Puzzling out the role of MIAT LncRNA in hepatocellular carcinoma.

A non-negligible part of our DNA has been proven to be transcribed into non-protein coding RNA and its intricate involvement in several physiological processes has been highly evidenced. The significant biological role of non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) has been variously reported. In the current review, the authors highlight the multifaceted role of myocardial infarction-associated transcript (MIAT), a well-known lncRNA, in hepatocellular carcinoma (HCC). Since its discovery, MIAT has been described as a regulator of carcinogenesis in several malignant tumors and its overexpression predicts poor prognosis in most of them. At the molecular level, MIAT is closely linked to the initiation of metastasis, invasion, cellular migration, and proliferation, as evidenced by several in-vitro and in-vivo models. Thus, MIAT is considered a possible theranostic agent and therapeutic target in several malignancies. In this review, the authors provide a comprehensive overview of the underlying molecular mechanisms of MIAT in terms of its downstream target genes, interaction with other classes of ncRNAs, and potential clinical implications as a diagnostic and/or prognostic biomarker in HCC.

Emerging Role of Circular RNAs in Hepatocellular Carcinoma Immunotherapy.

Hepatocellular carcinoma (HCC) is a highly fatal malignancy with limited therapeutic options and high recurrence rates. Recently, immunotherapeutic agents such as immune checkpoint inhibitors (ICIs) have emerged as a new paradigm shift in oncology. ICIs, such as programmed cell death protein 1 (PD-1) inhibitors, have provided a new source of hope for patients with advanced HCC. Yet, the eligibility criteria of HCC patients for ICIs are still a missing piece in the puzzle. Circular RNAs (circRNAs) have recently emerged as a new class of non-coding RNAs that play a fundamental role in cancer pathogenesis. Structurally, circRNAs are resistant to exonucleolytic degradation and have a longer half-life than their linear counterparts. Functionally, circRNAs possess the capability to influence various facets of the tumor microenvironment, especially at the HCC tumor-immune synapse. Notably, circRNAs have been observed to control the expression of immune checkpoint molecules within tumor cells, potentially impeding the therapeutic effectiveness of ICIs. Therefore, this renders them potential cancer-immune biomarkers for diagnosis, prognosis, and therapeutic regimen determinants. In this review, the authors shed light on the structure and functional roles of circRNAs and, most importantly, highlight the promising roles of circRNAs in HCC immunomodulation and their potential as promising biomarkers and immunotherapeutic regimen determinants.

3'-tRF-CysGCA overexpression in HEK-293 cells alters the global expression profile and modulates cellular processes and pathways.

tRNA fragments (tRFs) are small non-coding RNAs generated through specific cleavage of tRNAs and involved in various biological processes. Among the different types of tRFs, the 3'-tRFs have attracted scientific interest due to their regulatory role in gene expression. In this study, we investigated the role of 3'-tRF-CysGCA, a tRF deriving from cleavage in the T-loop of tRNACysGCA, in the regulation of gene expression in HEK-293 cells. Previous studies have shown that 3'-tRF-CysGCA is incorporated into the RISC complex and interacts with Argonaute proteins, suggesting its involvement in the regulation of gene expression. However, the general role and effect of the deregulation of 3'-tRF-CysGCA levels in human cells have not been investigated so far. To fill this gap, we stably overexpressed 3'-tRF-CysGCA in HEK-293 cells and performed transcriptomic and proteomic analyses. Moreover, we validated the interaction of this tRF with putative targets, the levels of which were found to be affected by 3'-tRF-CysGCA overexpression. Lastly, we investigated the implication of 3'-tRF-CysGCA in various pathways using extensive bioinformatics analysis. Our results indicate that 3'-tRF-CysGCA overexpression led to changes in the global gene expression profile of HEK-293 cells and that multiple cellular pathways were affected by the deregulation of the levels of this tRF. Additionally, we demonstrated that 3'-tRF-CysGCA directly interacts with thymopoietin (TMPO) transcript variant 1 (also known as LAP2α), leading to modulation of its levels. In conclusion, our findings suggest that 3'-tRF-CysGCA plays a significant role in gene expression regulation and highlight the importance of this tRF in cellular processes.

Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

Next-generation sequencing reveals altered gene expression and enriched pathways in triple-negative breast cancer cells treated with oleuropein and oleocanthal.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by poor prognosis and limited treatment options. Oleuropein and oleocanthal are bioactive chemicals found in extra-virgin olive oil; they have been shown to have anti-cancer potential. In this study, we examined the inhibitory effects of these two natural compounds, on MDA-MB-231 and MDA-MB-468 TNBC cell lines. The human TNBC MDA-MB-231 and MDA-MB-468 cell lines were treated with oleuropein or oleocanthal at ranging concentrations for 48 h. After determining the optimum concentration to reach IC50, using the sulforhodamine B assay, total RNA was extracted after 12, 24, and 48 h from treated and untreated cells. Poly(A)-RNA selection was conducted, followed by library construction and RNA sequencing. Differential gene expression (DEG) analysis was performed to identify DEGs between treated and untreated cells. Pathway analysis was carried out using the KEGG and GO databases. Oleuropein and oleocanthal considerably reduced the proliferation of TNBC cells, with oleocanthal having a slightly stronger effect than oleuropein. Furthermore, multi-time series RNA sequencing showed that the expression profile of TNBC cells was significantly altered after treatment with these compounds, with temporal dynamics and groups of genes consistently affected at all time points. Pathway analysis revealed several significant pathways associated with TNBC, including cell death, apoptotic process, programmed cell death, response to stress, mitotic cell cycle process, cell division, and cancer progression. Our findings suggest that oleuropein and oleocanthal have potential therapeutic benefits for TNBC and can be further investigated as alternative treatment options.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

High mRNA Expression Levels of Heat Shock Protein Family B Member 2 (HSPB2) Are Associated with Breast Cancer Patients' Relapse and Poor Survival.

Small heat shock proteins (sHSPs) are ubiquitous ATP-independent chaperones that contribute to the maintenance of proteome integrity and functionality. Recent evidence suggests that sHSPs are ubiquitously expressed in numerous types of tumors and have been proposed to be implicated in oncogenesis and malignant progression. Heat shock protein family B member 2 (HSPB2) is a member of the sHSPs, which is found to be expressed, among others, in human breast cancer cell lines and constitutes an inhibitor of apical caspase activation in the extrinsic apoptotic pathway. In this study, we investigated the potential prognostic significance of HSPB2 mRNA expression levels in breast cancer, which represents the most frequent malignancy in females and one of the three most common cancer types worldwide. To this end, malignant breast tumors along with paired non-cancerous breast tissue specimens were used. HSPB2 expression levels were quantified in these two cohorts using a sensitive and accurate SYBR green-based quantitative real-time polymerase chain reaction (q-RT-PCR). Extensive biostatistical analyses were performed including Kaplan-Meier and Cox regression survival analyses for the assessment of the results. The significant downregulation of HSPB2 gene expression was revealed in breast tumors compared to their adjacent non-cancerous breast tissues. Notably, high HSPB2 mRNA expression predicts poor disease-free survival and overall survival of breast cancer patients. Multivariate Cox regression analysis revealed that HSPB2 mRNA overexpression is a significant predictor of poor prognosis in breast cancer, independent of other clinicopathological factors. In conclusion, high HSPB2 mRNA expression levels are associated with breast cancer patients' relapse and poor survival.

A Cancer-Related microRNA Signature Shows Biomarker Utility in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy, arising from terminally differentiated B cells, namely plasma cells. miRNAs are small non-coding RNAs that participate in the post-transcriptional regulation of gene expression. In this study, we investigated the role of nine miRNAs in MM. CD138+ plasma cells were selected from bone marrow aspirates from MM and smoldering MM (sMM) patients. Total RNA was extracted and in vitro polyadenylated. Next, first-strand cDNA synthesis was performed using an oligo-dT-adapter primer. For the relative quantification of the investigated miRNAs, an in-house real-time quantitative PCR (qPCR) assay was developed. A functional in silico analysis of the miRNAs was also performed. miR-16-5p and miR-155-5p expression was significantly lower in the CD138+ plasma cells of MM patients than in those of sMM patients. Furthermore, lower levels of miR-15a-5p, miR-16-5p, and miR-222-3p were observed in the CD138+ plasma cells of MM patients with osteolytic bone lesions, compared to those without. miR-125b-5p was also overexpressed in the CD138+ plasma cells of MM patients with bone disease that presented with skeletal-related events (SREs). Furthermore, lower levels of miR-223-3p were associated with significantly worse overall survival in MM patients. In conclusion, we propose a miRNA signature with putative clinical utility in MM.

tRNA Derivatives in Multiple Myeloma: Investigation of the Potential Value of a tRNA-Derived Molecular Signature.

Multiple myeloma (MM) is a hematologic malignancy arising from the clonal proliferation of malignant plasma cells. tRNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNAs, deriving from specific enzymatic cleavage of tRNAs. To the best of our knowledge, this is one of few studies to uncover the potential clinical significance of tRFs in MM. Total RNA was extracted from CD138+ plasma cells of MM and smoldering MM patients, and in vitro polyadenylated. First-strand cDNA synthesis was performed, priming from an oligo-dT-adaptor sequence. Next, real-time quantitative PCR (qPCR) assays were developed for the quantification of six tRFs. Biostatistical analysis was performed to assess the results and in silico analysis was conducted to predict the function of one of the tRFs. Our results showed that elevated levels of five out of six tRFs are indicators of favorable prognosis in MM, predicting prolonged overall survival (OS), while two of them constitute potential molecular biomarkers of favorable prognosis in terms of disease progression. Moreover, three tRFs could be used as surrogate prognostic biomarkers along with the R-ISS staging system to predict OS. In conclusion, tRFs show molecular biomarker utility in MM, while their mechanisms of function merit further investigation.

The Role of Circulating MicroRNAs in Patients with Early-Stage Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is increasing in incidence and is still associated with a high rate of mortality. Only a minority of patients are diagnosed in the early stage. Radical surgery is the only potential curative procedure. However, radicality is reached in 20% of patients operated on. Despite the multidisciplinary approach in resectable tumors, early tumor recurrences are common. Options on how to select optimal candidates for resection remain limited. Nevertheless, accumulating evidence shows an important role of circulating non-coding plasma and serum microRNAs (miRNAs), which physiologically regulate the function of a target protein. miRNAs also play a crucial role in carcinogenesis. In PDAC patients, the expression levels of certain miRNAs vary and may modulate the function of oncogenes or tumor suppressor genes. As they can be detected in a patient's blood, they have the potential to become promising non-invasive diagnostic and prognostic biomarkers. Moreover, they may also serve as markers of chemoresistance. Thus, miRNAs could be useful for early and accurate diagnosis, prognostic stratification, and individual treatment planning. In this review, we summarize the latest findings on miRNAs in PDAC patients, focusing on their potential use in the early stage of the disease.

Circular RNAs: Emerging Regulators of the Major Signaling Pathways Involved in Cancer Progression.

Signal transduction is an essential process that regulates and coordinates fundamental cellular processes, such as development, immunity, energy metabolism, and apoptosis. Through signaling, cells are capable of perceiving their environment and adjusting to changes, and most signaling cascades ultimately lead to alterations in gene expression. Circular RNAs (circRNAs) constitute an emerging type of endogenous transcripts with regulatory roles and unique properties. They are stable and expressed in a tissue-, cell-, and developmental stage-specific manner, while they are involved in the pathogenesis of several diseases, including cancer. Aberrantly expressed circRNAs can mediate cancer progression through regulation of the activity of major signaling cascades, such as the VEGF, WNT/ß-catenin, MAPK, PI3K/AKT, and Notch signaling pathways, as well as by interfering with signaling crosstalk. Deregulated signaling can then function to induce angiogenesis, promote invasion, migration, and metastasis, and, generally, modulate the hallmarks of cancer. In this review article, we summarize the most recently described and intriguing cases of circRNA-mediated signaling regulation that are involved in cancer progression, and discuss the biomarker potential of circRNAs, as well as future therapeutic applications.

The Multifaceted Role and Utility of MicroRNAs in Indolent B-Cell Non-Hodgkin Lymphomas.

Normal B-cell development is a tightly regulated complex procedure, the deregulation of which can lead to lymphomagenesis. One common group of blood cancers is the B-cell non-Hodgkin lymphomas (NHLs), which can be categorized according to the proliferation and spread rate of cancer cells into indolent and aggressive ones. The most frequent indolent B-cell NHLs are follicular lymphoma and marginal zone lymphoma. MicroRNAs (miRNAs) are small non-coding RNAs that can greatly influence protein expression. Based on the multiple interactions among miRNAs and their targets, complex networks of gene expression regulation emerge, which normally are essential for proper B-cell development. Multiple miRNAs have been associated with B-cell lymphomas, as the deregulation of these complex networks can lead to such pathological states. The aim of the present review is to summarize the existing information regarding the multifaceted role of miRNAs in indolent B-cell NHLs, affecting the main B-cell subpopulations. We attempt to provide insight into their biological function, the complex miRNA-mRNA interactions, and their biomarker utility in these malignancies. Lastly, we address the limitations that hinder the investigation of the role of miRNAs in these lymphomas and discuss ways that these problems could be overcome in the future.

Multiple Myeloma Bone Disease: Implication of MicroRNAs in Its Molecular Background.

Multiple myeloma (MM) is a common hematological malignancy arising from terminally differentiated plasma cells. In the majority of cases, symptomatic disease is characterized by the presence of bone disease. Multiple myeloma bone disease (MMBD) is a result of an imbalance in the bone-remodeling process that leads to increased osteoclast activity and decreased osteoblast activity. The molecular background of MMBD appears intriguingly complex, as several signaling pathways and cell-to-cell interactions are implicated in the pathophysiology of MMBD. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the expression of their target mRNAs. Numerous miRNAs have been witnessed to be involved in cancer and hematological malignancies and their role has been characterized either as oncogenic or oncosuppressive. Recently, scientific research turned towards miRNAs as regulators of MMBD. Scientific data support that miRNAs finely regulate the majority of the signaling pathways implicated in MMBD. In this review, we provide concise information regarding the molecular pathways with a significant role in MMBD and the miRNAs implicated in their regulation. Moreover, we discuss their utility as molecular biomarkers and highlight the putative usage of miRNAs as novel molecular targets for targeted therapy in MMBD.

MicroRNAs: Tiny Regulators of Gene Expression with Pivotal Roles in Normal B-Cell Development and B-Cell Chronic Lymphocytic Leukemia.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs bearing regulatory potency. The implication of miRNAs in physiological cellular processes has been well documented so far. A typical process orchestrated by miRNAs is the normal B-cell development. A stage-specific expression pattern of miRNAs has been reported in the developmental procedure, as well as interactions with transcription factors that dictate B-cell development. Besides their involvement in normal hematopoiesis, miRNAs are severally implicated in hematological malignancies, a typical paradigm of which is B-cell chronic lymphocytic leukemia (B-CLL). B-CLL is a highly heterogeneous disease characterized by the accumulation of abnormal B cells in blood, bone marrow, lymph nodes, and spleen. Therefore, timely, specific, and sensitive assessment of the malignancy is vital. Several studies have attempted to highlight the remarkable significance of miRNAs as regulators of gene expression, biomarkers for diagnosis, prognosis, progression, and therapy response prediction, as well as molecules with potential therapeutic utility. This review seeks to outline the linkage between miRNA function in normal and malignant hematopoiesis by demonstrating the main benchmarks of the implication of miRNAs in the regulation of normal B-cell development, and to summarize the key findings about their value as regulators, biomarkers, or therapeutic targets in B-CLL.

Identification of Two Novel Circular RNAs Deriving from BCL2L12 and Investigation of Their Potential Value as a Molecular Signature in Colorectal Cancer.

The utility of circular RNAs (circRNAs) as molecular biomarkers has recently emerged. However, only a handful of them have already been studied in colorectal cancer (CRC). The purpose of this study was to identify new circRNAs deriving from BCL2L12, a member of the BCL2 apoptosis-related family, and investigate their potential as biomarkers in CRC. Total RNA extracts from CRC cell lines and tissue samples were reversely transcribed. By combining PCR with divergent primers and nested PCR followed by Sanger sequencing, we were able to discover two BCL2L12 circRNAs. Subsequently, bioinformatical tools were used to predict the interactions of these circRNAs with microRNAs (miRNAs) and RNA-binding proteins (RBPs). Following a PCR-based pre-amplification, real-time qPCR was carried out for the quantification of each circRNA in CRC samples and cell lines. Biostatistical analysis was used to assess their potential prognostic value in CRC. Both novel BCL2L12 circRNAs likely interact with particular miRNAs and RBPs. Interestingly, circ-BCL2L12-2 expression is inversely associated with TNM stage, while circ-BCL2L12-1 overexpression is associated with shorter overall survival in CRC, particularly among TNM stage II patients. Overall, we identified two novel BCL2L12 circRNAs, one of which can further stratify TNM stage II patients into two subgroups with substantially distinct prognosis.

A novel, mitochondrial, internal tRNA-derived RNA fragment possesses clinical utility as a molecular prognostic biomarker in chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults. The prognosis of CLL patients varies considerably. Transfer RNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNA fragments excised from mature tRNAs and pre-tRNAs located in nuclei as well as in mitochondria. In this study, the clinical utility of i-tRF-PheGAA, a novel mitochondrial tRF, was investigated in CLL. DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 91 CLL patients and 43 non-leukemic controls. Total RNA was isolated from each sample, polyadenylated at the 3' end and reversely transcribed. An in-house developed real-time quantitative PCR assay was developed and applied, and the results were biostatistically analyzed. For the normalization of the i-tRF-PheGAA expression levels, the expression of a small nucleolar RNA (RNU48) was used as reference. RESULTS: Mann-Whitney U test showed that i-tRF-PheGAA can distinguish between CLL samples and normal controls (p < 0.001). As determined by Kaplan-Meier survival analysis, overexpression of i-tRF-PheGAA was related to poor overall survival of the CLL patients (p < 0.001). Univariate bootstrap Cox regression analysis exhibited a higher hazard ratio of 7.95 (95% CI = 2.37-26.72, p < 0.001) for patients with positive i-tRF-PheGAA expression status. Multivariate bootstrap Cox regression analysis showed that the prognostic value of this tRF is independent of clinical stage, mutational status of the immunoglobulin heavy chain variable (IGHV) genetic locus, and CD38 expression status (p = 0.010). CONCLUSIONS: Our results show that i-tRF-PheGAA can serve as a molecular biomarker of poor prognosis in CLL, alongside with the existing factors for CLL prognosis.