Small RNA-seq and clinical evaluation of tRNA-derived fragments in multiple myeloma: Loss of mitochondrial i-tRFHisGTG results in patients' poor treatment outcome.

Despite the substantial progress in multiple myeloma (MM) therapy nowadays, treatment resistance and disease relapse remain major clinical hindrances. Herein, we have investigated tRNA-derived fragment (tRF) profiles in MM and precursor stages (smoldering MM/sMM; monoclonal gammopathy of undetermined significance/MGUS), aiming to unveil potential MM-related tRFs in ameliorating MM prognosis and risk stratification. Small RNA-seq was performed to profile tRFs in bone marrow CD138+ plasma cells, revealing the significant deregulation of the mitochondrial internal tRFHisGTG (mt-i-tRFHisGTG) in MM versus sMM/MGUS. The screening cohort of the study consisted of 147 MM patients, and mt-i-tRFHisGTG levels were quantified by RT-qPCR. Disease progression was assessed as clinical end-point for survival analysis, while internal validation was performed by bootstrap and decision curve analyses. Screening cohort analysis highlighted the potent association of reduced mt-i-tRFHisGTG levels with patients' bone disease (p = 0.010), osteolysis (p = 0.023) and with significantly higher risk for short-term disease progression following first-line chemotherapy, independently of patients' clinical data (HR = 1.954; p = 0.036). Additionally, mt-i-tRFHisGTG-fitted multivariate models led to superior risk stratification of MM patients' treatment outcome and prognosis compared to disease-established markers. Notably, our study highlighted mt-i-tRFHisGTG loss as a powerful independent indicator of post-treatment progression of MM patients, leading to superior risk stratification of patients' treatment outcome.

Hybrid-seq deciphers the complex transcriptional profile of the human BRCA1 DNA repair associated gene.

Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants (BRCA1 sv.7 - sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

miRNA-seq identification and clinical validation of CD138+ and circulating miR-25 in treatment response of multiple myeloma.

BACKGROUND: Despite significant advancements in multiple myeloma (MM) therapy, the highly heterogenous treatment response hinders reliable prognosis and tailored therapeutics. Herein, we have studied the clinical utility of miRNAs in ameliorating patients' management. METHODS: miRNA-seq was performed in bone marrow CD138+ plasma cells (PCs) of 24 MM and smoldering MM (sMM) patients to analyze miRNAs profile. CD138+ and circulating miR-25 levels were quantified using in house RT-qPCR assays in our screening MM/sMM cohort (CD138+ plasma cells n = 167; subcohort of MM peripheral plasma samples n = 69). Two external datasets (Kryukov et al. cohort n = 149; MMRF CoMMpass study n = 760) served as institutional-independent validation cohorts. Patients' mortality and disease progression were assessed as clinical endpoints. Internal validation was performed by bootstrap analysis. Clinical benefit was estimated by decision curve analysis. RESULTS: miRNA-seq highlighted miR-25 of CD138+ plasma cells to be upregulated in MM vs. sMM, R-ISS II/III vs. R-ISS I, and in progressed compared to progression-free patients. The analysis of our screening cohort highlighted that CD138+ miR-25 levels were correlated with short-term progression (HR = 2.729; p = 0.009) and poor survival (HR = 4.581; p = 0.004) of the patients; which was confirmed by Kryukov et al. cohort (HR = 1.878; p = 0.005) and MMRF CoMMpass study (HR = 1.414; p = 0.039) validation cohorts. Moreover, multivariate miR-25-fitted models contributed to superior risk-stratification and clinical benefit in MM prognostication. Finally, elevated miR-25 circulating levels were correlated with poor survival of MM patients (HR = 5.435; p = 0.021), serving as a potent non-invasive molecular prognostic tool. CONCLUSIONS: Our study identified miR-25 overexpression as a powerful independent predictor of poor treatment outcome and post-treatment progression, aiding towards modern non-invasive disease prognosis and personalized treatment decisions.

Unveiling the Human Gastrointestinal Tract Microbiome: The Past, Present, and Future of Metagenomics.

Over 1014 symbiotic microorganisms are present in a healthy human body and are responsible for the synthesis of vital vitamins and amino acids, mediating cellular pathways and supporting immunity. However, the deregulation of microbial dynamics can provoke diverse human diseases such as diabetes, human cancers, cardiovascular diseases, and neurological disorders. The human gastrointestinal tract constitutes a hospitable environment in which a plethora of microbes, including diverse species of archaea, bacteria, fungi, and microeukaryotes as well as viruses, inhabit. In particular, the gut microbiome is the largest microbiome community in the human body and has drawn for decades the attention of scientists for its significance in medical microbiology. Revolutions in sequencing techniques, including 16S rRNA and ITS amplicon sequencing and whole genome sequencing, facilitate the detection of microbiomes and have opened new vistas in the study of human microbiota. Especially, the flourishing fields of metagenomics and metatranscriptomics aim to detect all genomes and transcriptomes that are retrieved from environmental and human samples. The present review highlights the complexity of the gastrointestinal tract microbiome and deciphers its implication not only in cellular homeostasis but also in human diseases. Finally, a thorough description of the widely used microbiome detection methods is discussed.

The Repertoire of RNA Modifications Orchestrates a Plethora of Cellular Responses.

Although a plethora of DNA modifications have been extensively investigated in the last decade, recent breakthroughs in molecular biology, including high throughput sequencing techniques, have enabled the identification of post-transcriptional marks that decorate RNAs; hence, epitranscriptomics has arisen. This recent scientific field aims to decode the regulatory layer of the transcriptome and set the ground for the detection of modifications in ribose nucleotides. Until now, more than 170 RNA modifications have been reported in diverse types of RNA that contribute to various biological processes, such as RNA biogenesis, stability, and transcriptional and translational accuracy. However, dysfunctions in the RNA-modifying enzymes that regulate their dynamic level can lead to human diseases and cancer. The present review aims to highlight the epitranscriptomic landscape in human RNAs and match the catalytic proteins with the deposition or deletion of a specific mark. In the current review, the most abundant RNA modifications, such as N6-methyladenosine (m6A), N5-methylcytosine (m5C), pseudouridine (Ψ) and inosine (I), are thoroughly described, their functional and regulatory roles are discussed and their contributions to cellular homeostasis are stated. Ultimately, the involvement of the RNA modifications and their writers, erasers, and readers in human diseases and cancer is also discussed.

Unraveling the Concealed Transcriptomic Landscape of PTEN in Human Malignancies.

Background: Phosphatase and tensin homolog, widely known as PTEN, is a major negative regulator of the PI3K/AKT/mTOR signaling pathway, involved in the regulation of a variety of important cellular processes, including cell proliferation, growth, survival, and metabolism. Since most of the molecules involved in this biological pathway have been described as key regulators in cancer, the study of the corresponding genes at several levels is crucial. Objective: Although previous studies have elucidated the physiological role of PTEN under normal conditions and its involvement in carcinogenesis and cancer progression, the transcriptional profile of PTEN has been poorly investigated. Methods: In this study, instead of conducting the "gold-standard" direct RNA sequencing that fails to detect less abundant novel mRNAs due to the decreased sequencing depth, we designed and implemented a multiplexed PTEN-targeted sequencing approach that combined both short- and long-read sequencing. Results: Our study has highlighted a broad spectrum of previously unknown PTEN mRNA transcripts and assessed their expression patterns in a wide range of human cancer and non-cancer cell lines, shedding light on the involvement of PTEN in cell cycle dysregulation and thus tumor development. Conclusion: The identification of the described novel PTEN splice variants could have significant implications for understanding PTEN regulation and function, and provide new insights into PTEN biology, opening new avenues for monitoring PTEN-related diseases, including cancer.

The Transition from Cancer "omics" to "epi-omics" through Next- and Third-Generation Sequencing.

Deciphering cancer etiopathogenesis has proven to be an especially challenging task since the mechanisms that drive tumor development and progression are far from simple. An astonishing amount of research has revealed a wide spectrum of defects, including genomic abnormalities, epigenomic alterations, disturbance of gene transcription, as well as post-translational protein modifications, which cooperatively promote carcinogenesis. These findings suggest that the adoption of a multidimensional approach can provide a much more precise and comprehensive picture of the tumor landscape, hence serving as a powerful tool in cancer research and precision oncology. The introduction of next- and third-generation sequencing technologies paved the way for the decoding of genetic information and the elucidation of cancer-related cellular compounds and mechanisms. In the present review, we discuss the current and emerging applications of both generations of sequencing technologies, also referred to as massive parallel sequencing (MPS), in the fields of cancer genomics, transcriptomics and proteomics, as well as in the progressing realms of epi-omics. Finally, we provide a brief insight into the expanding scope of sequencing applications in personalized cancer medicine and pharmacogenomics.

Structural characterization and expression analysis of novel MAPK1 transcript variants with the development of a multiplexed targeted nanopore sequencing approach.

Mitogen-activated protein kinases (MAPKs) represent a protein family firmly involved in many signaling cascades, regulating a vast spectrum of stimulated cellular processes. Studies have shown that alternatively spliced isoforms of MAPKs play a crucial role in determining the desired cell fate in response to specific stimulations. Although the implication of most MAPKs transcript variants in the MAPK signaling cascades has been clarified, the transcriptional profile of a pivotal member, MAPK1, has not been investigated for the existence of additional isoforms. In the current study we developed and implemented targeted long-read and short-read sequencing approaches to identify novel MAPK1 splice variants. The combination of nanopore sequencing and NGS enabled the implementation of a long-read polishing pipeline using error-rate correction algorithms, which empowered the high accuracy of the results and increased the sequencing efficiency. The utilized multiplexing option in the nanopore sequencing approach allowed not only the identification of novel MAPK1 mRNAs, but also elucidated their expression profile in multiple human malignancies and non-cancerous cell lines. Our study highlights for the first time the existence of ten previously undescribed MAPK1 mRNAs (MAPK1 v.3 - v.12) and evaluates their relative expression levels in comparison to the main MAPK1 v.1. The optimization and employment of qPCR assays revealed that MAPK1 v.3 - v.12 can be quantified in a wide spectrum of human cell lines with notable specificity. Finally, our findings suggest that the novel protein-coding mRNAs are highly expected to participate in the regulation of MAPK pathways, demonstrating differential localizations and functionalities.

Decoding the concealed transcriptional signature of the apoptosis-related BCL2 antagonist/killer 1 (BAK1) gene in human malignancies.

BCL2 antagonist/killer (BAK) is a multidomain pro-apoptotic effector protein, encoded by the human BAK1 gene, which has emerged as a key checkpoint in the apoptotic machinery. Disassembly of BAK's tertiary structure, such as the truncation of the α1 helix, leads to deregulation of the pro-apoptotic functions and reduction of the protein's stability, thus being implicated in human malignancies. Although many studies have already clarified the vital role of BAK in cellular mechanisms, its pre-mRNA maturation process under cancerous and physiological human cells is neglected. In the present work, we developed and employed a custom multiplexed nanopore sequencing approach that enabled the identification and structural characterization of previously undescribed BAK1 mRNA transcripts (BAK1 v.2-v.11). The described novel mRNAs are derived from multiple types of alternative splicing events, including exon skipping and intron retentions. The implemented multiplexed long-read sequencing approach provided the detailed expression profile of the novel mRNAs in a wide panel of human malignancies and at the same time allowed their relative quantification as compared to the annotated BAK1 v.1. The validation of each novel transcript was carried out with qPCR-based assays. Our results strongly support that most of the novel BAK1 mRNAs harbor open reading frames with conserved BH domains that provide new insights into the correlated mechanisms of apoptosis suppression and cancer. The current study highlights for the first time the hidden aspects of BAK1's transcriptional landscape in both physiological and cancerous human cells and distinguishes the amino acid sequence of the putative BAK isoforms that may possess key apoptosis-related functions not only in diseases, but also under normal cellular conditions.

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.

A versatile 5' RACE-Seq methodology for the accurate identification of the 5' termini of mRNAs.

BACKGROUND: Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5' ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5' rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5' ends of cDNAs. In the current study, we developed a 5' RACE-seq method by coupling a custom template-switching and 5' RACE assay with targeted nanopore sequencing, to accurately unveil 5' termini of mRNA targets. RESULTS: The optimization of the described 5' RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5' RACE. Collectively, our results support the existence of two distinct 5' termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5' RACE-seq methodology to investigate the 5' UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5' UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones. CONCLUSIONS: In this work we present an in-house developed 5' RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5' UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.

miRNA-seq and clinical evaluation in multiple myeloma: miR-181a overexpression predicts short-term disease progression and poor post-treatment outcome.

BACKGROUND: Despite significant advances in multiple myeloma (MM) therapy, disease relapse and treatment resistance remain major obstacles in clinical management. Herein, we have studied the clinical utility of miRNAs in improving patients' risk-stratification and prognosis. METHODS: miRNA-seq was performed in CD138+ plasma cells of MM, smoldering multiple myeloma (sMM) and monoclonal gammopathy of undetermined significance (MGUS) patients. The screening MM cohort consisted of 138 patients. miRNA levels of CD138+ plasma cells were quantified by RT-qPCR following 3'-end RNA polyadenylation. Disease progression and patients' death were used as clinical end-point events. Internal validation was conducted by bootstrap analysis. Clinical net benefit on disease prognosis was assessed by decision curve analysis. Kruykov et al. 2016 served as validation cohort (n = 151). RESULTS: miRNA-seq highlighted miR-181a to be upregulated in MM vs. sMM/MGUS, and R-ISS III vs. I patients. Screening and validation cohorts confirmed the significantly higher risk for short-term progression and worse survival of the patients overexpressing miR-181a. Multivariate models integrating miR-181a with disease established markers led to superior risk-stratification and clinical benefit for MM prognosis. CONCLUSIONS: CD138+ overexpression of miR-181a was strongly correlated with inferior disease outcome and contributed to superior prediction of MM patients early progression, supporting personalised prognosis and treatment decisions.

A comprehensive nanopore sequencing methodology deciphers the complete transcriptional landscape of cyclin-dependent kinase 4 (CDK4) in human malignancies.

Cyclin-dependent kinase 4 (CDK4) is a member of the cyclin-dependent kinases, a family of protein kinases with outstanding roles in signaling pathways, transcription regulation, and cell division. Defective or overactivated CDK4/cyclin D1 pathway leads to enhanced cellular proliferation, thus being implicated in human cancers. Although the biological role of CDK4 has been extensively studied, its pre-mRNA processing mechanism under normal or pathological conditions is neglected. Thus, the identification of novel CDK4 mRNA transcripts, especially protein-coding ones, could lead to the identification of new diagnostic and/or prognostic biomarkers or new therapeutic targets. In the present study, instead of using the 'gold standard' direct RNA sequencing application, we designed and employed a targeted nanopore sequencing approach, which offers higher sequencing depth and enables the thorough investigation of new mRNAs of any target gene. Our study elucidates for the first time the complex transcriptional landscape of the human CDK4 gene, highlighting the existence of previously unknown CDK4 transcripts with new alternative splicing events and protein-coding capacities. The relative expression levels of each novel CDK4 transcript in human malignancies were elucidated with custom qPCR-based assays. The presented wide spectrum of CDK4 transcripts (CDK4 v.2-v.42) is only the first step to distinguish and assemble the missing pieces regarding the exact functions and implications of this fundamental kinase in cellular homeostasis and pathophysiology.

Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members.

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes (KRAS, NRAS and HRAS), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

Nanopore Sequencing Unveils Diverse Transcript Variants of the Epithelial Cell-Specific Transcription Factor Elf-3 in Human Malignancies.

The human E74-like ETS transcription factor 3 (Elf-3) is an epithelium-specific member of the ETS family, all members of which are characterized by a highly conserved DNA-binding domain. Elf-3 plays a crucial role in epithelial cell differentiation by participating in morphogenesis and terminal differentiation of the murine small intestinal epithelium, and also acts as an indispensable regulator of mesenchymal to epithelial transition, underlying its significant involvement in development and in pathological states, such as cancer. Although previous research works have deciphered the functional role of Elf-3 in normal physiology as well as in tumorigenesis, the present study highlights for the first time the wide spectrum of ELF3 mRNAs that are transcribed, providing an in-depth analysis of splicing events and exon/intron boundaries in a broad panel of human cell lines. The implementation of a versatile targeted nanopore sequencing approach led to the identification of 25 novel ELF3 mRNA transcript variants (ELF3 v.3-v.27) with new alternative splicing events, as well as two novel exons. Although the current study provides a qualitative transcriptional profile regarding ELF3, further studies must be conducted, so the biological function of all novel alternative transcript variants as well as the putative protein isoforms are elucidated.

A 3' tRNA-derived fragment produced by tRNALeuAAG and tRNALeuTAG is associated with poor prognosis in B-cell chronic lymphocytic leukemia, independently of classical prognostic factors.

OBJECTIVE: 3' tRNA-derived fragments (3' tRFs) are important epigenetic regulators in normal and pathological conditions. In this study, we aimed to explore the potential value of a 3' tRF as a prognostic and/or screening biomarker for B-cell chronic lymphocytic leukemia (B-CLL). METHODS: Publicly available next-generation sequencing data from 20 B-CLL cases were analyzed, followed by prediction of targets of the most abundantly and ubiquitously expressed 3' tRFs, leading to selection of tRF-LeuAAG/TAG . PBMCs were isolated from blood samples of 91 B-CLL patients and 43 non-leukemic donors, followed by total RNA extraction, in-vitro polyadenylation, and first-strand cDNA synthesis. Next, a real-time quantitative PCR (qPCR) assay was developed for the accurate quantification of tRF-LeuAAG/TAG and applied in all samples, prior to biostatistical analysis. RESULTS: High tRF-LeuAAG/TAG levels are associated with inferior overall survival (OS) of B-CLL patients. The unfavorable significance of tRF-LeuAAG/TAG was independent of established prognostic factors in B-CLL. Stratified Kaplan-Meier OS analysis uncovered the unfavorable prognostic role of high tRF-LeuAAG/TAG levels for patients in Binet A or Rai I stage, negative CD38 expression, mutated, or unmutated IGHV genomic locus. CONCLUSION: Our approach revealed the independent prognostic value of a particular 3' tRF, derived from tRNALeuAAG and tRNALeuTAG (tRF-LeuAAG/TAG ) in B-CLL.

Unraveling novel survivin mRNA transcripts in cancer cells using an in-house developed targeted high-throughput sequencing approach.

The human baculoviral IAP repeat containing 5 (BIRC5), also known as survivin, is a conserved member of the inhibitor of apoptosis protein (IAPs) family, which is normally expressed during embryonic and fetal development. Although the expression levels of survivin are low in terminally differentiated cells and/or tissues, they can be found notably increased in certain pathological conditions including malignant tumors. Conventional cloning and sequencing techniques have already confirmed that alternative splicing events of the survivin pre-mRNA result in five distinct alternative transcript variants. In the present study, however, we implemented an innovative, in-house developed, targeted DNA-seq assay to identify novel survivin alternative transcript variants with increased depth and coverage that high-throughput sequencing approaches offer. Bioinformatics analysis of the derived NGS datasets unveiled several novel splice junctions between annotated exons of survivin gene as well as the existence of a novel exon of 117 nt, spanning between the annotated exons 3 and 3B. Validation of the NGS findings with PCR-based assays, using variant-specific primers, led to the identification of fourteen novel survivin alternative splice variants (BIRC5 v.4 - v.17), which demonstrate wide expression profiles in a broad established panel of human cell lines. Although the presented novel findings provide a crystal-clear overview of the survivin mRNAs that are actually generated from the pre-mRNA, future studies should focus on the impending necessity of characterizing the biological function of all novel alternative transcript variants as well as the putative protein isoforms. Such studies will further contribute to our understanding of how the balance between survivin isoforms regulate malignant cell proliferation and apoptosis, providing novel diagnostic, prognostic and predictive biomarkers as well as therapeutic targets.

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.

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.

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.