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Abstract Several non‐coding RNAs are known to be associated with the pathobiology and progression of multiple myeloma (MM). ciRS‐7 (also known as CDR1‐AS), a key oncogenic circular RNA (circRNA) that sponges miR‐7‐5p and other cancer‐related microRNAs, was recently found to be downregulated in malignant plasma cells resistant to immunomodulatory drugs. Considering that various circRNAs have a strong potential as molecular biomarkers, we aimed to investigate the expression of ciRS‐7 in plasma cell disorders, assess its prognostic importance in MM, and compare these findings with those of individuals with smoldering MM (SMM) and monoclonal gammopathy of unknown significance (MGUS). This study included 171 patients (110 newly diagnosed MM, 34 SMM, and 27 MGUS cases), from which bone marrow aspirate samples were collected for CD138+ plasma cell selection. Total RNA was reversely transcribed using random hexamer primers, and the expression levels of ciRS‐7 were quantified using an in‐house‐developed protocol that includes pre‐amplification and real‐time quantitative polymerase chain reaction. ciRS‐7 levels were found to significantly differ among CD138+ plasma cells of MM, SMM, and MGUS patients. ROC analysis indicated that ciRS‐7 expression effectively distinguishes between MM and SMM patients. Moreover, high levels of ciRS‐7 were associated with unfavorable prognosis in MM, independently of MM patients’ age and Revised International Staging System stage. Additionally, in silico analysis predicted the binding of 85 microRNAs to ciRS‐7. In conclusion, this study provides novel insights into the role of ciRS‐7 as a promising molecular marker able to distinguish MM from SMM and predict prognosis in MM.

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.

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.

Abstract Circular RNAs (circRNAs) are associated with the pathobiology of multiple myeloma (MM). Recent findings regarding circCCT3 support its involvement in the development and progression of MM, through microRNA sponging. Thus, we aimed to examine the expression of circCCT3 in smoldering and symptomatic MM and to assess its clinical importance. Three cell lines from plasma cell neoplasms were cultured and bone marrow aspirate (BMA) samples were collected from 145 patients with MM or smoldering MM. Next, CD138+ enrichment was performed in BMA samples, followed by total RNA extraction and reverse transcription. Preamplification of circCCT3 and GAPDH cDNA was performed. Finally, a sensitive assay for the relative quantification of circCCT3 using nested real‐time quantitative polymerase chain reaction was developed, optimized, and implemented in the patients' samples and cell lines. MM patients exhibited significantly higher intracellular circCCT3 expression in their CD138+ plasma cells, compared to those from SMM patients. In addition, MM patients overexpressing circCCT3 had longer progression‐free and overall survival intervals. The favorable prognostic significance of high circCCT3 expression in MM was independent of disease stage (either International Staging System [ISS] or revised ISS [R‐ISS]) and age of MM patients. Interestingly, circCCT3 expression could serve as a surrogate molecular biomarker of prognosis in MM patients, especially those of R‐ISS stage II. In conclusion, our study sheds new light on the significance of circCCT3 as a promising molecular marker for predicting MM patients' prognosis.

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.

Colorectal cancer (CRC) is the second most lethal cause of cancer-related deaths in Europe. Fragments of tRNAPro are conserved among vertebrates, characterized by pleiotropic regulatory functions and have been found to discriminate colorectal tumors from normal colorectal mucosa. In the current study, we investigated the prognostic utility of 5′-tiRNA-ProTGG levels in CRC. For this purpose, total RNA was extracted from 155 malignant colorectal tumors and 74 adjacent non-cancerous tissue specimens, polyadenylated and reverse-transcribed using an oligo-dT adapter as primer. Real-time quantitative PCR (qPCR) was used to assess the levels of 5′-tiRNA-ProTGG. Kaplan-Meier survival analysis demonstrated that high 5′-tiRNA-ProTGG levels predict both poor disease-free survival (DFS) and overall survival (OS) of CRC patients. Of note, high 5′-tiRNA-ProTGG levels retain their unfavorable prognostic value in patients with rectal cancer and/or moderately differentiated CRC (grade II). More importantly, multivariate cox regression analysis highlighted that the overexpression of 5′-tiRNA-ProTGG constitutes an adverse prognostic factor predicting short-term relapse of CRC patients independently of the established prognosticators in CRC. Finally, bioinformatics analysis unveiled a potentially critical role of 5′-tiRNA-ProTGG regarding the maintenance of cellular homeostasis, signaling, cell communication, and cellular motility.

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.