Urbanavičiūtė-Orentė, Rūta

Glioma is malignant central nervous system tumor, and the most malignant glioma, glioblastoma (GBM) is one of the deadliest tumors that can cause death approximately to 93% of patients within 5 years after diagnosis. This study aimed to determine the expression of non-coding Y RNAs in glioma cell model after Temozolomide (TMZ) exposure and in patients’ tumor samples to assess RNY involvement in glioma therapy resistance in vitro, and diagnostic and prognostic significance in clinical cohort. Glioblastoma cell lines U251, A172, and LN229 were treated for 2 weeks with the alkylating agent Temozolomide. A number of 32 glioma patient tumor tissues were obtained for analysis: 23 patients had glioblastoma multiforme (grade 4, GBM) and 7 patients had diffuse astrocytoma (grade 2, DA). Total RNA was isolated using TRIzol reagent, and cDNA was synthesised from RNA using reverse transcriptase enzymes. Gene expression analysis of RNY1, 3, 4, and 5 was performed by real-time PCR with SYBR Green detection, enabling quantitative assessment of transcript levels. GAPDH, 18S rRNA, and ACTB were used as reference genes for data normalisation. A statistically significant upregulation of RNY1 and RNY4 was observed in grade 4 and grade 2 gliomas as compared to normal brain tissue, respectively. RNY4 expression was also higher in patients aged up to 55 years. In treated A172 cells, RNY1 expression was approximately 5-fold higher than in the control cells. In U251 cells treated with TMZ, RNY3 expression was 1.8-fold higher compared with the control. In contrast, in treated LN229 cells, RNY5 expression was approximately 7.7-fold higher than in the control cells. It was found that RNY1 and RNY4 may represent potential diagnostic biomarkers for gliomas, as their expression in tumor specimens differed significantly between grade 2 and grade 4 tumors. Treatment of different glioblastoma cell lines induced gene- and cell line–specific alterations in RNY expression. The marked increase of RNY1 expression in A172 cells suggests that this gene may be involved in cellular response mechanisms to treatment. Temozolomide-treated cells may be linked to the development and progression of glioma.

Įvadas Glioblastoma (GBM) yra viena iš labiausiai gąsdinančių diagnozių, kurią gali išgirsti pacientas. Net ir taikant įvairius gydymo būdus, įskaitant chirurginę rezekciją, spindulinę terapiją ir chemoterapiją, šiais smegenų navikais sergančių pacientų 5 metų išgyvenamumas siekia vos 5 %. Mažas gydymo efektyvumas atsiranda dėl glioblastomų heterogeniškumo ir didelio atsparumo terapijai [1]. Smegenų navikai skirstomi į I–IV laipsnius pagal tam tikrų histologinių požymių nebuvimą arba buvimą. Didžioji dauguma piktybinių gliomų atsiranda atsitiktinai, tačiau apie 5 % yra susijusios su genetiniais sindromais, tokiais kaip Gardnerio ir Turcot sindromas, arba piktybinių smegenų navikų paveldėjimas šeimoje. Šie navikai sudaro 60 % visų pirminių smegenų navikų, vidutiniškai pasitaikantys 6 iš 100 000 žmonių per metus [2]. YRNR (su Ro susijusi Y) nekoduojančių RNR klasė, buvo identifikuotos kaip įvairių piktybinių navikų biožymenys. Šiuo metu žinomos keturios labai konservatyvios žmogaus YRNR (RNY1, RNY3, RNY4 ir RNY5). YRNR dydis yra 80–110 nt, o dėl komplementarių 5′ ir 3′ galų jos turi kilpinės formos struktūrą. Jos yra funkciškai svarbios DNR replikacijai ir Ro60 (autoimuninių reakcijų aptinkamas baltymas) slopinimui. Nustatyta, kad RNY1 ir RNY3 slopinimas sumažina ląstelių proliferaciją. Taip pat buvo įrodyta, kad RNY5 fragmentai tarpląstelinėse pūslelėse sukelia ląstelių mirtį ir tokiu būdu gali padėti vėžio ląstelėms optimizuoti mikroaplinką proliferacijai ir invazijai [3]. Y-RNR raiškos lygio pokyčiai gali rodyti naviko buvimą ir gali būti naudojami kaip neinvazinės diagnostikos priemonė [4]. Tad šio darbo tikslas buvo ištirti YRNR molekulių raišką gliomos pacientų navikiniuose mėginiuose, siekiant įvertinti jų galimą diagnostinę ir prognostinę reikšmę gliomų vystymesi. Tyrimo uždaviniai:

1. Ištirti ir įvertinti YRNR molekulių raišką skirtingo piktybiškumo laipsnio gliomos pacientų navikiniuose mėginiuose taikant tikro laiko PGR metodą.
2. Įvertinti YRNR raiškos sąsajas su gliomos pacientų išgyvenimo trukme.
3. Įvertinti kiekvienos tiriamos YRNR raiškos sąsajas su gliomos pacientų klinikinėmis charakteristikomis. Metodai Buvo atliktas navikų susmulkinimas šaltos homogenizacijos metodu naudojant skystą azotą. Visuminės RNR išskyrimas naudojant „Trizol“ reagentą, kDNR sintezė iš RNR naudojant atvirkštinės transkriptazės fermentus. Genų raiškos analizė atlikta naudojant tikro laiko PGR metodą su SYBR Green detekcija, kuri leidžia kiekybiškai įvertinti genų raišką. Naudoti GAPDH ir 18sRNA referentiniai genai duomenų normalizavimui. Rezultatai buvo statistiškai įvertinti naudojant Kolmogorovo-Smirnovo testą. Vėliau buvo taikomas Mann-Whitney kriterijus kai duomenų pasiskirstymas nebuvo Gausinis bei Stjudento t kriterijus – kuomet duomenų skirstiniai buvo normalieji. Pacientų išgyvenimo trukmės analizė buvo atlikta naudojant Kaplano-Mejerio testą. Vertinant sąsajas tarp YRNR molekulių raiškos ir gliomos pacientų klinikinių charakteristikų pasitelkėme Chi-kvadrato (χ2 ) kriterijų. Statistiškai reikšmingais rezultatais buvo laikomi, kai p ≤ 0,05. Rezultatai Statistiškai reikšmingai padidėjusi RNY1 bei RNY3 raiška buvo stebima antroje stadijoje, lyginant šių molekulių raišką IV laipsnio gliomos navikiniuose mėginiuose. RNY3 ir RNY4 raiška buvo padidėjusi pacientams, kuriems mažiau nei 55 metai, o padidėjusi RNY1 raiška buvo susijusi su ilgesne pacientų išgyvenimo trukme. Išvados
4. Nustatyta, kad RNY1 ir RNY4 galimai yra potencialūs gliomų diagnostiniai žymenys, kurių raiška navikiniuose mėginiuose statistiškai reikšmingai skyrėsi tarp II laipsnio bei glioblastomos pacientų (p<0,05).
5. RNY1 turi prognostinį potencialą gliomų tyrimuose, kadangi jų raiška parodė sąsajas su pacientų išgyvenimo trukme (p<0,05).
6. Visos keturios tirtos YRNR molekulės galimai dalyvauja gliomagenezės procesuose, kadangi buvo stebimos visų tirtų molekulių raiškos sąsajos su gliomos pacientų klinikinėmis charakteristikomis: RNY1 – su gliomų piktybiškumo laipsniu; RNY3 – pacientų išgyvenimo trukme; RNY4 – amžiumi bei lytimi; RNY5 – amžiumi.

Introduction. Glioblastoma (GBM), the most widespread and aggressive primary brain tumor with survival time typically less than two years [1]. Therefore, a deeper understanding of the molecular processes underlying the onset and progression of GBM is also required to identify more effective therapeutic and diagnostic strategies. RNA methylation, a post-transcriptional modification, has emerged as a crucial regulator of gene expression, RNA stability, and protein translation, thus influencing cellular processes, including those implicated in oncogenesis [2]. This study focuses on several modifications: m6A and m7G, dynamically controlled by methyltransferases (”writers”), demethylases (”erasers”), and “readers”. Furthermore, the transcription factor SOX2, essential for stem cell maintenance and implicated in tumor initiation and progression, is frequently overexpressed in gliomas [3,4]. Investigating a modified form lacking the C-terminal domain provides insights into SOX2’s effects on glioma cell survival and proliferation. This study explores the potential of RNA methylation regulators and SOX2 as novel targets in glioma diagnosis and therapy. Aim. To investigate the expression and functional significance of specific RNA methylation regulators as well as modified SOX2 variant in glioma, evaluating their potential as diagnostic and therapeutic biomarkers. Methods. Gene expression levels were quantified in both tumor tissue samples from 54 glioma patients and U87 and U87 SOX2ΔC glioblastoma cell lines using qRT-PCR. Cell migration was assessed using a wound-healing assay. Publicly available RNA sequencing data from experiments involving ALKBH5 knockdown were analyzed bioinformatically to identify differentially expressed genes and affected biological pathways. Results. Analysis of patient tumor samples revealed significantly reduced expression of ALKBH5 (p<0.01), FTO (p<0.0001), and BUD23 (p<0.002) in higher-grade gliomas. In contrast, increased expression of these RNA methylation regulators was positively associated with patient survival (ALKBH5 p<0.02; FTO p<0.001 BUD23, p<0.003), suggesting their potential as valuable prognostic biomarkers. In modified U87 cell line, SOX2ΔC overexpression led to a significant increase in ALKBH5 expression (p=0.03) and a decrease in BUD23 expression (p=0.03), demonstrating a regulatory link between SOX2 and these methylation regulators. Over 53% of transcripts showed significant transcriptome changes as a result of ALKBH5 knockdown, including genes essential for cell division, and DNA replication and repair. Notably, some of these dysregulated genes, such as TOP2A are established targets in other cancer therapies, suggesting their potential repurposing for glioma treatment. Conclusions. ALKBH5, FTO, and BUD23 are promising prognostic biomarkers. Targeting these methylation regulators and SOX2 offers potential for improved glioma treatment and early diagnosis.

Gliomas are aggressive primary brain tumors with high morbidity and mortality [1]. Despite advances in oncology, their complex biology limits effective treatment options, making gliomas a key focus of cancer research. Epigenetic modifications, including RNA methylation (m6A and 5mC), have been implicated in tumor progression, yet their specific roles in gliomas remain unclear [2]. This study investigates m6A and 5mC RNA methylation profiles in gliomas and their potential as biomarkers for tumor malignancy and clinical outcomes. Methods: Total RNA was extracted from glioma tissue samples (n = 30; grade II = 20, grade IV = 8) and cell cultures (n = 8; commercial glioblastoma cell lines = 4, glioma stem cell cultures = 2, neural stem cell cultures = 3) using TRIzol reagent. Poly(A) RNA was isolated with magnetic oligo(dT) beads, and m6A/5mC modifications were quantified via ELISA-based colorimetric and fluorometric assays. Statistical analyses, including Kolmogorov-Smirnov, unpaired t-test, Mann-Whitney U test, and Kaplan-Meier survival analysis, assessed correlations between methylation levels, tumor grade, and clinical characteristics. Results: m6A levels were significantly lower in grade II gliomas and neural stem cell lines and highest in grade IV gliomas and GBM cell lines (p < 0.05). Female patients exhibited significantly higher m6A levels compared to males (p = 0.011), but no association with survival was observed. 5mC levels were significantly higher in tumor samples than in cell lines (p < 0.05), and elevated 5mC was correlated with shorter survival in grade IV gliomas (p = 0.0299). Conclusion: Distinct m6A and 5mC RNA methylation profiles were observed in gliomas, suggesting their involvement in tumor progression. The association between 5mC levels and survival highlights its potential prognostic value, warranting further investigation into RNA methylation as a biomarker for glioma malignancy.

Background. Glioblastomas are the most aggressive and lethal primary brain tumours, with a median survival of 14.6 months [1]. RNA methylation is a post-transcriptional modification that regulates RNA translation, splicing, and stability, which may contribute to cancer initiation and progression [2]. With the rise of personalised medicine need, it is believed that aberrant RNA methylation patterns present a novel opportunity for targeted gene therapy and early diagnostics. The expression of the transcription factor SOX2 is associated with a poor prognosis and is essential for glioma stemness and malignancy [3]. Investigating a modified SOX2 variant lacking the C-terminal domain, which is crucial for transcriptional activation, reveals its regulatory impact on glioma cell survival and proliferation. Integrating SOX2 activity with RNA methylation may uncover potential biomarkers that could facilitate liquid biopsy development, providing a minimally invasive approach to diagnose and treat gliomas, monitor progression and effectiveness of the treatment [4]. Aim. This study aims to identify novel biomarkers for early glioma diagnostic and targeted gene therapy. Methods. Samples of copy DNA from tumour biopsies of 54 glioma patients were analysed with de-identified clinical data. mRNA extracted from U87 and U87 SOX2ÄC cell lines was used for cDNA synthesis. Gene expression (RT-PCR) and cell migration (wound-healing assay) were assessed. Bioinformatic analysis of publicly available RNA sequencing data explored the impact of ALKBH5 knockdown on gene expression and related pathways. Statistical significance was determined at p 0.05. Results. This study found that ALKBH5, FTO, and BUD23 expression levels correlated with glioma malignancy and patient survival, suggesting their potential as diagnostic biomarkers. Higher gene expression linked to longer survival. Significant associations were observed between these genes and clinical factors, including IDH mutation status, age, and gender. Furthermore, we demonstrate that SOX2ÄC influences ALKBH5 and BUD23 expression, indicating its involvement in glioma development. Knockdown of ALKBH5 significantly altered gene expression profiles and impacted crucial biological pathways, suggesting potential therapeutic benefits. Additionally, novel glioma therapy targets were identified based on their functional relevance and prior use in other cancers. Conclusion. This study suggests that ALKBH5, FTO, and BUD23 may serve as novel glioma biomarkers with prognostic value. Further research into targeting these genes and investigation of SOX2’s role in RNA methylation may improve glioma treatment strategies.

Introduction. Glioblastoma (GBM), the most common WHO grade IV glioma, is known for its aggressive nature and low average patient survival time (averaging <15 months) [1]. Glioblastoma accounts for 57% of all glioma cases and 48% of primary malignant central nervous system neoplasms [1]. RNA methylation is a new focus of research as a possible contributing factor, which influences the cell cycle, the structure and stability of RNA molecules [2]. With the development of personalized medicine, RNA methylation may be used in targeted gene therapy to alter gene expression and correct abnormal RNA methylation patterns. These regulators may serve as early diagnostic biomarkers to detect cancer in early stages. Transcription factor SOX2 is associated with tumor aggressiveness and poor prognosis, and is important for glioma development and growth [3]. Investigation of a modified SOX2 variant without the C-terminal domain, a region crucial for transcriptional activation, offers valuable insights into its impact on glioma cell survival and proliferation. These findings could support the development of non-invasive diagnostic and monitoring techniques, allowing for early glioma detection, assessment of disease progression, and evaluation of treatment effectiveness. This study investigates how methylation regulators ALKBH5, FTO, METTL1, and BUD23, along with a modified SOX2 variant, contribute to glioma initiation and progression, potentially revealing new therapeutic targets. Aim. To explore the functions of RNA methylation modifications and SOX2ΔC in glioma and assess their potential as diagnostic and therapeutic biomarkers. Methods. cDNA samples from tumor biopsies of 54 glioma patients were examined along with de-identified clinical information. Quantitative RT-PCR was performed to measure gene expression levels in both patient samples and modified glioblastoma U87 SOX2ΔC cell lines. Additionally, cell migration assay was performed. Bioinformatic analysis of RNA sequencing data was conducted to identify significant changes in gene expression and biological pathways following ALKBH5 knockdown. Results. We discovered that ALKBH5, FTO, and BUD23 gene expression increased with glioma malignancy, suggesting them as potential targets for therapy or diagnostic tools. Patients with higher levels of these genes showed longer survival. The study revealed associations between gene expression and factors such as IDH mutation, gender, age, and survival time, suggesting potential applications in personalized treatment. Additionally, SOX2ΔC impacted ALKBH5 and BUD23 expression, suggesting its potential role in regulating cancer development. Furthermore, the study discovered that ALKBH5 plays a critical role in gene expression. RNA sequencing analysis of ALKBH5 knockdown revealed significant transcriptomic alterations, with over half of all detected transcripts showing differential expression, confirming its critical role in gene expression. Additionally, ALKBH5 knockdown impacted important biological and oncogenic pathways, such as homologous recombination, mismatch repair, DNA replication, and the cell cycle, supporting its role in glioblastoma progression. Lastly, novel therapeutic targets were identified based on their functional relevance to gliomas and known implications in other malignancies. Conclusions. This study identifies RNA methylation regulators ALKBH5, FTO, and BUD23, as well as SOX2ΔC, as promising biomarkers and potential therapeutic targets for glioblastoma, offering valuable insights for the development of personalized treatments.

Introduction Glioblastomas are the most aggressive brain tumors (median survival 14.6 months) [1]. RNA methylation affects the cell cycle, structure, and stability of RNA molecules therefore plays a crucial role in cancer initiation and progression [2]. This modification is a promising avenue for gene therapy, targeting aberrant methylation patterns to personalize treatment. Transcription factor SOX2 is associated with glioma aggressiveness, poor prognosis, and growth [3]. Studying a modified form of SOX2 without C-terminal domain offers insights into its effects on glioma survival and proliferation. Combining SOX2 activity with RNA methylation uncovers new biomarkers and therapeutic targets for liquid biopsies, providing a less-invasive method to monitor glioma progression and treatment efficacy [4]. Demethylases ALKBH5 and FTO are linked to pro-oncogenic functions, promoting cell proliferation and selfrenewal, while BUD23 methyltransferase contributes to glioblastoma progression, suggesting their potential as biomarkers [5,6]. Aim This study aims to explore RNA methylation-associated biomarkers in gliomas to assess their diagnostic and therapeutic potential, enhancing personalized glioma management. Methods The research was conducted at the Lithuanian University of Health Sciences, Neuroscience Institute, Molecular Neurooncology Laboratory. In this study samples of cDNA from tumor biopsies of 54 patients with glioma were used, along with de-identified and encrypted clinical data. mRNA was extracted from U87 and U87 SOX2ΔC cell lines using TRIzol, followed by cDNA synthesis. Gene expression levels analyzed using RT-PCR and cell migration via wound-healing assay. Bioinformatic analysis was conducted using publicly available RNA sequencing data to evaluate the effects of ALKBH5 knockdown on gene expression and associated pathways using the open-access "Galaxy" platform. Statistical analyses were conducted using GraphPad Prism (p ≤ 0.05). Results ALKBH5, FTO, and BUD23 gene expression correlates with glioma malignancy, potentially serving as diagnostic markers. Higher gene expression linked to longer survival. Our study revealed associations between these genes and IDH mutation, gender, age, and survival. Additionally, we observed that SOX2ΔC influences ALKBH5 and BUD23 expression, suggesting its role in cancer development. ALKBH5 knockdown significantly altered gene expression and impacted key biological pathways, highlighting potential therapeutic benefits. Lastly, analysis identified novel glioma therapy targets based on their relevance to glioma function and use in other cancers. Conclusions Our findings demonstrate the potential of ALKBH5, FTO, and BUD23 as novel glioma biomarkers. Targeting these genes, along with exploring the role of SOX2 in regulating RNA methylation, may offer promising avenues for improved glioma treatment, possess prognostic and predictive value.

Increasing evidence suggests that the beneficial abilities of neural stem cells (NSCs) can be attributed to their paracrine secretion of extracellular vesicles (EVs). These EVs may offer promising therapies for neurological disorders due to their capacity to induce neuroprotection and neurogenesis, migrate to damaged brain areas, including tumours, and modulate inflammation and the aggregation of misfolded proteins. [...].

Introduction. Glioma, a highly aggressive brain tumor with a median survival of just 14.6 months, lacks effective treatment options, leading to high mortality rates [1]. A new focus of research is RNA methylation and its regulating genes that are believed to be potential targets for early diagnosis and targeted gene therapy, offering the possibility of earlier cancer detection and more effective treatment for patients [2]. While gliomas were traditionally thought to develop from astrocytes, there is a theory that stem cells can acquire astrocyte-like properties [3]. The pluripotency and ability of these cells to maintain an undifferentiated state prevent the complete eradication of tumors, contributing to the high mortality of this cancer [4]. The transcription factor SOX2 is considered essential for the development of stem cells, and its expression increases in cases of glioblastoma [5-6]. However, there is limited data on the influence of such biomolecules determining cell stemness on the epitranscriptome and the epigenetic changes occurring in gliomas that drive their development or progression. Therefore, understanding RNA methylation regulators' molecular mechanisms and their impact on cellular processes is crucial for treatment tailoring. Aim. To investigate the differences in the expression of ALKBH5 and FTO demethylases, as well as BUD23 and METTL1 methyltransferases, in postoperative glioma tumor tissues samples with varying degrees of malignancy, and in a modified glioblastoma U87 cell line. To determine the effects of inhibition of the ALKBH5 methylation regulator on biological processes using RNA-sequencing data analysis. Methods. The methodology involves extracting iRNA using Trizol, synthesizing cDNA from RNA using reverse transcriptase enzymes. Additionally, primer design was performed using bioinformatics tools such as the "Ensemble" database and the "PerlPrimer" program. Gene expression analysis was conducted using real-time PCR with SYBR Green detection, allowing for quantitative assessment of gene expression. Results were statistically evaluated using the Kolmogorov-Smirnov test, Mann-Whitney criterion, t-test, KaplanMeier method, and chi-square criterion. Statistically significant results were considered when the p-value was ≤ 0.05. Furthermore, RNA sequencing data analysis from “NCBI SRA” database was performed to assess the impact of disrupting the methylation process by suppressing ALKBH5 gene expression on glioblastoma cells, their gene expression, and biological processes. Results. The ALKBH5, FTO, and BUD23 gene expressions were directly linked to the malignancy level of brain tumors, suggesting their potential as biomarkers or targets for glioma treatment. Patients with higher expression levels of these genes tended to survive longer. However, METTL1 expression showed no such association with tumor aggressiveness or patient survival. Additionally, significant correlations were observed between the expression of these genes and various factors such as IDH mutation, patient gender, age, and survival duration. The SOX2ΔC protein's expression significantly impacted the regulation of RNA methylation modifiers, particularly ALKBH5 and BUD23. Furthermore, suppressing ALKBH5 gene expression in glioblastoma cells led to significant changes in the expression of numerous genes involved in crucial biological processes such as DNA replication, mismatch repair, Fanconi anemia, cell cycle and homologous recombination, suggesting a profound influence on tumor biology.

In recent years, epigenetic modifications of non-coding RNAs (ncRNAs) have received increasing attention. Various new modifications are discovered, as well as the proteins that write, read, and erase them, so-called “writers” (enzymes that deposit modifications), “erasers” (enzymes that remove modifications) and “readers” (proteins that recognize and bind epigenetic modifications). Epigenetic regulation of genes involved in cell proliferation, survival, and differentiation is believed to be involved, at least in part, in the initiation, development, and malignancy of various types of tumors. Gliomas - quite common, malignant brain tumors with poor prognosis - are no exception. Numerous publications have discussed the role of N6-methyladenosine (m6A) and 5-methylcytosine (m5C) modifications and their regulatory proteins in mentioned brain tumors. However, data on various other modifications, e.g., pseudouridine (Ψ), N7-methylguanosine (m7G), 5- hydroxymethylcytosine (hm5C), 5-N1-methyladenosine (m1A) and others, as well as their regulatory proteins in gliomas are rarely found. Since the link between these modifications and the proteins that regulate them in other types of tumors have been at least partially described, the aim of this research was to investigate expression differences of the coding genes of various "writers", "readers" and "erasers", influencing ncRNA modifications in different malignancy grade glioma patients’ tumor samples. Several ncRNA modifications were selected for the analysis: pseudouridine (Ψ), N7-methylguanosine (m7G), 5-hydroxymethylcytosine (hm5C), 5-N1-methyladenosine (m1A). Twelve coding genes’ of the proteins regulating these modifications – FTO, ALKBH3, ALKBH5, BUD23, METTL1, DKC1, TET1, TET2, TET3, TRMT6, YTHDF1, YTHDC1 - as well as two reference genes’ - GAPDH and βActin - expression were evaluated using qRT-PCR method. The study group consisted of 54 patients, diagnosed with different malignancy grade astrocytoma. Results were also compared with reference human brain (RHB) samples. In the majority of analyzed cases, gene expression was related to the degree of tumor malignancy and overall survival was significantly longer in astrocytoma patients with higher than median gene expression of analyzed genes.