The (lamin B1), and HTRA1 (high temperature

The effect of single-walled carbon nanotubes (SWCNTs) on the expression
of a subset of immune response, apoptosis and cell proliferation –associated genes
was studied in normal human astrocytes (line NHA/TS). In the cells treated with
SWCNTs (2, 10 and 50 ng/ml of medium for 24 h) we observed a strong
dose-dependent down-regulation of the expression of a cell surface glycoproteins
HLA-DRA (major histocompatibility complex, class II, DR alpha) and HLA-DRB1. At
the same time, the expression of HLA-F (major histocompatibility complex, class
I, F), LMNB1 (lamin B1), and HTRA1 (high temperature requirement A1) genes as
well as the level of miR-190b and miR-7 was up-regulated in NHA/TS subjected to
different concentrations of SWCNTs. After 24 h of treatment with SWCNTs we
detected a dose-dependent suppression of PHLDA2 (pleckstrin homology-like
domain, family A, member 2) gene expression in these cells. Obtained
data show that SWCNTs
may affect an immune response, in particular through suppression of HLA-DRA and HLA-DRB1 gene expressions
and that miR-190b and miR-7 possibly participated in this suppression. Deregulation
of lamin B1 expression indicates the possibility of alterations in genome
stability following treatment of astrocytes with SWCNTs. Thus, more caution is needed in biomedical
application of SWCNTs.

Keywords: Single-walled
carbon nanotubes, HLA-DRA expression, Normal human astrocytes

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Introduction

Carbon nanotubes have distinctive and remarkable material properties,
different from bulk materials with the same chemical composition, and potential
technological applications, including those in biology and medicine (He et al., 2013; Prylutska et al., 2013; Guo et al., 2017;
Harvey et al., 2017). These nanotubes have recently emerged as a new
option for cancer treatment, bioengineering, as well as gene therapy, but
inconsistent data on cytotoxicity and limited control over functionalized
carbon nanotubes behavior currently restrict predictability of such applications
(Firme and Bandaru, 2010). Carbon nanotubes have a
highly hydrophobic surface and a non-biodegradable nature that contributes to
their reduced biocompatibility, limiting their biomedical applications, with
growing concerns about their chronic toxicity (Prylutska et al., 2008; Tejral et
al., 2009; Yang et al., 2009; Minchenko et al., 2016; Kobayashi et al., 2017). It was
reported that different variants of these nanotubes exhibit different toxicity both in vitro and in vivo and
that intratracheal administration of single-walled carbon nanotubes (SWCNTs)
resulted in inflammation (Uo et al., 2011). The toxicity of carbon nanotubes is attributed to
their physicochemical properties, including structure, length and aspect ratio,
surface area, degree of aggregation, extent of oxidation, surface topology,
bound functional groups, concentration, and dose offered to cells or organisms (Tejral et al., 2009; Uo et al., 2011). It is
interesting to note that carbon
nanotubes can elicit toxicity through numerous mechanisms: membrane damage, DNA
damage, oxidative stress, changes in mitochondrial activities, altered
intracellular metabolic routes, and protein synthesis Yuan et al., 2011, 2012; Shvedova
et al., 2012; Ahmadi et al., 2017; Guo et al., 2017). Recently, it was
shown that functionalized SWCNTs induce oxidative stress in human circulating leukocytes (Kermanizadeh et al., 2018). However,
their cytotoxicity is significantly controversial, with a large number of
studies reporting altered toxic responses to SWCNTs both in vitro and in vivo (Uo et al., 2011).

Previously, we have shown that SWCNTs affect
the expression of genes related to cell cycle control and proliferation in U87 glioma
cells (Minchenko
et al., 2016).
In particular, a strong time and
dose-dependent suppressive effect was shown for CCND2 gene expression. A less prominent, but still
statistically significant effect was shown for PFKFB3, PFKFB4,
and PARVB genes. Thus, our
previous results demonstrated that treatment of cells with SWCNTs is far from neutral. To the
contrary, it led to noteworthy changes in the expression of some key cell cycle
regulator genes. In
this study we investigate the effect of SWCNTs on the expression of genes associated
with immune response, cell proliferation and apoptosis in normal human
astrocytes (line NHA/TS). Following genes were selected: HLA-DRA (major histocompatibility complex, class II, DR
alpha), HLA-DRB1, HLA-F (major histocompatibility complex, class I, F), LMNB1 (lamin B1), HTRA1 (high-temperature requirement A
serine peptidase 1), PHLDA2 (pleckstrin homology-like domain,
family A, member 2) and PLOD2 (procollagen-lysine, 2-oxoglutarate
5-dioxygenase 2) genes as well as micro RNA miR-190b and miR-7.
The HLA-DRA and HLA-DRB1 genes encode the alpha and beta1 subunits of
HLA-DR, which create a heterodimer. Both subunits anchored in the membrane and their co-regulated expression is mediated by the MHCII RNA operon and
is controlled by FOXP1 (Pisapia et al., 2013; Brown et al., 2016). It presents peptides
derived from extracellular proteins and thus plays an important role in the
immune response. HLA-DR gene expression is reduced in cancer and in sepsis and
correlates with impaired TNF? response (Cajander
et al., 2013; Leite et al., 2014; Winkler et al., 2017). Moreover, miR-7 and
miR-190b, which have relation to cancer cell proliferation and invasion and interact with
3′-untranslated region of IGF1 and some other mRNA, also recognize specific
sites in the HLA-DRA and HLA-DRB1
mRNAs (Hung et al., 2014; Tang et al., 2014). Human leukocyte antigen HLA-F is a
non-classical HLA-class I molecule, which has attracted attention as an
important immunosuppressive molecule. Its expression is correlated with tumor
cell invasion and metastasis (Xu et al., 2013; Ishigami et al.,
2015). HLA-F expression was found to be enhanced in
gastric adenocarcinoma, breast cancer, esophageal squamous cell carcinoma,
hepatocellular carcinoma, and neuroblastoma (Morandi
et al., 2013; Zhang et al., 2013; Harada et al., 2015; Martínez-Canales et al.,
2017). HLA-F is also associated with susceptibility to type 1 diabetes: its overexpression in islet cells is
a hallmark in the immunopathogenesis of type 1 diabetes (Richardson et al., 2016).

Nuclear lamina protein
LMNB1 constitutes one of the major structural proteins in the lamina mesh. It is involved in the maintenance of
nuclear stability, chromatin structure, and regulation of gene expression (Young et al., 2014; Camps et al., 2015). This
protein participates in a senescence program (Hernandez-Segura
et al., 2017). Cellular senescence is a state of irreversibly arrested
proliferation, often induced by genotoxic stress and senescent cells
participate in a variety of physiological and pathological conditions,
including tumor suppression (Hernandez-Segura et al.,
2017). It was also shown that fragment of the nuclear protein lamin B1
can non-covalently attach to SWCNTs and deliver this nanostructure to the
nucleus due to its exposed nuclear localization sequence (Boyer et al., 2016).

High-temperature
requirement A serine peptidase 1 (HTRA1), which is also known as serine
protease with IGF-binding domain PRSS11, is a stress responsive enzyme, that regulates the availability of
insulin-like growth factors (IGFs) by cleaving IGF-binding proteins. HTRA1 is also associated with malignancy (Zhu et al.,
2010; Xia et al., 2013; Minchenko et al., 2015). It was also shown that microRNA miR-30e
and miR-181d contribute to the dynamic regulation of HTRA1 expression and
Radial Glia cell proliferation (Nigro et al., 2012). Recently
it was shown that calcium phosphate nanoparticles can transfer HTRA1 protein into MG-63
cells but the uptake pathway for dissolved HTRA1 and HTRA1-loaded
nanoparticles is
different (Rotan et al., 2017).

The PHLDA2 gene is associated
with glioma and other
cancer types (Altinoz et al., 2016). Overexpression of this gene inhibits trophoblast proliferation, migration
and invasion, and induces apoptosis (Jia et al., 2016). The PLOD2 is involved in the formation
of the stabilized collagen cross-link and thus is associated with the stiffness
of the extracellular matrix. It was also shown to play a role in the
development of different types of cancers such as hepatocellular carcinoma,
breast cancer and sarcoma (Miyamoto et al., 2016; Du
et al., 2017). Interestingly, PLOD2 is directly regulated by microRNAs:
miR-26a-5p and miR-26b-5p (Miyamoto et al., 2016; Du
et al., 2017).

In this study we aim to investigate the effect
of SWCNTs on
the expression of a subset of genes associated with immune response and some proliferation
related genes in normal human astrocytes for analysis of these nanoparticles
genotoxicity.

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