirmed by qPCR, which might support the proliferative effect of estrogens in ovarian cells.

Suppressing the PI3K/AKT Pathway by miR-30d-5p Mimic Sensitizes Ovarian Cancer Cells to Cell Death Induced by High-Dose Estrogen.

MicroRNAs are short non-coding RNA molecules that are involved in tumor development and are considered to be promising candidates in cancer therapy. Here, we studied the role of miR-30s in the pathophysiology of ovarian cancer. According to our results miR-30a-5p, miR-30d-5p, and miR-30e-5p were overexpressed in the estrogen receptor α (ERα)-expressing PEO1 cell line compared to A2780 that lacks this receptor. Furthermore, the expression of miR-30a-5p, miR-30d-5p, and miR-30e-5p were induced in response to high-dose estrogen treatment in PEO1 where intensive cell death was observed according to the induction of apoptosis and autophagy. Lacking or blocking ERα function reduced tolerance to high-dose estrogen that suggests the importance of ERα-mediated estrogen response in the maintenance of proliferation. MiR-30d-5p mimic reduced cell proliferation in both A2780 and PEO1. Furthermore, it decreased the tolerance of PEO1 cells to high-dose estrogen by blocking the ERα-mediated estrogen response. This was accompanied by decreased SOX4 expression that is thought to be involved in the regulation of the PI3K/AKT pathway. Blocking this pathway by AZD8835 led to the same results. MiR-30d-5p or AZD8835 sensitized PEO1 cells to tamoxifen. We suggest that miR-30d-5p might be a promising candidate in the therapy of ovarian cancer.

Analysis of Circulating miRNA Profile in Plasma Samples of Glioblastoma Patients.

(1) Background: Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis. Treatment options are limited, clinicians lack efficient prognostic and predictive markers. Circulating miRNAs-besides being important regulators of cancer development-may have potential as diagnostic biomarkers of GBM. (2) Methods: In this study, profiling of 798 human miRNAs was performed on blood plasma samples from 6 healthy individuals and 6 patients with GBM, using a NanoString nCounter Analysis System. To validate our results, five miRNAs (hsa-miR-433-3p, hsa-miR-362-3p, hsa-miR-195-5p, hsa-miR-133a-3p, and hsa-miR-29a-3p) were randomly chosen for RT-qPCR detection. (3) Results: In all, 53 miRNAs were significantly differentially expressed in plasma samples of GBM patients when data were filtered for FC 1 and FDR 0.1. Target genes of the top 39 differentially expressed miRNAs were identified, and we carried out functional annotation and pathway enrichment analysis of target genes via GO and KEGG-based tools. General and cortex-specific protein-protein interaction networks were constructed from the target genes of top miRNAs to assess their functional connections. (4) Conclusions: We demonstrated that plasma microRNA profiles are promising diagnostic and prognostic molecular biomarkers that may find an actual application in the clinical practice of GBM, although more studies are needed to validate our results.

The Role of Exosomes in Cancer Progression.

Early detection, characterization and monitoring of cancer are possible by using extracellular vesicles (EVs) isolated from non-invasively obtained liquid biopsy samples. They play a role in intercellular communication contributing to cell growth, differentiation and survival, thereby affecting the formation of tumor microenvironments and causing metastases. EVs were discovered more than seventy years ago. They have been tested recently as tools of drug delivery to treat cancer. Here we give a brief review on extracellular vesicles, exosomes, microvesicles and apoptotic bodies. Exosomes play an important role by carrying extracellular nucleic acids (DNA, RNA) in cell-to-cell communication causing tumor and metastasis development. We discuss the role of extracellular vesicles in the pathogenesis of cancer and their practical application in the early diagnosis, follow up, and next-generation treatment of cancer patients.

Circulating Cell-Free Nucleic Acids: Main Characteristics and Clinical Application.

Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.

The Cell-Free Expression of MiR200 Family Members Correlates with Estrogen Sensitivity in Human Epithelial Ovarian Cells.

Exposure to physiological estrogens or xenoestrogens (e.g., zearalenone or bisphenol A) increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes (GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells.

Circulating epithelial-mesenchymal transition-associated miRNAs are promising biomarkers in ovarian cancer.

Ovarian cancer is the fifth most common cause of cancer death among women that is mostly due to the difficulty of early diagnosis. Circulating miRNAs proved to be reliable biomarkers in various cancers. We screened 9 miRNAs, which are involved in epithelial-mesenchymal transition, in the plasma samples of patients with malignant (n = 28) or non-malignant (n = 12) ovarian tumors and disease-free healthy volunteers (n = 60) by qRT-PCR. The expression levels of miR200a, miR200b, miR200c, miR141, miR429, miR203a, miR34b (p < 0.001) and miR34a (p < 0.01) were significantly higher in the malignant samples than in healthy controls. MiR203a, miR141 (p < 0.01), miR200a and miR429 (p < 0.05) levels were also higher in malignant compared to non-malignant samples. ROC-AUC was the highest in the case of miR200c: 0.861 (95%CI = 0.776-0.947). Spearman's rank correlation analysis revealed positive correlation between the plasma levels of the studied miRNAs that was the highest between miR200b and miR200c (rs = 0.774; p < 0.001). Target analysis also suggested tight interaction between these miRNAs in the regulation of cancer development. The agreement of diagnostic tests based on miRNA levels and the standard CA125 or HE4 was weak according to Cohen's kappa values. We conclude that miR200 family members, miR34b and miR203a might be promising complementary biomarkers in ovarian cancer.

Mutation in afsR Leads to A-Factor Deficiency in Streptomyces griseus B2682.

BACKGROUND/AIMS: A-factor, a γ-butyrolactone autoregulator, in Streptomyces griseus is involved in the regulation of differentiation and antibiotic production. Here we studied the S. griseus B2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the afsR gene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. METHODS: Wild afsR was cloned to the pHJL401 shuttle vector and was transformed to the S. griseus AFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. RESULTS: Transformation of AFN by a wild afsR restored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of afsR to the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. CONCLUSIONS: AfsR has an effect on the regulation of A-factor production in S. griseus. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.

Autolytic enzymes are responsible for increased melanization of carbon stressed Aspergillus nidulans cultures.

Melanization of carbon stressed Aspergillus nidulans cultures were studied. Melanin production showed strong positive correlation with the activity of the secreted chitinase and ß-1,3-glucanase. Deletion of either chiB encoding an autolytic endochitinase or engA encoding an autolytic ß-1,3-endoglucanase, or both, almost completely prevented melanization of carbon stressed cultures. In contrast, addition of Trichoderma lyticase to cultures induced melanin production. Synthetic melanin could efficiently inhibit the purified ChiB chitinase activity. It could also efficiently decrease the intensity of hyphal fragmentation and pellet disorganization in Trichoderma lyticase treated cultures. Glyphosate, an inhibitor of L-3,4-dihydroxyphenylalanine-type melanin synthesis, could prevent melanization of carbon-starved cultures and enhanced pellet disorganization, while pyroquilon, a 1,8-dihydroxynaphthalene-type melanin synthesis inhibitor, enhanced melanization, and prevented pellet disorganization. We concluded that cell wall stress induced by autolytic cell wall hydrolases was responsible for melanization of carbon-starved cultures. The produced melanin can shield the living cells but may not inhibit the degradation and reutilization of cell wall materials of dead hyphae. Controlling the activity of autolytic hydrolase production can be an efficient approach to prevent unwanted melanization in the fermentation industry, while applying melanin synthesis inhibitors can decrease the resistance of pathogenic fungi against the chitinases produced by the host organism.

γ-Glutamyl transpeptidase (GgtA) of Aspergillus nidulans is not necessary for bulk degradation of glutathione.

Aspergillus nidulans exhibited high γ-glutamyl transpeptidase (γGT) activity in both carbon-starved and carbon-limited cultures. Glucose repressed, but casein peptone increased γGT production. Null mutation of creA did not influence γGT formation, but the functional meaB was necessary for the γGT induction. Deletion of the AN10444 gene (ggtA) completely eliminated the γGT activity, and the mRNA levels of ggtA showed strong correlation with the observed γGT activities. While ggtA does not contain a canonical signal sequence, the γGT activity was detectable both in the fermentation broth and in the hyphae. Deletion of the ggtA gene did not prevent the depletion of glutathione observed in carbon-starved and carbon-limited cultures. Addition of casein peptone to carbon-starved cultures lowered the formation of reactive species (RS). Deletion of ggtA could hinder this decrease and resulted in elevated RS formation. This effect of γGT on redox homeostasis may explain the reduced cleistothecia formation of ΔggtA strains in surface cultures.

Interactions between naturally occurring antifungal agents.

Pairwise interactions between four antifungal compounds were studied. The ß-1,3-glucan synthase inhibitor echinocandin B (ECB) showed synergistic effect with the cell wall hydrolase ChiB chitinase and EngA ß-1,3-glucanase on Saccharomyces cerevisiae, Candida albicans, Aspergillus rugulosus and A. fumigatus. The antifungal protein of Penicillium chrysogenum (PAF) did not influence the antifungal activity of ChiB or EngA, but showed antagonistic effect with ECB on A. nidulans, A. rugulosus and A. fumigatus. PAF had no significant effect on the growth of the tested yeasts as it was expected and did not influence significantly the antifungal activity of ECB, ChiB or EngA against yeasts.

bZIP transcription factors affecting secondary metabolism, sexual development and stress responses in Aspergillus nidulans.

The eukaryotic basic leucine zipper (bZIP) transcription factors play critical roles in the organismal response to the environment. Recently, a novel YAP-like bZIP, restorer of secondary metabolism A (RsmA), was found in a suppressor screen of an Aspergillus nidulans secondary metabolism (SM) mutant in which overexpression of rsmA was found to partially remediate loss of SM in Velvet Complex mutants. The Velvet Complex is a conserved fungal transcriptional heteromer that couples SM with sexual development in fungi. Here we characterized and contrasted SM in mutants of RsmA and four other A. nidulans bZIP proteins (NapA, ZipA, ZipB and ZipC) with predicted DNA binding motifs similar to RsmA. Only two overexpression mutants exhibited both SM and sexual abnormalities that were noteworthy: OE : : rsmA resulted in a 100-fold increase in sterigmatocystin and a near loss of meiotic spore production. OE : : napA displayed decreased production of sterigmatocystin, emericellin, asperthecin, shamixanthone and epishamixanthone, coupled with a shift from sexual to asexual development. Quantification of bZIP homodimer and heterodimer formation using fluorescence resonance energy transfer (FRET) suggested that these proteins preferentially self-associate.

Transcriptome changes initiated by carbon starvation in Aspergillus nidulans.

Carbon starvation is a common stress for micro-organisms both in nature and in industry. The carbon starvation stress response (CSSR) involves the regulation of several important processes including programmed cell death and reproduction of fungi, secondary metabolite production and extracellular hydrolase formation. To gain insight into the physiological events of CSSR, DNA microarray analyses supplemented with real-time RT-PCR (rRT-PCR) experiments on 99 selected genes were performed. These data demonstrated that carbon starvation induced very complex changes in the transcriptome. Several genes contributing to protein synthesis were upregulated together with genes involved in the unfolded protein stress response. The balance between biosynthesis and degradation moved towards degradation in the case of cell wall, carbohydrate, lipid and nitrogen metabolism, which was accompanied by the production of several hydrolytic enzymes and the induction of macroautophagy. These processes provide the cultures with long-term survival by liberating nutrients through degradation of the cell constituents. The induced synthesis of secondary metabolites, antifungal enzymes and proteins as well as bacterial cell wall-degrading enzymes demonstrated that carbon-starving fungi should have marked effects on the micro-organisms in their surroundings. Due to the increased production of extracellular and vacuolar enzymes during carbon starvation, the importance of the endoplasmic reticulum increased considerably.

Antifungal activity of extracellular hydrolases produced by autolysing Aspergillus nidulans cultures.

Carbon-starving Aspergillus nidulans cultures produce high activities of versatile hydrolytic enzymes and, among these, ChiB endochitinase and EngA ß-1,3-endoglucanase showed significant antifungal activity against various fungal species. Double deletion of engA and chiB diminished the antifungal activity of the fermentation broths and increased conidiogenesis and long-term viability of A. nidulans, but decreased the growth rate on culture media containing weak carbon sources. Production of ChiB and EngA can influence fungal communities either directly due to their antifungal properties or indirectly through their effects on vegetative growth. Our data suggest saprophytic fungi as promising future candidates to develop novel biocontrol technologies.

Extracellular proteinase formation in carbon starving Aspergillus nidulans cultures--physiological function and regulation.

Extracellular proteinase formation in carbon depleted cultures of the model filamentous fungus Aspergillus nidulans was studied to elucidate its regulation and possible physiological function. As demonstrated by gene deletion, culture optimization, microbial physiological and enzymological experiments, the PrtA and PepJ proteinases of A. nidulans did not appear to play a decisive role in the autolytic decomposition of fungal cells under the conditions we tested. However, carbon starvation induced formation of the proteinases observable in autolytic cultures. Similar to other degradative enzymes, production of proteinase was regulated by FluG-BrlA asexual developmental signaling and modulated by PacC-dependent pH-responsive signaling. Under the same carbon starved culture conditions, alterations of CreA, MeaB or heterotrimeric G protein mediated signaling pathways caused less significant changes in the formation of extracellular proteinases. Taken together, these results indicate that while the accumulation of PrtA and PepJ is tightly coupled to the initiation of autolysis, they are not essential for autolytic cell wall degradation in A. nidulans. Thus, as Aspergillus genomes contain a large group of genes encoding proteinases with versatile physiological functions, selective control of proteinase production in fungal cells is needed for the improved industrial use of fungi.

MeaB-dependent nutrition sensing regulates autolysis in carbon starved Aspergillus nidulans cultures.

Carbon starvation induced autolysis is an active process of self-digestion and is under complex regulation in Aspergillus nidulans. In this study we investigated how autolysis depends on the composition of the culture medium, especially on the presence of yeast extract. We demonstrated that the rate of autolytic cell wall degradation as well as the extracellular chitinase and proteinase productions significantly decreased in the presence of this nutrient. The effect of yeast extract on carbon starved cultures was independent of loss-of-function mutations in the carbon and nitrogen regulatory genes creA and areA and in the heterotrimeric G protein signalling genes fadA and ganB. In contrast, the nitrogen regulating transcription factor MeaB was involved in the yeast-extract-mediated repression of autolysis. Reverse transcriptase - polymerase chain reaction (RT-PCR) experiments demonstrated that MeaB affects the FluG-BrlA sporulation regulatory pathway by affecting transcription of brlA, a gene also initiating the autolytic cell wall degradation in this fungus.

Regulation of autolysis in Aspergillus nidulans.

In terms of cell physiology, autolysis is the centerpiece of carbon-starving fungal cultures. In the filamentous fungus model organism Aspergillus nidulans, the last step of carbon-starvation-triggered autolysis was the degradation of the cell wall of empty hyphae, and this process was independent of concomitantly progressing cell death at the level of regulation. Autolysis-related proteinase and chitinase activities were induced via FluG signaling, which initiates sporulation and inhibits vegetative growth in surface cultures of A. nidulans. Extracellular hydrolase production was also subjected to carbon repression, which was only partly dependent on CreA, the main carbon catabolite repressor in this fungus. These data support the view that one of the main functions of autolysis is supplying nutrients for sporulation, when no other sources of nutrients are available. The divergent regulation of cell death and cell wall degradation provides the fungus with the option to keep dead hyphae intact to help surviving cells to absorb biomaterials from dead neighboring cells before these are released into the extracellular space. The industrial significance of these observations is also discussed in this paper.

Heterotrimeric G protein mediated regulation of proteinase production in Aspergillus nidulans.

Extracellular proteinase production induced by carbon starvation was studied in a series of heterotrimeric G protein signaling pathway mutants of Aspergillus nidulans. All the mutants tested--including deltafadA (Galpha), deltasfaD (Gbeta), deltagpgA (Ggamma) and deltasfgA (regulator of FadA signaling)--showed an elevated proteinase production after glucose depletion. Our results strongly support the view that during growth, FadA/SfaD/GpgA G protein signaling inhibits proteinase production via both Galpha and Gbetagamma subunits, and all conditions, which are not sufficient to support vegetative growth and, hence, inhibit this type of G protein signaling, elevate extracellular proteinase activities.