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Objective. Nanographene oxide, an oxidation derivative of graphene, is considered to be one of the nanomaterials attractive for biomedical applications, although this nanomaterial is toxic. The increasing exploitation of graphene-based materials necessitates a comprehensive evaluation of the potential impact of these materials on the human health. Moreover, it is necessary to investigate in detail the mechanisms of its toxic effect on living cells particularly at the genome level. The present study aimed to evaluate the impact of low doses of nanographene oxide on the expression of key regulatory genes in normal human astrocytes. Methods. Normal human astrocytes, line NHA/TS, were exposed to low doses of nanographene oxide (1 and 4 ng/ml) for 24 h. RNA was extracted from the cells and used for cDNA synthesis. The expression levels of NAMPT, TSPAN13, BCAR3, BRCA1, PTGS2, P4HA1, and P4HA2 mRNAs as well as microRNAs were measured by quantitative polymerase chain reaction. Results. It was found that the low doses of nanographene oxide induced a dysregulation in the expression of the key regulatory genes in normal human astrocytes in dose-dependent (1 and 4 ng/ml) and gene-specific manner. Nanographene oxide also strongly suppressed the expression of NAMPT, BCAR3, and TSPAN13 genes and significantly up-regulated BRCA1, PTGS2, P4HA1, and P4HA2 ones with a more significant effect in P4HA1 and P4HA2 genes. The expression of miR-96-5p and miR-145-5p was also down-regulated in astrocytes treated with nanographene oxide in a dose-dependent manner. Conclusion. The data obtained demonstrate that the low doses of nanographene oxide disturbed the genome functions by changing the expression levels of key regulatory genes in gene-specific and dose-dependent manner. Moreover, a higher dose of nanographene oxide induced more pronounced changes in expression of genes indicating for both genotoxic and neurotoxic possible effects in the normal human astrocytes.

Objective. Single-walled carbon nanotubes (SWCNTs) are able to cross the blood-brain barrier, penetrate through the cell membrane, and accumulate in the cell nucleus, which purposefully allows their use in the health sciences as imaging probes and drug carriers in the cancer therapy. The aim of this study was to investigate the effect of low doses of SWCNTs on the expression of microRNAs associated with the cell proliferation and the brain development in zebrafish (Danio rerio) embryos. Methods. The zebrafish embryos (72 h post fertilization) were exposed to low doses of SWCNTs (2 and 8 ng/ml of medium) for 24 or 72 h. The microRNAs (miR-19, miR-21, miR-96, miR-143, miR-145, miR-182, and miR-206) expression levels were measured by quantitative polymerase chain reaction analysis. Results. It was found that low doses of SWCNTs elicited dysregulation in the expression of numerous cell proliferation and brain development-related microRNAs (miR-19, miR-21, miR-96, miR-143, miR-145, miR-182, and miR-206) in dose- (2 and 8 ng/ml of medium) as well as malformations in the zebrafish embryos brain development in a time-dependent (24 and 72 h) manner. Conclusion. Taken together, the present data indicate that the low doses of SWCNTs disturbed the genome functions and reduced the miR-19, miR-21, miR-96, miR-143, miR-145, miR-182, and miR-206 expression levels in dose- and time-dependent manners and interrupted the brain development in the zebrafish embryos indicating for both the genotoxic and the neurotoxic interventions.

Graphical abstract, Highlights • Low doses of SWCNTs affect the expression of numerous genes in normal astrocytes. • Low doses of SWCNTs interrupt the expression of various microRNAs in astrocytes. • SWCNTs affect the gene expressions through induction of endoplasmic reticulum stress. • Low doses of SWCNTs disturb the genome stability showing the genotoxic effects. • SWCNTs at low doses may have potential exposure-related risks for human health., The unique properties of single-walled carbon nanotubes (SWCNTs) make them viable candidates for versatile implementation in the biomedical devices. They are able to cross the blood–brain barrier, enter cells and accumulate in cell nuclei. We studied the effect of these carbon nanoparticles on the expression of genes associated with endoplasmic reticulum stress and proliferation, cell viability and cancerogenesis as well as microRNAs in normal human astrocytes. We have shown that treatment of normal human astrocytes by small doses of SWCNTs (2 and 8 ng/ml of medium for 24 hrs) affect the expression of DNAJB9, IGFBP3, IGFBP6, CLU, ZNF395, KRT18, GJA1, HILPDA, and MEST mRNAs as well as several miRNAs, which have binding sites at 3′-UTR of these mRNAs. These changes in the expression profile of individual mRNAs introduced by SWCNTs are dissimilar in magnitude and direction and are the result of both transcriptional and posttranscriptional mechanisms of regulation. It is possible that these changes in gene expressions are mediated by the endoplasmic reticulum stress introduced by carbon nanotubes and reflect the disturbance of the genome stability. In conclusion, the low doses of SWCNTs disrupt the functional integrity of the genome and possibly exhibit a genotoxic effect.

Objective. The aim of the present investigation was to study the expression of genes encoding homeobox proteins ZEB2 (zinc finger E-box binding homeobox 2), TGIF1 (TGFB induced factor homeobox 1), SPAG4 (sperm associated antigen 4), LHX1 (LIM homeobox 1), LHX2, LHX6, NKX3-1 (NK3 homeobox 1), and PRRX1 (paired related homeobox 1) in U87 glioma cells in response to glucose deprivation in control glioma cells and cells with knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), the major pathway of the endoplasmic reticulum stress signaling, for evaluation of it possible significance in the control of glioma growth through ERN1 signaling and chemoresistance. Methods. The expression level of homeobox family genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation condition by real-time quantitative polymerase chain reaction. Results. It was shown that the expression level of ZEB2, TGIF1, PRRX1, and LHX6 genes was up-regulated in control glioma cells treated by glucose deprivation. At the same time, the expression level of three other genes (NKX3-1, LHX1, and LHX2) was down-regulated. Furthermore, ERN1 knockdown of glioma cells significantly modified the effect glucose deprivation condition on the expression almost all studied genes. Thus, treatment of glioma cells without ERN1 enzymatic activity by glucose deprivation condition lead to down-regulation of the expression level of ZEB2 and SPAG4 as well as to more significant up-regulation of PRRX1 and TGIF1 genes. Moreover, the expression of LHX6 and NKX3-1 genes lost their sensitivity to glucose deprivation but LHX1 and LHX2 genes did not change it significantly. Conclusions. The results of this investigation demonstrate that ERN1 knockdown significantly modifies the sensitivity of most studied homeobox gene expressions to glucose deprivation condition and that these changes are a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to glioma cell growth and possibly to their chemoresistance.

Objective. The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations. Methods. The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction. Results. It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3’-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells. Conclusions. Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.

Objective. The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation. Methods. The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction. Results. It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition. Conclusions. Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.