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THE NATURE OF CHANGES IN mRNA EXPRESSION AND THE RELATIVE CONTENT OF SELENOPROTEINS IN A MOUSE MODEL WITH TAA-INDUCED FIBROSIS AND HEPATOCELLULAR CARCINOMA (HCC)
E.G. Varlamova*, E.A. Turovsky*
Federal State Institution of Science Institute of Cell Biophysics, Russian Academy of Sciences, 3 Institutskaya St., Pushchino, Moscow Region, 142290, Russia.
* Corresponding authors: [email protected], [email protected]
Abstract. This work is devoted to the study of mRNA expression patterns and the relative content of selenoproteins in mice with TAA-induced liver fibrosis and TAA-induced HCC. The main objective of this study is to evaluate the activation or suppression of selenoprotein synthesis during HCC progression and directly in the tumor in one TAA-treated mouse model, which is a pilot study and allows us to closely approximate the situation observed during HCC progression in vivo. It was found that as HCC progresses, there is an increase in the mRNA expression of thioredoxin reductases TXNRD1 and TXNRD2, deiodinase DIO3, glutathione peroxidases GPX1, GPX2, GPX4, and an inverse correlation in the expression of mRNA was characteristic of GPX3. In addition, the mRNA expression of endoplasmic reticulum resident selenoproteins: SELENOM, SELENON, SELENOT and SELENOS changed significantly. Also, in tumor liver samples and directly in the tumor itself, an increase in the expression of the selenoprotein SELENOP was recorded. The information obtained from the results of this work will significantly complement the existing data on the role of mammalian selenoproteins in various liver pathologies and in oncogenesis in general.
Keywords: selenoproteins, liver fibrosis, hepatocellular carcinoma.
List of Abbreviations
AKT - Protein kinase B ATF-4 - transcription factor with a bZIP leucine zipper domain, a member of the ATF/CREB family of b-ZIP proteins
BIM - proapoptotic protein of the BCL-2 family, interacting with the antiapoptotic proteins BCL-2 and BCL-XL
CHOP - proapoptotic transcription factor, member of the DNA-binding transcription factor CAAT/enhancer-binding protein family DIO - deiodinases
ER-stress - stress of Endoplasmic Reticu-lum
GADD34 - growth retardation and DNA damage gene-34
GPX - glutathione peroxidases HCC - hepatocellular carcinoma KD - knockdown
MAP3K5 - mitogen-activated protein kinase kinase 5
MAPK-8 - mitogen-activated protein kinase 8 NF-kB - universal transcription factor that controls the expression of immune response, apoptosis and cell cycle genes
OST - oliosaccharyltransferase complex PERK - PKR-like ER kinase, also known as PEK, EIF2AK3
PUMA - p53 up-modulator of apoptosis ROS - Reactive oxygen species RYR1 - ryanodine receptor SELENO - selenoprotein Se - selenium TAA - thioacetamide TXNRD - thioredoxin reductases UPR - cellular response to an increase in misfolded proteins
Introduction
There are several hypotheses about the anticancer activity of the important trace element selenium (Se), but the main mechanism of cytotoxicity is the generation of oxidative stress through the intracellular redox cycling of selenide, a Se metabolite, with oxygen and cellular thiols, which leads to the formation of non-stoi-chiometric amounts of superoxide and cellular disulfides.
However, Se is a component of 25 mammalian selenoproteins and can exert its oncogenic
and anti-oncogenic functions through them. To date, a large number of works have been accumulated indicating the various functions of sele-noproteins in processes associated with carcino-genesis of various etiologies (Varlamova & Cheremushkina, 2017; Kuznetsova et al., 2018; Zhang et al., 2023; Varlamova et al., 2016; Varlamova et al., 2017; Short & Williams, 2017; Davis, 2012; Varlamova, 2018). At the same time, it has been repeatedly demonstrated that, depending on the activity of selenoproteins, their functions can differ significantly in various oncological diseases and using the example of a wide variety of cancer cell lines (Tsuji et al.., 2015; Irons et al., 2010; Hatfield et al., 2009).
Early observational studies have shown that people with adequate levels of Se in their diet or body tissues have a lower risk of developing cancer, and plasma Se levels may decline before some cancers develop (Sundstrom et al., 1984; Schwartz, 1975). However, a clinical study showed that the risk of developing cancer in people with the highest quintile of serum Se was half that of people with the lowest quintile (Willett et al., 1983). However, some clinical studies have concluded that Se supplementation does not reduce the overall incidence of cancers such as lung, bladder and prostate cancer, with the exception of liver cancer (Lotan et al., 2012; Reid et al., 2002; Lippman et al., 2009).
Se concentration in the liver reflects the level of intestinal absorption. Despite the fact that the main selenoprotein synthesized by the liver is SELENOP, which enters the bloodstream and supplies Se to other tissues and organs (Leiter et al., 2022; Gharipour et al., 2018; Hill et al., 2012), it is also important to study the functions of all other selenoproteins in such serious liver pathologies as fibrosis and HCC.
This work is devoted to the study of seleno-protein mRNA expression patterns in the liver using models of TAA-induced fibrosis and hepatocellular carcinoma (HCC) in mice; in addition, their expression was assessed directly in tumor tissue. It is known that the liver is the largest gland that regulates a large number of diverse physiological processes, therefore it is extremely important to develop effective drugs for the treatment of various liver pathologies,
which include viral and non-viral hepatitis, fi-brosis, steatosis, cirrhosis, HCC, etc.
The results obtained in this work will contribute to the understanding of the role of each of the selenoproteins in fibrosis and HCC, as well as their activity in tumor tissue. The main advantage of this work is that it is the first work to evaluate mRNA expression within a single model in vivo, which gives us insight into how the expression patterns of these selenoproteins change as liver pathologies progress.
Materials and Methods
Materials
Thioacetamide, 98%, (Sigma-Aldrich, #172502), qPCRmix-HS SYBR (Evrogen #PK147S), MMLV RT kit (Evrogen, #SK021), ExtractRNA (Evrogen, #BC032). The following reagents from «Evrogen», Russian Federation were used in the work: synthesis of gene-specific oligonucleotides, ExtractRNA reagent (#BC032), MMLV-RT kit for cDNA synthesis (#SK021) qPCRmix-HS SYBR mixture for PCR in real (#PK147S), DNA length markers (#NL001, #NL002). The following primary antibodies were used in the work: anti-GAPDH (Thermo FS, #MA5-35235), anti-TXNRD1 (Thermo FS, #PA5-28886), anti-DIO3 (Thermo FS, #PA5-26537), anti-GPX1 (Thermo FS, #702762), anti-GPX2 (Thermo FS, #PA5-145241), anti-GPX3 (Thermo FS, #PA5-145241), anti-GPX4 (Thermo FS, #MA5-32827), anti-SELENOM (Thermo FS, #PA5-72639), anti-SELENOP (Thermo FS, #PA5-112707), anti-SELENON (Thermo FS, #PA5-69493) and mouse anti-rabbit Ab (Abcam, #99697) were used as secondary antibodies. PVDF membranes (Thermo FS, #LC2005) were used for protein transfer. Protein Concentrators (Thermo FS, #88517) were used to concentrate proteins in tissue lysates.
Animals and injection protocol
All animal experiments were performed in accordance with the experimental protocols approved by the Bioethics Committee of the Institute of Cell Biophysics (Approval ID: 1/092022, date: 2022-9-08). Experiments were performed in accordance with the Rules of La-
boratory Practice for the Care and Use of Laboratory Animals and Directive 2010/63 EU of the European Parliament on the protection of animals used for scientific purposes. The work used male mice of the C57BL/6J line (weight 15 g, 2-3 weeks), which were purchased from the Stolbovaya branch of the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency" of Russia. All animals are certified in accordance with the regulations on quality control of laboratory animals, nurseries and experimental biological clinics (vivariums).
To induce liver fibrosis, male C57BL/6J mice (weight 15 g, 2-3 weeks) were intraperito-neally injected with TAA (150 |ig/g mouse weight) twice a week for 3 months. This protocol was developed by us and used previously (Varlamova et al., 2023). To induce liver cancer, mice were injected with TAA (150 |ig/g mouse weight) twice a week for 6 months. The number of animals in each experimental group was 10. The experimental protocol is schematically presented in Fig. 1.
RNA isolation, reverse transcription, RT-PCR
RNA isolation was carried out using the Ex-tractRNA reagent (Evrogen), intended for the isolation of total RNA from biological samples. This reagent is a monophasic solution of phenol and guanidine isothiocyanate. Tissue samples
were homogenized by pipetting in 1 ml of Ex-tractRNA reagent, and then total RNA was isolated according to the manufacturer's protocol. The quality of RNA isolation was checked using electrophoresis in a 1% agarose gel, as well as using a spectrophotometer at a wavelength of 260 nm. To prevent contamination of RNA samples with genomic DNA, they were treated with DNase I at 37 °C for 1 h, after which the enzyme was inactivated by adding 50 mM EDTA to the mixture and heating to 60 °C for 10 min.
The reverse transcription reaction was carried out according to the protocol and using a first-strand cDNA synthesis reagent kit containing murine leukemia virus (MMLV) reverse transcriptase. The content of total RNA (0.5-2 p,g) was controlled by performing a parallel amplification reaction using primers specific to the reference gene.
The resulting cDNA was used as a template for RT-PCR using the qPCRmix-HS SYBR mixture containing the intercalating dye SYBR Green I. The amplification reaction was carried out at the following temperature conditions: 95 °C - 1 min; 95 °C - 10 sec, 60 °C - 10 sec, 72 °C - 15 sec (35 cycles). The relative level of gene expression (RUE - the level of expression of the gene under study relative to the expression of the reference gene) in each cell line was determined by the formula. The change in the level of mRNA expression of the studied proteins, before and after treatment, was deter-
Fig. 1. Schematic protocol for intraperitoneal injection of TAA into mice to induce liver fibrosis and HCC
mined by the formula TUE = 2-AA Ct, where AACt is the difference in ACt values for each gene before and after cell treatment. Each experimental cycle was repeated three or more times. When performing RT-PCR, reference gene encoding glyceraldehyde-3-phosphate de-hydrogenase was used. The sequences of all primers used in RT-PCR are given in Table 1.
Western blotting
Tissue samples were homogenized in lysis buffer (20mM Tris, 150mM NaCl, 2mM EDTA, 1mM PMSF, 1% Triton-X100), incubated on ice for 15 min, then centrifuged for 30 min at 20,000 g at 4 °C, and the supernatant fraction was concentrated. 70 p,g of total protein was added per well for each sample. Proteins were separated by PAGE electrophoresis in a 12.5% polyacrylamide gel, after which the proteins were electro-transferred to a PVDF membrane. The membranes were blocked in 5% BSA for 5 h at room temperature or 15 h at 4 °C, then the membranes were incubated with primary antibodies in 3% BSA for 2 h at room temperature or 15 h at 4 °C. After thoroughly washing the membranes in 1x PBST, they were incubated with secondary antibodies for 2 h at room temperature, then washed thoroughly, im-munoreactive bands were visualized by determining peroxidase activity using DAB staining (0.05% DAB in 1x PBS + 10 ^l 30% peroxide hydrogen).
Statistical analysis
Microsoft Excel and GraphPadPrism 5 software were used to analyze the data, create graphs and process statistics. Protein evaluation was carried out in different samples according to the Lowry method. The protein concentration was calculated from a standard curve constructed using 1 mg/ml BSA solution. Values were given in the work as the mean ± standard deviation of at least three independent experiments. Differences were considered significant at P < 0.05. Protein expression was quantified using ImageJ software. Origin 8.5 (Microcal Software Inc., Northampton, MA) and Prism 5 (GraphPad Software, La Jolla, CA) were used for plotting and statistical processing. The sig-
nificance of differences between groups of experiments was determined using Student's t-test, and within groups - Student's t-test. Differences were considered significant: *** at p < 0.001, ** at p < 0.01, * at p < 0.05, n/s -differences were not significant.
Results
The mRNA expression levels of thioredoxin reductases TXNRD1 and TXNRD2 are upregu-lated in HCC in the liver and tumor
As can be seen in Fig. 2A, real-time PCR results revealed that TXNRD1 mRNA expression was increased more than 10-fold in cancerous liver and 16-fold in the tumor itself. In addition, a slight increase in mRNA expression (2-fold) was characteristic of TXNRD2 in cancerous liver and tumor. However, in fibrous liver, the mRNA expression of both thioredoxin reduc-tases did not differ from the control (healthy liver). For TXNRD3, in all studied samples we did not record any significant differences compared to the control. Since TXNRD1 mRNA changes most significantly, we additionally checked how its relative content changes in all studied samples. Presented in Fig. 2.B and C results confirm the real-time PCR data. There is an increase in the relative amount of TXNRD1 in cancerous liver and tumor.
The mRNA expression patterns of four glu-tathione peroxidases vary differently in fibrotic, cancerous livers and tumors
According to the results of PCR analysis, it can be stated that the expression of GPX1 mRNA increases 3 times in cancerous liver and tumor, while its expression does not change with fibrosis (Fig. 3A). The expression of GPX2 mRNA increased in all studied samples: threefold in fibrotic and cancerous livers, and fivefold in tumor tissue. For GPX3, the mRNA expression patterns were opposite: maximum expression was observed in fibrosis (almost 7fold increase); in cancerous liver, a 3-fold increase in expression was observed. In the tumor, the level of GPX3 mRNA expression did not differ from control. GPX4 was characterized by an increase in mRNA expression by 2.5-3 times in all studied samples. Since signif-
Fig. 2. The nature of changes in thioredoxin reductase mRNA expression patterns and relative amounts TXNRD3 in mice with TAA-induced fibrosis and HCC. A - results of real-time PCR; B - immunoblotting results; C - quantitative immunoblotting calculation. The level of expression in control cells was taken as 1. Statistical significance was assessed using paired t-test. Comparison of experimental groups with control is indicated by black asterisks. Unlabeled columns - significant values. n/s - data not significant (p > 0.05), * p < 0.05, ** p < 0.01, n = 3
Fig. 3. The nature of changes in deiodinase mRNA expression patterns and relative amounts DIO3 in mice with TAA-induced fibrosis and HCC. A - results of real-time PCR; B - immunoblotting results; C - quantitative immunoblotting calculation. The level of expression in control cells was taken as 1. Statistical significance was assessed using paired t-test. Comparison of experimental groups with control is indicated by black asterisks. Unlabeled columns - significant values. n/s - data not significant (p > 0.05), * p < 0.05, ** p < 0.01, n = 3
Fig. 4. The nature of changes in glutathione peroxidases mRNA expression patterns and relative amounts of glutathione peroxidases in mice with TAA-induced fibrosis and HCC. A - results of real-time PCR; B -immunoblotting results; C - quantitative immunoblotting calculation. The level of expression in control cells was taken as 1. Statistical significance was assessed using paired t-test. Comparison of experimental groups with control is indicated by black asterisks. Unlabeled columns - significant values. n/s - data not significant (p > 0.05), * p < 0.05, ** p < 0.01, n = 3
icant changes in expression were found for GPX2 and 3, we decided to check how the relative abundance of these enzymes changes in the studied samples. In Fig. 3 B and C it is clear that Western blotting results correlate with PCR results.
Different patterns of mRNA expression of the deiodinase family selenoproteins in fibrotic, cancerous livers and tumors
Real-time PCR results indicate that DIO1 mRNA expression does not change depending on the stage of liver disease and in the tumor, while DIO2 is characterized by an almost 2-fold decrease in the expression level in cancerous liver. The absence of significant differences in DIO2 mRNA expression was observed in fibrosis and in tumor tissue. The patterns of DIO3 mRNA expression in the studied samples changed in the opposite way: in fibrotic liver, no significant differences from the control were observed, while in cancerous liver and tumor, an increase in mRNA expression was observed
by 9 and 10.5 times, respectively (Fig. 4A). The Western blot results confirmed the PCR data for DIO3 (Fig. 4 B and C).
Different patterns of mRNA expression of other selenoproteins
Significant changes in the expression of mRNA in the studied samples were characteristic of a number of other selenoproteins, for example, the patterns of mRNA expression of SE-LENOM, which is an ER resident, increases more than 2 times in fibrosis, in a cancerous liver does not significantly differ from the control, and in a tumor increases almost 10 times (Fig. 5A).
In addition, a slight increase in mRNA expression in tumor tissue was also characteristic of two other ER selenoproteins: SELENOT and SELENOS. For the selenoprotein SELENON, which is also an ER resident, it was found that the expression of its mRNA in fibrotic liver is increased twofold, in cancerous liver by 3.5 times, and in tumor tissue by 4.5 times (Fig. 5A).
Fig. 5. The nature of changes in selenoproteins mRNA expression patterns and relative amounts of some selenoproteins in mice with TAA-induced fibrosis and HCC. A - results of real-time PCR; B - immunoblot-ting results; C - quantitative immunoblotting calculation. The level of expression in control cells was taken as 1. Statistical significance was assessed using paired t-test. Comparison of experimental groups with control is indicated by black asterisks. Unlabeled columns - significant values. n/s - data not significant (p > 0.05), * p < 0.05, ** p < 0.01, n = 3
The Western blot results performed for the two selenoproteins SELENOM and SELENON correlate with real-time PCR data (Fig. 5B and C).
For another selenoprotein SELENOP, a decrease in mRNA expression by almost 3 times was observed in fibrotic liver, while in cancerous liver and tumor there was a sharp increase of more than 4 times, which corresponds to the results of immunoblotting presented in Fig. 4B and C. At the same time, a decrease in mRNA expression levels was observed in fibrous tissue (Fig. 5A), which was also confirmed by the results of Western blotting (Fig. 5B and C).
Discussion
In recent decades, the anticancer properties of Se-containing compounds of various natures have been actively studied. Among them, sodium selenite, methylselenic acid, selenomethionine, etc. occupy a special place (Goltyaev et al., 2020; Kuznetsova et al., 2018; Dauplais et al., 2024). However, Se-based nanoparticles and nanocomposites have significant ad-
vantages due to their low toxicity, high biocom-patibility, and bioactivity (Varlamova & Turovsky, 2021; Varlamova, 2024; Sampath et al., 2024). In addition, selenium nanoparticles doped with known anticancer drugs are widely used in preclinical practice and have significant prospects for use in clinical practice (Xia et al., 2020; Goltyaev & Varlamova, 2023; Varlamova et al., 2022). Much attention is also paid to the study of the role of selenoproteins in the regulation of carcinogenesis. It has been repeatedly proven that mammalian selenoproteins play an important role in carcinogenesis, and their expression changes significantly in cancer of various etiologies (Varlamova & Cheremu-shkina, 2017; Kuznetsova et al., 2018; Zhang et al., 2023; Varlamova et al., 2016; Varlamova et al., 2017; Short & Williams, 2017; Davis, 2012; Varlamova, 2018; Rua et al., 2023; Peters et al., 2018; Reszka, 2012; Callejon-Leblic et al., 2021; Varlamova et al., 2019; Diamond, 2019). To date, 25 selenoproteins have been identified in mammals, but the functions of many of them
have not yet been sufficiently studied, so the studies performed in this work are very relevant.
It is known that thioredoxin reductases are one of the main redox regulators in mammalian cells and essential selenoproteins (Iwasawa et al., 2011; Fernandes et al., 2009; Soini et al., 2001; Cañas et al., 2012; Cadenas et al., 2010; Esen et al., 2015; Fu et al., 2017). We showed that in mice with HCC, a significant increase in TXNRD1 was observed in the tumor tissue and liver (Fig. 1 A and B). This thioredoxin reduc-tase is known to be activated in many human malignancies and acts as a prognostic factor for many tumors, such as oral squamous cell carcinoma (Iwasawa et al., 2011), lung cancer (Fernandes et al., 2009; Soini et al., 2001), breast cancer (Cañas et al., 2012; Cadenas et al., 2010), astrocytomas (Esen et al., 2015). In addition, this enzyme may regulate the progression of HCC. Multivariate analysis showed that TXNRD1 is an independent prognostic bi-omarker for patients with HCC (Fu et al., 2017). In our work, TXNRD1 mRNA expression was increased only in cancer, but not in fi-brosis, which can be explained by metabolic reprogramming occurring in cancer. Improper activation of cellular signaling pathways and metabolism leads to increased oxidative stress, which requires activation of the antioxidant system, a key component of which is TXNRD1. In addition, it has been repeatedly shown that overexpression of TXNRD1 can reduce colony size and number and plays a crucial role in promoting cell proliferation and metastasis in HCC (Huang et al., 2022). TXNRD1 has been repeatedly shown to promote tumor growth, DNA replication, and oncogenicity (Yoo et al., 2006; Yoo et al., 2007), and knockdown of this selenoprotein increased the sensitivity of some cancer cells to chemotherapeutic drugs (Poerschke & Moos, 2011; Tobe et al., 2012). Thus, TXNRD1 may induce ROS production, promoting the development of HCC and drug resistance (Fu et al., 2017).
We also observed a slight upward trend in mRNA expression in cancerous livers and tumors for another member of the thioredoxin re-ductase family, TXNRD2 (Fig. 1A). It is a mi-
tochondrial thioredoxin reductase that, along with TXNRD1, is a key component of signaling pathways that control cell differentiation and cellular responses to stress. The data obtained in this work regarding the expression of TXNRD2 mRNA are consistent with many other works (Bu et al., 2021; Gencheva & Arnér, 2022; Yang et al., 2020; Dagnell et al., 2018), which further demonstrates the increase in the amount of TXNRD2 transcript in tumor tissue and liver in HCC.
The second reduction system in mammalian cells is the glutathione system, which usually complements the thioredoxin system. According to our data, significant changes in the mRNA expression of all glutathione peroxidases were observed in all studied samples (Fig. 2 A and B). It is well known that glutathione peroxidases are selenium-dependent enzymes that participate in the detoxification of ROS by catalyzing the reduction of hydrogen peroxide, lipid peroxidase due to reduced glutathione (Brigelius-Flohé & Flohé, 2020; Pei et al., 2023; Brigelius-Flohé & Maiorino, 2013).
The most common and widely expressed in the cytosol and mitochondria of various tissue types is GPX1 (Lubos et al., 2011). The role of this enzyme in tumorigenesis is controversial, since on the one hand, overexpression of GPX1 reduces the growth of cancer cells, inhibits the transmission of pro-survival signals through the AKT pathway and weakens the expression of pro-inflammatory mediators (Capdevila et al., 1995; Handy et al., 2009; Liu et al., 2004). On the other hand, in a skin cancer model, it was shown that GPX1 overexpression correlated with the number of tumors and their growth rate (Klionsky et al., 2021; Yang et al., 2015). This work established an increase in GPX1 mRNA expression as liver cancer progresses and does not change with fibrosis. It is known that tumors often have higher levels of basal oxidative stress, and overexpression may enhance protection against ROS-based apoptosis. Our data regarding the increase in GPX2 mRNA expression as HCC progresses are consistent with other studies (Huang et al., 2017; Emmink et al., 2014; Suzuki et al., 2013). It has been shown that high expression of GPX2 in HCC
cells can increase their resistance to anticancer drugs (Tan et al., 2023). In addition, GPX2 was found to be upregulated in HCC tumor tissues and associated with early hepatocarcinogenesis (Suzuki et al., 2013).
A decrease in GPX3 mRNA expression as cancer progresses, as found in our work, was also demonstrated in other solid tumors (Kaiser et al., 2013; Qu et al., 2013). Of particular importance, promoter methylation occurs during HCC progression, suggesting that epigenetic modifications of GPX3 and reduction in its expression contribute to HCC development. This study showed that DNA methylation was observed in 46 of 60 HCC cases (Cao et al., 2015). Most likely, the decrease in GPX3 mRNA expression as HCC progresses, as established in our work, is also associated with hypermethyl-ation of the promoter of this selenoprotein. In addition, it was found that a decrease in GPX3 mRNA expression in serum can be used as a tumor marker (Yu et al., 2007; Yi et al., 2019).
This work also demonstrated that in liver pathologies, the levels of mRNA expression of two of the three existing deiodinases, DIO2 and DIO3, changed. We found that the expression of DIO2 mRNA increases more than three times in fibrous liver and decreases in cancerous liver, and in the tumor does not significantly differ from the control. Our data are consistent with the finding that DIO2 mRNA expression is rarely associated with neoplastic transformation and is upregulated in benign hyperfunctioning thyroid nodules and in thyroid tumors (Kim et al., 2003; Meyer et al., 2008). In addition, it has been shown that the functional activity of DIO2 is present in the tissues of the anterior pituitary gland, both in adenomas with varying secretory activity and in normal tissues (Baur et al., 2002; Itagaki et al., 1990). It has also been established that DIO2 is a prognostic marker for cutaneous squa-mous cell carcinoma (Alam & Ratner, 2001). However, there is no data regarding the expression of this enzyme in various liver pathologies, so at this stage it is difficult to explain the reason for such patterns of DIO2 expression in fibrosis and cancer.
Our results suggest that the mRNA expression and relative abundance of DIO3 changes in
the opposite manner to DIO2 (Fig. 3A, B and C). It is known that this enzyme is reactivated under certain physiopathological conditions in which cell proliferation increases, including cancer (Dentice et al., 2007; Hernandez et al., 1998; Dentice et al., 2012). Interestingly, a similar pattern of antagonistic expression of both de-iodinases was recorded previously (Dentice et al., 2012). It is known that the Wnt/p-catenin/T-cell factor signaling pathway transcriptionally induces the overexpression of DIO3 and simultaneously suppresses the expression of DIO2. In the opposite direction, P-catenin knockdown (KD) reduces DIO3 expression and simultaneously increases DIO2 expression (Nappi et al., 2021). It is possible that there is also an increase in P-catenin signaling in liver cancer, leading to upregulation of DIO3.
The expression of the selenoprotein SELE-NOP, according to the data obtained, increased in the cancerous liver and in the tumor, but in the fibrotic liver decreased by more than three times. This selenoprotein is synthesized in the liver, after which it enters the bloodstream and supplies other organs with Se (Leiter et al., 2022; Gharipour et al., 2018; Hill et al., 2012). It is believed that SELENOP plays a dual role in the liver: its level decreases during acute inflammation, which may be the case with fibrosis, as we have shown previously (Zhang et al., 2023). At the same time, low intake of Se into peripheral tissues can aggravate oxidative stress and inflammation (Polyzos et al., 2019). During long-term chronic inflammation and oxidative stress, SELENOP expression is upregulated and it tends to balance the response to oxidative stress and inflammation. Although there are a number of studies indicating that the concentration of Se and SELENOP in the liver and bloodstream decreases in direct proportion to the severity of liver disease: in fibrosis and cirrhosis its expression is higher than in HCC (Polyzos et al., 2019; Nangliya et al., 2015; Casaril et al., 1989; Polyzos et al., 2020). In any case, the available information is extremely insufficient to talk about the specific role of SELENOP in these liver pathologies.
We also found that in mice with fibrosis the expression of SELENOM mRNA increases
twofold, while in the tumor itself it increases 10-fold, which is consistent with earlier studies (Jia et al., 2020; Varlamova et al., 2022). SE-LENOM in different tissues can regulate the activity of two antioxidant enzymes in different ways (Huang et al., 2016; Reeves et al., 2010; Varlamova et al., 2022). In addition, overexpression of SELENOM in HT22 and C8-D1A cells increases the concentration of cytosolic calcium in response to oxidative stress and, possibly, is involved in the regulation of apoptosis, blocking or delaying it (Hwang et al., 2008). Studies of the role of SELENOM in carcino-genesis are limited by data on the expression of this protein in hepatocellular carcinoma (HCC) cells (Guerriero et al., 2014). This study demonstrated overexpression of SELENOM in two liver cancer cell lines, HepG2 and Huh7, which is consistent with our in vivo model data obtained in this paper. It was also found that miR-138-5p overexpression is able to inhibit SELENOM expression, resulting in increased levels of hydrogen peroxide, maldialdehyde, and reduced catalase and superoxide dismutase activity (Chi et al., 2019).
SELENOM-KD in human glioblastoma cells, according to our previous results, contributed to increased expression of a number of key pro-apoptotic genes, two selenoproteins SELENOT and SELENOK and two glutathione peroxidases GPX1 and 2, as well as thioredoxin reductase 3 (TXNRD3). In addition, a significant increase in the expression of the transcription factor ATF-4 was observed, which may indicate the activation of the PERK signaling pathway UPR and an increase in ER-stress (Varlamova et al., 2022). Similar results on increased expression of proapoptotic genes with reduced SELENOM activity were demonstrated in HT22 hippocampal cells and C8-D1A cerebellar cells (Reeves et al., 2010). Thus, we can conclude that overexpression of SELENOM in a tumor entails undesirable consequences associated with a decrease in the level of antioxidant enzymes, a decrease in the expression of pro-apoptotic genes, markers of ER-stress, and abnormal regulation of calcium homeostasis.
In addition, two ER-resident selenoproteins, SELENOT and SELENOS, had similar mRNA
expression profiles during the development of HCC in mice. Their expression levels were doubled only in tumor tissue. SELENOT is known to interact with the components of the OST complex (KSR2, STT3A, and OST 48) and participate in the formation of a mixed di-sulfide bond between the glycoprotein moving through the translocon (Hamieh et al., 2017). SELENOT-KD contributed more than a twofold decrease in the expression of a number of proapoptotic genes (CHOP, PUMA, BIM and GADD34). At the same time, a significant increase in mRNA expression (more than three times) was typical for the genes of two MAPK kinases MAP3K5 and MAPK-8, but the expression of ER-stress markers remained practically unchanged (Varlamova et al., 2022). In addition, SELENOT-KD contributed to the functional disorders of the ER, reducing its ability to deposit Ca2+ ions (Grumolato et al., 2008).
A slight increase in the expression of SELENOS mRNA in tumor tissue can be aimed at preventing prolonged ER-stress, maintaining calcium homeostasis, reducing ROS, and controlling the normal functioning of mitochondria, as was shown in the example of Hepa 1-6 hepatoma cells (Li et al., 2018). Using HepG2 cells as an example, it was also shown that suppression of SELENOS mRNA expression leads to apoptotic death of these cancer cells. It has also been shown that SELENOS plays an important role in protecting HepG2 cells from ER-stress caused by pharmacological stress induc-ers (Zeng et al., 2008). SELENOS is also involved in the regulation of lipopolysaccharide-induced inflammatory response in HepG2 cells (Barnes & Karin, 1997).
In this work it was established that in all studied samples there was an increase in the expression of mRNA of the endoplasmic re-ticulum resident selenoprotein SELENON. This protein is a transmembrane glycoprotein containing an EF motif in its structure (Petit et al. , 2003), the C-terminal sequence is located in the lumen of the ER (Lescure et al., 2009). Its expression has been shown to increase during the proliferation of cells such as fibroblasts and myoblasts (Petit et al., 2003).
It is believed that the expression of SELE-NON can be regulated during ER stress in the cell, since 5 sites for binding to the transcription factor NF-kB were found in its promoter region (Baylin et al., 1998), as well as the binding site for the redox-sensitive factor AP-1 (Stoytcheva & Berry, 2009). Thus, the expression of the SELENON gene is most likely regulated by various cellular stressors (ROS, ER stress, cytokines) (Arbogast & Ferreiro, 2010), which, as a rule, increase during cancer development. In addition, it has been shown that SELENON may play an important role in protecting cells from oxidative stress and maintaining Ca2+ homeostasis, interacts with the ryanodine receptor RYR1, and can also neutralize hydrogen peroxide-induced inhibition of SERCA2b (Arbogast & Ferreiro, 2010; Appenzeller-Herzog & Simmen, 2016). Perhaps, in this way, this selenoprotein pro-
tects cancerous and fibrotic liver cells from death and enhances their proliferation.
Conclusions
In this work, for the first time, using one animal model, we show how the mRNA expression patterns of mammalian selenoproteins change as TAA-induced liver pathologies develop. Thus, an analysis of the expression levels of selenoproteins was carried out in mice with liver fibrosis, in livers with tumors, and in the tumors themselves. This is very important for understanding in which specific pathological processes selenoproteins can actively participate, which will serve as a significant addition to the already wide list of their functions in liver tumorigenesis.
Funding: the study was supported by the Russian Science Foundation grant No. 23-25-00030, https://rscf.ru/project/23-25-00030/.
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