Editorial for the Special Issue "Molecular Biomarkers in Colorectal Adenocarcinoma".

Colorectal cancer (CRC) is one of the most common malignancies, with an elevated mortality rate [...].

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.

Heat shock protein beta 3 (HSPB3) is an unfavorable molecular biomarker in colorectal adenocarcinoma.

Small heat shock proteins (sHSPs) participate in numerous cellular functions including cell signaling, differentiation, and apoptosis. Deregulation of the physiological expression level of sHSPs has been associated with several malignancies. Heat shock protein beta 3 (HSPB3) is the third member of the sHSP family in human and is mainly expressed in skeletal and smooth muscles. In this study, we investigated the potential prognostic significance of HSPB3 expression in colorectal adenocarcinoma, the most frequent type of colorectal cancer. For this purpose, we isolated total RNA from 188 colorectal adenocarcinoma specimens and 68 paired noncancerous ones. After reverse transcription of 2 µg total RNA, we quantified HSPB3 levels by using an in-house-developed real-time quantitative polymerase chain reaction method, based on the SYBR Green chemistry. Comparison of HSPB3 levels among 68 pairs of colorectal tumors and their adjacent noncancerous mucosae uncovered the downregulation of HSPB3 expression in the majority of malignant colorectal tumors. More importantly, high HSPB3 expression is associated with poor relapse-free survival (RFS) and overall survival (OS) of patients with colorectal adenocarcinoma. Multivariable Cox regression analysis revealed that HSPB3 overexpression could serve as an adverse prognostic biomarker in colorectal adenocarcinoma, independent of tumor location, histological grade, and TNM stage. Patients' stratification according to tumor location, histological grade, and TNM stage revealed that high HSPB3 messenger RNA expression retains its unfavorable prognostic potential regarding OS, in particular groups of patients with substantially different prognosis. In conclusion, high HSPB3 expression is associated with poor RFS and OS of patients with colorectal adenocarcioma, independently of clinicopathological prognosticators.

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.

Revised Exon Structure of l-DOPA Decarboxylase (DDC) Reveals Novel Splice Variants Associated with Colorectal Cancer Progression.

Colorectal cancer (CRC) is a highly heterogenous malignancy with an increased mortality rate. Aberrant splicing is a typical characteristic of CRC, and several studies support the prognostic value of particular transcripts in this malignancy. l-DOPA decarboxylase (DDC) and its derivative neurotransmitters play a multifaceted role in physiological and pathological states. Our recent data support the existence of 6 DDC novel exons. In this study, we investigated the existence of additional DDC novel exons and transcripts, and their potential value as biomarkers in CRC. Next-generation sequencing (NGS) in 55 human cell lines coupled with Sanger sequencing uncovered 3 additional DDC novel exons and 20 splice variants, 7 of which likely encode new protein isoforms. Eight of these transcripts were detected in CRC. An in-house qPCR assay was developed and performed in TNM II and III CRC samples for the quantification of transcripts bearing novel exons. Extensive biostatistical analysis uncovered the prognostic value of specific DDC novel exons for patients' disease-free and overall survival. The revised DDC exon structure, the putative protein isoforms with distinct functions, and the prognostic value of novel exons highlight the pivotal role of DDC in CRC progression, indicating its potential utility as a molecular biomarker in CRC.

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.

Alternative Splicing in Human Physiology and Disease.

Since the discovery of alternative splicing in the late 1970s, a great number of alternatively spliced transcripts have emerged; this number has exponentially increased with the advances in transcriptomics and massive parallel sequencing technologies [...].

High Expression of a tRNAPro Derivative Associates with Poor Survival and Independently Predicts Colorectal Cancer Recurrence.

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.

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.

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.

Next-generation sequencing reveals alternative L-DOPA decarboxylase (DDC) splice variants bearing novel exons, in human hepatocellular and lung cancer cells.

The human L-DOPA decarboxylase (DDC) is an enzyme that displays a pivotal role in metabolic processes. It is implicated in various human disorders, including hepatocellular and lung cancer. Several splice variants of DDC have previously been described, most of which encode for protein isoforms of this enzyme. In the present study, we used next-generation sequencing (NGS) technology along with nested touchdown PCR and Sanger sequencing to identify new splice variants bearing novel exons of the DDC gene, in hepatocellular and lung cancer cell lines. Using an in-house-developed algorithm, we discovered seven novel DDC exons. Next, we determined the structure of ten novel DDC transcripts, three of which contain an open reading frame (ORF) and probably encode for three previously unknown protein isoforms of this enzyme. Future studies should focus on the elucidation of their role in cellular physiology and cancer pathobiology.

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.

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.

Circular RNAs: A New Piece in the Colorectal Cancer Puzzle.

Colorectal cancer (CRC) is the third most fatal type of malignancy, worldwide. Despite the advances accomplished in the elucidation of its molecular base and the existing CRC biomarkers introduced in the clinical practice, additional research is required. Circular RNAs (circRNAs) constitute a new RNA type, formed by back-splicing of primary transcripts. They have been discovered during the 1970s but were characterized as by-products of aberrant splicing. However, the modern high-throughput approaches uncovered their widespread expression; therefore, several questions were raised regarding their potential biological roles. During the last years, great progress has been achieved in the elucidation of their functions: circRNAs can act as microRNA sponges, transcription regulators, and interfere with splicing, as well. Furthermore, they are heavily involved in various human pathological states, including cancer, and could serve as diagnostic and prognostic biomarkers in several diseases. Particularly in CRC, aberrant expression of circRNAs has been observed. More specifically, these molecules either inhibit or promote colorectal carcinogenesis by regulating different molecules and signaling pathways. The present review discusses the characteristics and functions of circRNA, prior to analyzing the multifaceted role of these molecules in CRC and their potential value as biomarkers and therapeutic targets.

The role of circular RNAs in therapy resistance of patients with solid tumors.

Circular RNAs (circRNAs) are a type of single-stranded RNA molecules forming a covalently closed, continuous structure, lacking 5'-3' polarity and polyadenylated tails. Recent advances in high-throughput sequencing technologies have revealed that these molecules are abundant, resistant to degradation and often expressed in a tissue- or developmental stage-specific manner. circRNAs are produced by back-splicing circularization of primary transcripts and exhibit a variety of functions, including regulation of transcription, translation and cellular localization. This review focuses on differentially expressed circRNAs conferring therapy resistance or sensitivity of solid tumors, such as carcinomas, sarcomas and lymphomas. Deregulated circRNAs can participate in the development of resistance to treatment by modulating regulatory pathways and cellular processes, including the mitogen-activated protein kinase pathway, epithelial-mesenchymal transition, apoptosis and autophagy.

High clusterin (CLU) mRNA expression levels in tumors of colorectal cancer patients predict a poor prognostic outcome.

OBJECTIVES: Clusterin (CLU) is a multifunctional intra-/extra-cellular molecular chaperone with indications of serving as a promising prognostic biomarker for colorectal cancer (CRC). Several studies have examined the potential prognostic value of the CLU protein in CRC; however, our research follows an alternative approach, focusing on the CLU mRNA expression. DESIGN AND METHODS: Total RNA from 172 cancerous tissue specimens and 39 paired non-cancerous ones was isolated and 2 µg of this were subjected to reverse transcription with an oligo-dT primer. The single stranded DNA, which was synthesized, was amplified with an in-house developed highly sensitive and precise qPCR method, using specific pair of primers for the CLU molecule. Finally, an extensive biostatistical analysis took place for the assessment of the results. RESULTS: Patients with tumors expressing high CLU mRNA levels had a higher probability for poor outcome (relapse and death), comparing to those with CLU mRNA-negative tumors. This association between CLU mRNA expression status and both disease-free survival (DFS) and overall survival (OS) is evident in Cox regression analysis and is also depicted in the Kaplan-Meier survival curves. Consistently, the aforementioned associations and the CLU mRNA expression levels are significantly enhanced as CRC tumors progress from TNM stage I to IV, further supporting the functional implication of CLU in tumorigenesis. CONCLUSIONS: High CLU mRNA levels in CRC tumors can act as a new adverse prognostic biomarker of DFS and OS for CRC, independent of clinicopathological and biological features of the patient.

Identification of a novel, internal tRNA-derived RNA fragment as a new prognostic and screening biomarker in chronic lymphocytic leukemia, using an innovative quantitative real-time PCR assay.

Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Several studies have identified various prognostic biomarkers in CLL. In this study, we investigated the potential value of an internal fragment of the tRNAs bearing the Glycine anticodon CCC (i-tRF-GlyCCC), which is a small non-coding RNA, as a prognostic and screening biomarker in CLL. For this purpose, blood samples were collected from 90 CLL patients and 43 non-leukemic blood donors. Peripheral blood mononuclear cells (PBMCs) were isolated, total RNA was extracted and in-vitro polyadenylated, and first-strand cDNA was synthesized using an oligo-dT-adaptor primer. A real-time quantitative PCR assay was developed and applied for the quantification of i-tRF-GlyCCC in our samples. The biostatistical analysis revealed that i-tRF-GlyCCC levels are significantly lower in PBMCs of CLL patients, compared to PBMCs of non-leukemic controls, and that i-tRF-GlyCCC could be considered as a screening biomarker. Kaplan-Meier overall survival (OS) analysis revealed reduced OS for CLL patients with positive i-tRF-GlyCCC expression (P = 0.001). Multivariate Cox regression confirmed its independent unfavorable prognostic power with regard to OS. In conclusion, i-tRF-GlyCCC may constitute a promising molecular biomarker in CLL, for screening and prognostic purposes.