The role of circular RNAs in driving cancer advancement in low-oxygen conditions.

<p dir="ltr">Neuronal development is a complex process regulated by precise gene expression programs involving chromatin organization, epigenetic modifications and RNA processing. Understanding these regulatory mechanisms is essential for elucidating how neural cells acquire and maintain their identity. In this thesis, the roles of RNA-binding protein HNRNPU and circular RNAs in regulating neuronal development were investigated using in vitro cell cultures and cortical organoids as model systems.</p><p dir="ltr">In Study I, the focus was on examining the effects of HNRNPU deficiency on neural differentiation, RNA splicing, and 3D genome organization using a 2D in vitro model. HNRNPU deficiency was shown to cause significant disruptions in these processes. Genes that are localized in chromatin regions with differential 3D conformation and genes encoding mRNAs with differential splicing in HNRNPU- deficient cells were associated with neurodevelopmental disorders. Additionally, HNRNPU deficiency was shown to result in increased proportion of neural progenitors within differentiating neural populations. These disrupted trajectories highlight the importance of HNRNPU in maintaining genomic integrity and neural stem cell commitment to differentiation.</p><p dir="ltr">The molecular interactome of HNRNPU during neural differentiation was investigated in Study II. Key regulatory networks associated with HNRNPU were identified using ribonucleoprotein immunoprecipitation, mass spectrometry and RNA sequencing. HNRNPU was found to, for example, interact with mammalian SWI/SNF chromatin remodeling complex, bringing new insights into the regulatory mechanisms behind chromatin organization defects seen in relation to HNRNPU deficiency. Identifying HNRNPU binding to mRNAs encoding DNA methylation regulators led to measuring global DNA methylation levels after HNRNPU silencing, revealing significant hypomethylation. This finding linked HNRNPU deficiency to the distinct methylation episignatures reported in individuals with HNRNPU- related neurodevelopmental disorders.</p><p dir="ltr">The expression of circular RNAs during neural differentiation was explored in Study III. Key circular RNAs with increasing expression patterns were identified and validated. Interestingly, several circular RNAs were formed from linear mRNAs encoding proteins with synaptic functions, elucidating their possible regulatory roles and contributions to neurodevelopmental processes. Additionally, the exons within these circular RNAs had an increased frequency of genetic variation, and RNA-binding protein SFPQ was identified as a key regulator of these circular RNAs. The findings from this study provide an important resource for further research focusing on the roles of circular RNAs in neuronal development.</p><p dir="ltr">In Unpublished results, the effects of different types of mutations affecting the HNRNPU-locus were investigated using a 3D cortical organoid in vitro model. New insights into the early gene expression regulation and epigenetic control exerted by HNRNPU were uncovered during neuronal development through single-cell RNA sequencing and DNA methylation arrays. Preliminary results revealed differences from different types of genetic variants in the HNRNPU-locus.</p><p dir="ltr">Collectively, these studies enhance our understanding of the genetic and molecular basis of neurodevelopmental disorders. By elucidating the roles of HNRNPU and circular RNAs in neuronal development, we provide valuable knowledge of the roles of RNA-binding proteins and non-coding RNAs in orchestrating gene expression control during neuronal development.</p><h3>List of scientific papers</h3><p dir="ltr">I. Mastropasqua F.#, <b>Oksanen M.</b>#, Soldini C., Alatar S., Arora A., Ballarino R., Molinari M., Agostini F., Poulet A., Watts M.E., Rabkina I., Becker M., Li D., Anderlid B.M., Isaksson J., Lundin Remnelius K., Molsem M., Jacob Y., Falk A., Crosetto N., Bienko M., Santini E., Borgkvist A., Bölte S., Tammimies K. Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis. Biol Open. 2023 Oct 15;12(10):bio060113. <a href="https://doi.org/10.1242/bio.060113" rel="noreferrer" target="_blank">https://doi.org/10.1242/bio.060113</a></p><p dir="ltr">II. <b>Oksanen M.</b>, Mastropasqua F., Mazan-Mamczarz K., Martindale J.L., Ye X., Arora A., Banskota N., Gorospe M., Tammimies K. Molecular interactome of HNRNPU reveals regulatory networks in neuronal differentiation and DNA methylation. BioRxiv. [Submitted; Preprint]<br><a href="https://doi.org/10.1101/2025.02.19.638869">https://doi.org/10.1101/2025.02.19.638869<br></a><br></p><p dir="ltr">III. Watts M.E., <b>Oksanen M.</b>, Lejerkrans S., Mastropasqua F., Gorospe M., Tammimies K. Circular RNAs arising from synaptic host genes during human neuronal differentiation are modulated by SFPQ RNA-binding protein. BMC Biol. 2023 May 26;21(1):127. <a href="https://doi.org/10.1186/s12915-023-01627-w" rel="noreferrer" target="_blank">https://doi.org/10.1186/s12915-023-01627-w</a></p><p dir="ltr"># Denotes equal contribution</p>

<p dir="ltr">Neuronal development is a complex process regulated by precise gene expression programs involving chromatin organization, epigenetic modifications and RNA processing. Understanding these regulatory mechanisms is essential for elucidating how neural cells acquire and maintain their identity. In this thesis, the roles of RNA-binding protein HNRNPU and circular RNAs in regulating neuronal development were investigated using in vitro cell cultures and cortical organoids as model systems.</p><p dir="ltr">In Study I, the focus was on examining the effects of HNRNPU deficiency on neural differentiation, RNA splicing, and 3D genome organization using a 2D in vitro model. HNRNPU deficiency was shown to cause significant disruptions in these processes. Genes that are localized in chromatin regions with differential 3D conformation and genes encoding mRNAs with differential splicing in HNRNPU- deficient cells were associated with neurodevelopmental disorders. Additionally, HNRNPU deficiency was shown to result in increased proportion of neural progenitors within differentiating neural populations. These disrupted trajectories highlight the importance of HNRNPU in maintaining genomic integrity and neural stem cell commitment to differentiation.</p><p dir="ltr">The molecular interactome of HNRNPU during neural differentiation was investigated in Study II. Key regulatory networks associated with HNRNPU were identified using ribonucleoprotein immunoprecipitation, mass spectrometry and RNA sequencing. HNRNPU was found to, for example, interact with mammalian SWI/SNF chromatin remodeling complex, bringing new insights into the regulatory mechanisms behind chromatin organization defects seen in relation to HNRNPU deficiency. Identifying HNRNPU binding to mRNAs encoding DNA methylation regulators led to measuring global DNA methylation levels after HNRNPU silencing, revealing significant hypomethylation. This finding linked HNRNPU deficiency to the distinct methylation episignatures reported in individuals with HNRNPU- related neurodevelopmental disorders.</p><p dir="ltr">The expression of circular RNAs during neural differentiation was explored in Study III. Key circular RNAs with increasing expression patterns were identified and validated. Interestingly, several circular RNAs were formed from linear mRNAs encoding proteins with synaptic functions, elucidating their possible regulatory roles and contributions to neurodevelopmental processes. Additionally, the exons within these circular RNAs had an increased frequency of genetic variation, and RNA-binding protein SFPQ was identified as a key regulator of these circular RNAs. The findings from this study provide an important resource for further research focusing on the roles of circular RNAs in neuronal development.</p><p dir="ltr">In Unpublished results, the effects of different types of mutations affecting the HNRNPU-locus were investigated using a 3D cortical organoid in vitro model. New insights into the early gene expression regulation and epigenetic control exerted by HNRNPU were uncovered during neuronal development through single-cell RNA sequencing and DNA methylation arrays. Preliminary results revealed differences from different types of genetic variants in the HNRNPU-locus.</p><p dir="ltr">Collectively, these studies enhance our understanding of the genetic and molecular basis of neurodevelopmental disorders. By elucidating the roles of HNRNPU and circular RNAs in neuronal development, we provide valuable knowledge of the roles of RNA-binding proteins and non-coding RNAs in orchestrating gene expression control during neuronal development.</p><h3>List of scientific papers</h3><p dir="ltr">I. Mastropasqua F.#, <b>Oksanen M.</b>#, Soldini C., Alatar S., Arora A., Ballarino R., Molinari M., Agostini F., Poulet A., Watts M.E., Rabkina I., Becker M., Li D., Anderlid B.M., Isaksson J., Lundin Remnelius K., Molsem M., Jacob Y., Falk A., Crosetto N., Bienko M., Santini E., Borgkvist A., Bölte S., Tammimies K. Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis. Biol Open. 2023 Oct 15;12(10):bio060113. <a href="https://doi.org/10.1242/bio.060113" rel="noreferrer" target="_blank">https://doi.org/10.1242/bio.060113</a></p><p dir="ltr">II. <b>Oksanen M.</b>, Mastropasqua F., Mazan-Mamczarz K., Martindale J.L., Ye X., Arora A., Banskota N., Gorospe M., Tammimies K. Molecular interactome of HNRNPU reveals regulatory networks in neuronal differentiation and DNA methylation. BioRxiv. [Submitted; Preprint]<br><a href="https://doi.org/10.1101/2025.02.19.638869">https://doi.org/10.1101/2025.02.19.638869<br></a><br></p><p dir="ltr">III. Watts M.E., <b>Oksanen M.</b>, Lejerkrans S., Mastropasqua F., Gorospe M., Tammimies K. Circular RNAs arising from synaptic host genes during human neuronal differentiation are modulated by SFPQ RNA-binding protein. BMC Biol. 2023 May 26;21(1):127. <a href="https://doi.org/10.1186/s12915-023-01627-w" rel="noreferrer" target="_blank">https://doi.org/10.1186/s12915-023-01627-w</a></p><p dir="ltr"># Denotes equal contribution</p>

CircSTK40 contributes to recurrent implantation failure via modulating the HSP90/AKT/FOXO1 axis

Long and Repeat-Rich Intronic Sequences Favor Circular RNA Formation under Conditions of Reduced Spliceosome Activity.

SummaryCircular RNAs (circRNAs) have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, the underlying regulatory mechanism of circRNAs biogenesis remains largely unknown. Here, we integrated in-depth multi-omics data including epigenome, transcriptome, and non-coding RNA and identified candidate circRNAs in six cellular contexts. Next, circRNAs were divided into two classes (high versus low) with different expression levels. Machine learning models were constructed that predicted circRNA expression levels based on 11 different histone modifications and host gene expression. We found that the models achieve great accuracy in predicting high versus low expressed circRNAs. Furthermore, the expression levels of host genes of circRNAs, H3k36me3, H3k79me2, and H4k20me1 contributed greatly to the classification models in six cellular contexts. In summary, all these results suggest that epigenetic modifications, particularly histone modifications, can effectively predict expression levels of circRNAs.

Biogenesis and Functions of Circular RNAs Come into Focus.

Circular RNAs (circRNAs) are endogenous, single-stranded, most frequently non-coding RNA (ncRNA) molecules that play a significant role in gene expression regulation. Circular RNAs can affect microRNA functionality, interact with RNA-binding proteins (RBPs), translate proteins by themselves, and directly or indirectly modulate gene expression during different cellular processes. The affected expression of circRNAs, as well as their targets, can trigger a cascade of events in the genetic regulatory network causing pathological conditions. Recent studies have shown that altered circular RNA expression patterns could be used as biomarkers in psychiatric diseases, including schizophrenia (SZ); moreover, circular RNAs together with other cell molecules could provide new insight into mechanisms of this disorder. In this review, we focus on the role of circular RNAs in the pathogenesis of SZ and analyze their biomarker and therapeutic potential in this disorder.

Although there is much controversy over the use of methylprednisolone (MP), it is one of the main drugs used in the treatment of acute spinal cord injury (SCI). The induction of the proliferation and differentiation of endogenous neural progenitor cells (NPCs) is considered a promising mode of treatment for SCI. However, the effects of MP on spinal cord-derived endogenous NPCs in a low oxygen enviroment remain to be delineated. Thus, the aim of this study was to investigate the potential effects of MP on NPCs cultured under low oxygen conditions in vitro and to elucidate the molecular mechanisms involved. Fetal rat spinal cord-derived NPCs were harvested and divided into 4 groups: 2 groups of cells cultured under normal oxygen conditions and treated with or without MP, and 2 groups incubated in 3% O2 (low oxygen) treated in a similar manner. We found that MP induced suppressive effects on NPC proliferation even under low oxygen conditions (3% O2). The proportion of nestin-positive NPCs decreased from 51.8±2.46% to 36.17±3.55% following the addition of MP and decreased more significantly to 27.20±2.68% in the cells cultured in 3% O2. In addition, a smaller number of glial fibrillary acidic protein (GFAP)-positive cells and a greater number of microtubule-associated protein 2 (MAP2)-positive cells was observed following the addition of MP under both normal (normoxic) and low oxygen (hypoxic) conditions. In response to MP treatment, hypoxia-inducible factor-1α (HIF-1α) and the Notch signaling pathway downstream protein, Hes1, but not the upstream Notch-1 intracelluar domain (NICD), were inhibited. After blocking NICD with a γ-secretase inhibitor (DAPT) MP still inhibited the expression of Hes1. Our results provide insight into the molecular mechanisms responsible for the MP-induced inhibition of proliferation and its effects on differentiation and suggest that HIF-1α and Hes1 play an important role in this effect.

Hypoxia, characterized by reduced oxygen levels, plays a pivotal role in cancer progression, profoundly influencing tumor behavior and therapeutic responses. A hallmark of solid tumors, hypoxia drives significant metabolic adaptations in cancer cells, primarily mediated by hypoxia-inducible factor-1α (HIF-1α), a key transcription factor activated in low-oxygen conditions. This hypoxic environment promotes epithelial-mesenchymal transition (EMT), enhancing cancer cell migration, metastasis, and the development of cancer stem cell-like properties, which contribute to therapy resistance. Moreover, hypoxia modulates the expression of circular RNAs (circRNAs), leading to their accumulation in the tumor microenvironment. These hypoxia-responsive circRNAs regulate gene expression and cellular processes critical for cancer progression, making them promising candidates for diagnostic and prognostic biomarkers in various cancers. This review delves into the intricate interplay between hypoxic circRNAs, microRNAs, and RNA-binding proteins, emphasizing their role as molecular sponges that modulate gene expression and signaling pathways involved in cell proliferation, apoptosis, and metastasis. It also explores the relationship between circRNAs and the tumor microenvironment, particularly how hypoxia influences their expression and functional dynamics. Additionally, the review highlights the potential of circRNAs as diagnostic and prognostic tools, as well as their therapeutic applications in innovative cancer treatments. By consolidating current knowledge, this review underscores the critical role of circRNAs in cancer biology and paves the way for future research aimed at harnessing their unique properties for clinical advancements. Specifically, this review examines the biogenesis, expression patterns, and mechanistic actions of hypoxic circRNAs, focusing on their ability to act as molecular sponges for microRNAs and their interactions with RNA-binding proteins. These interactions impact key signaling pathways related to tumor growth, metastasis, and drug resistance, offering new insights into the complex regulatory networks governed by circRNAs under hypoxic stress.

Circular RNA ACTN4 promotes intrahepatic cholangiocarcinoma progression by recruiting YBX1 to initiate FZD7 transcription

Cutaneous melanoma (CM) is a very aggressive disease, often characterized by unresponsiveness to conventional therapies and high mortality rates worldwide. The identification of the activating BRAFV600 mutations in approximately 50% of CM patients has recently fueled the development of novel small‐molecule inhibitors that specifically target BRAFV600 ‐mutant CM. In addition, a major progress in CM treatment has been made by monoclonal antibodies that regulate the immune checkpoint inhibitors. However, although target‐based therapies and immunotherapeutic strategies have yielded promising results, CM treatment remains a major challenge. In the last decade, accumulating evidence points to the aberrant expression of different types of noncoding RNAs (ncRNAs) in CM. While studies on microRNAs have grown exponentially leading to significant insights on CM biology, the role of circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) in this tumor is less understood, and much remains to be discovered. Here, we summarize and critically review the available evidence on the molecular functions of circRNAs and lncRNAs in BRAFV600 ‐mutant CM and CM immunogenicity, providing recent updates on their functional role in targeted therapy and immunotherapy resistance. In addition, we also include an evaluation of several algorithms and databases for prediction and validation of circRNA and lncRNA functional interactions.

The importance of circular RNAs in malignant tumors causes more attention in researchers. Circular RNA_LARP4 is identified as a tumor suppressor in gastric cancer, but the role of circular RNA_LARP4 in prostate cancer (PCa) remains unclear. Our work aims to uncover whether and how circular RNA_LARP4 functions in the PCa development. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to determine the level of circular RNA_LARP4 in PCa tissues and cell lines. The patients' prognosis was analyzed. Circular RNA_LARP4 lentivirus was constructed for transfection of PCa cells. Cell migrated and invaded ability was detected through wound healing assay and transwell assay. Western blot assay was performed to analyze the protein level of FOXO3A. The low circular RNA_LARP4 expression was associated with poor prognosis of PCa patients. The circular RNA_LARP4 was lowly expressed in PCa tissues compared with adjacent samples. The expression of circular RNA_LARP4 was downregulated in PCa cell lines. The cell migrated and invaded ability of PCa cells was inhibited after circular RNA_LARP4 was overexpressed. Furthermore, FOXO3A expression was increased via the overexpression of circular RNA_LARP4. Circular RNA_LARP4 could suppress cell migration and invasion of PCa by upregulating FOXO3A.

Recently, the progression of gastric cancer (GC), as one of the most ordinary malignant tumors, has been reported to be associated with circular RNAs. This study aimed to identify the role of circular RNA_LARP4 in GC. We performed real-time quantitative polymerase chain reaction (RT-qPCR) in 46 paired GC patients and GC cell lines to detect the expression of circular RNA_LARP4. Moreover, the role of circular RNA_LARP4 in GC proliferation was identified through proliferation assay and colony formation assay, while the role of circular RNA_LARP4 in GC metastasis was measured through scratch wound assay and transwell assay. Furthermore, the potential targets of circular RNA_LARP4 were predicted through bioinformatics methods and further identified by western blot assay and RT-qPCR. Circular RNA_LARP4 expression was remarkably lower in GC tissues compared with that in adjacent samples. Besides, cell proliferation of GC was inhibited after overexpression of circular RNA_LARP4, while cell migration and invasion of GC was inhibited after overexpression of circular RNA_LARP4. Furthermore, Upstream frameshift 1 (UPF1) was predicted as the potential target of circular RNA_LARP4 and was upregulated via overexpression of circular RNA_LARP4 in GC. Circular RNA_LARP4 inhibits GC cell proliferation and metastasis via targeting UPF1 in vitro, which might provide a new tumor suppressor in GC development.

Role of circular RNAs in regulating tumor microenvironment, epithelial mesenchymal transition, and resistance to cancer therapy.

Simple SummaryKeratinocyte carcinomas include BCC and cSCC and represent the most frequent cancer among fair-skinned people. Tumor development is associated with mutations and dysregulation of many genes involved in biological processes such as cell proliferation, differentiation and apoptosis. The expression of these genes is controlled in many ways, including transcriptional and post-transcriptional control by circular RNAs. In recent studies, a number of circular RNAs have been identified that are dysregulated in BCC and cSCC. Biological functions relevant to tumor development were shown for some of these circRNAs, which may represent biomarkers for disease progression and targets for novel treatment approaches.Keratinocyte carcinomas (KC) include basal cell carcinomas (BCC) and cutaneous squamous cell carcinomas (cSCC) and represents the most common cancer in Europe and North America. Both entities are characterized by a very high mutational burden, mainly UV signature mutations. Predominately mutated genes in BCC belong to the sonic hedgehog pathway, whereas, in cSCC, TP53, CDKN2A, NOTCH1/2 and others are most frequently mutated. In addition, the dysregulation of factors associated with epithelial to mesenchymal transition (EMT) was shown in invasive cSCC. The expression of factors associated with tumorigenesis can be controlled in several ways and include non-coding RNA molecules, such as micro RNAs (miRNA) long noncoding RNAs (lncRNA) and circular RNAs (circRNA). To update findings on circRNA in KC, we reviewed 13 papers published since 2016, identified in a PubMed search. In both BCC and cSCC, numerous circRNAs were identified that were differently expressed compared to healthy skin. Some of them were shown to target miRNAs that are also dysregulated in KC. Moreover, some studies confirmed the biological functions of individual circRNAs involved in cancer development. Thus, circRNAs may be used as biomarkers of disease and disease progression and represent potential targets of new therapeutic approaches for KC.