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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.

Alternative splicing is commonly involved in carcinogenesis, being highly implicated in differential expression of cancer-related genes. Recent studies have shown that the human CEACAM19 gene is overexpressed in malignant breast and ovarian tumors, possessing significant biomarker attributes. In the present study, 3' rapid amplification of cDNA ends (3' RACE) and next-generation sequencing (NGS) were used for the detection and identification of novel CEACAM19 transcripts. Bioinformatical analysis of our NGS data revealed novel splice junctions between previously annotated exons and ultimately new exons. Next, fifteen novel CEACAM19 transcripts were identified with Sanger sequencing. Additionally, their expression profile was investigated in a wide panel of human cell lines, using nested PCR with variant-specific primers. The broad expression pattern of the CEACAM19 gene, along with the fact that its overexpression has previously been associated with ovarian and breast cancer progression, indicate the potential of novel CEACAM19 transcripts as putative diagnostic and/or prognostic biomarkers., (Copyright © 2020. Published by Elsevier Inc.)

The kallikrein-related peptidase 15 (KLK15) gene is a member of the largest cluster of serine proteases in the human genome. Exhibiting trypsin-like activity, KLK15 is most likely involved in the activation of prostate-specific antigen (PSA; also known as KLK3), an established biomarker for the diagnosis and screening of prostate cancer. High mRNA expression levels of KLK15 have already been reported in ovarian and prostate cancer, in contrast with breast cancer, where KLK15 has been proposed as a biomarker of favorable prognosis. In this study, we exploited the next-generation sequencing (NGS) technology along with 3' rapid amplification of cDNA ends (3' RACE) to discover alternative KLK15 splice variants. Extensive computational analysis of the obtained NGS data revealed the existence of novel splice junctions, thus supporting the existence of novel KLK15 transcripts. Six novel KLK15 splice variants were identified and verified by Sanger sequencing. Two of them (KLK15 v.11 and v.12) contain an open reading frame and are hence predicted to encode two novel KLK15 protein isoforms. Expression analysis of each KLK15 splice variant in sixteen cDNA pools from malignant cell lines and in normal cell lines (HEK293, HaCaT, and BJ cells) revealed very different expression profiles of particular KLK15 transcripts. Moreover, the new KLK15 splice variants were shown to be expressed in breast, ovarian, prostate, urinary bladder, colon, and renal tissue specimens. Due to the prominent clinical value of KLK15 mRNA expression, the novel KLK15 transcripts appear as candidate cancer biomarkers for diagnostic and/or prognostic purposes and, therefore, merit further investigation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The human RNASEK gene encodes Ribonuclease κ, an endoribonuclease that belongs to a highly conserved protein family of metazoans. Recent evidence suggests that the mRNA levels of the RNASEK gene possess biomarker attributes in patients with prostate cancer. In the present study, we used 3' RACE and next-generation sequencing (NGS) to detect and identify novel RNASEK transcripts. Computational analysis of the NGS data revealed new alternative splicing events that support the existence of novel RNASEK alternative transcripts. As a result, eight RNASEK splice variants were discovered and their expression profile was analyzed with the use of nested PCR in a wide panel of human cell lines, originating from several cancerous and/or normal human tissues. Based on in silico analysis, six of the eight novel RNASEK transcripts are predicted to encode new protein isoforms, while the remaining two splice variants could be considered as nonsense-mediated mRNA decay (NMD) candidates., (Copyright © 2019. Published by Elsevier Inc.)

The human L-DOPA decarboxylase (DDC) is a gene that has been in the center of research attention in many laboratories the last decades, due to its major implication in various disorders, including many types of cancer. In the current work, we used in-house developed RACE and high-throughput sequencing approaches, in order to detect and identify novel DDC transcripts. Bioinformatic analysis revealed new alternative splicing events that support the existence of novel DDC transcripts. As a result, a total of 14 DDC splice variants were identified and their expression profile was investigated in a wide panel of human cancer cell lines. From all 14 novel DDC transcripts that were identified, 9 transcripts are predicted to encode new protein isoforms, while the remaining 5 are nonsense-mediated mRNA decay (NMD) candidates. Our results demonstrate that the human DDC gene undergoes complex processing leading to the figuration of multiple mRNA isoforms in cancer cells., (Copyright © 2019. Published by Elsevier B.V.)

Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional properties. Several KLK transcripts have been found aberrantly expressed in numerous human malignancies, confirming their prognostic or/and diagnostic values. However, the process of alternative splicing can now be studied in-depth due to the development of Next-Generation Sequencing (NGS). In the present study, we used NGS to discover novel transcripts of the KLK1 and KLK2 genes, after nested touchdown PCR. Bioinformatics analysis and PCR experiments revealed a total of eleven novel KLK transcripts (two KLK1 and nine KLK2 transcripts). In addition, the expression profiles of each novel transcript were investigated with nested PCR experiments using variant-specific primers. Since KLKs are implicated in human malignancies, qualifying as potential biomarkers, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer., (Copyright © 2018. Published by Elsevier Inc.)

Next-generation sequencing (NGS) technology is highly expected to help researchers disclose the complexity of alternative splicing and understand its association with carcinogenesis. Alternative splicing alterations are firmly associated with multiple malignancies, in terms of functional roles in malignant transformation, motility, and/or metastasis of cancer cells. One perfect example illustrating the connection between alternative splicing and cancer is the human protein arginine methyltransferase 1 (PRMT1) gene, previously cloned from members of our research group and involved in a variety of processes including transcription, DNA repair, and signal transduction. Two splice variants of PRMT1 (variants v.1 and v.2) are downregulated in breast cancer. In addition, PRMT1 v.2 promotes the survival and invasiveness of breast cancer cells, while it could serve as a biomarker of unfavorable prognosis in colon cancer patients. The aim of this study was the molecular cloning of novel alternative splice variants of PRMT1 with the use of 3' RACE coupled with NGS technology. Extensive bioinformatics and computational analysis revealed a significant number of 19 novel alternative splicing events between annotated exons of PRMT1 as well as one novel exon, resulting in the discovery of multiple PRMT1 transcripts. In order to validate the full sequence of the novel transcripts, RT-PCR was carried out with the use of variant-specific primers. As a result, 58 novel PRMT1 transcripts were identified, 34 of which are mRNAs encoding new protein isoforms, whereas the rest 24 transcripts are candidates for nonsense-mediated mRNA decay (NMD)., (Copyright © 2019. Published by Elsevier B.V.)

The adaptor-related protein (AP) complexes play important roles in cargo selection and vesicle formation, and hence in intracellular membrane trafficking. Five different AP complexes are currently known, each consisting of four subunits, known as adaptins. AP-2, the most thoroughly characterized of the five AP complexes, facilitates clathrin-mediated endocytosis. In this study, we describe the discovery and molecular cloning of seventy-seven novel alternatively spliced transcripts of the human AP2A1 gene, which encodes the αA adaptin of the AP-2 complex. For this purpose, we have used Next-Generation Sequencing (NGS), a powerful tool for studying alternative splicing. In brief, we subcultured fifty-five established human cell lines, originating from several distinct cancerous and normal tissues, extracted total RNA, and synthesized first-strand cDNA. Next, we used nested touchdown PCR to amplify the whole coding region of the AP2A1 transcripts of each cell line, mixed all PCR products, and proceeded to NGS library construction, template preparation, and semiconductor sequencing. Extensive bioinformatic analysis revealed thirteen novel splice junctions of previously annotated exons, as also verified via nested PCR with primers targeting these splice junctions. Moreover, consecutive nested PCRs led to the determination of the primary structure of seventy-seven novel AP2A1 transcripts, all of which were shown to comprise at least one premature translation termination codon, thus representing nonsense-mediated mRNA decay (NMD) candidates. NMD is a mechanism that cells use to control gene expression. Consequently, alterations in the levels of these potentially non-coding AP2A1 transcripts could lead to a decrease in the number of AP2A1 mRNA molecules, when needed. Undoubtedly, the exact role of these new APA1 splice variants merits elucidation., (Copyright © 2018. Published by Elsevier B.V.)

Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. In this study, we describe the molecular cloning of four novel splice variants of the human KLK11 and KLK12 genes, discovered by combining 3' rapid amplification of cDNA ends (3' RACE), next-generation sequencing (NGS) technology, advanced bioinformatic analysis and Sanger sequencing. Expression analysis of these new transcripts in cell lines originating from 17 cancerous and two normal tissues revealed the expression pattern of each transcript. These novel KLK11 and KLK12 splice variants represent new potential cancer biomarkers.

The human SR-related CTD associated factor 1 (SCAF1) gene is a new member of the human SR (Ser/Arg-rich) superfamily of pre-mRNA splicing factors, which has been discovered and cloned by members of our lab. SCAF1 interacts with the CTD domain of the RNA polymerase II polypeptide A and is firmly involved in pre-mRNA splicing. Although it was found to be expressed widely in multiple human tissues, its mRNA levels vary a lot. The significant relation of SCAF1 with cancer has been confirmed by many studies, since SCAF1 mRNA transcript was found to be overexpressed in breast and ovarian tumors, confirming its significant prognostic value as a cancer biomarker in both these human malignancies. In this study, we describe the discovery and cloning of fifteen novel transcripts of the human SCAF1 gene (SCAF1 v.2 - v.16), using nested PCR and NGS technology. In detail, extensive bioinformatic analysis revealed that these novel SCAF1 splice variants comprise a total of nine novel alternative splicing events between the annotated exons of the gene, thus producing seven novel SCAF1 transcripts with open-reading frames, which are predicted to encode novel SCAF1 isoforms and eight novel SCAF1 transcripts with premature termination codons that are likely long non-coding RNAs. Additionally, a novel 3' UTR was discovered and cloned using nested 3' RACE and was validated with Sanger sequencing. In order to validate the NGS findings as well as to investigate the expression profile of each novel transcript, RT-PCR experiments were carried out with the use of variant-specific primers. Since SCAF1 is implicated in many human malignancies, qualifying as a potential biomarker, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity and affecting cell cycle control, proliferation, apoptosis, angiogenesis, invasion, and metastasis. In this study, we describe the discovery and molecular cloning of thirty novel transcripts of the human KLK5, KLK6, KLK7, KLK8 and KLK9 genes, using 3' rapid amplification of cDNA ends (3' RACE) and NGS technology, as well as their expression analysis in many established cell lines, originating from several distinct cancerous and normal tissues. Extensive bioinformatic analysis revealed novel splice variants of these five members of the KLK family, comprising entirely new exons, previously unknown boundaries of the already annotated exons (extensions and truncations) as well as alternative splicing events between these exons. Nested RT-PCR in a panel of human cell lines originating from seventeen cancerous and two normal tissues with the use of variant-specific pairs of primers was carried out for expression analysis of these novel splice variants, and Sanger sequencing of the respective amplicons confirmed our NGS results. Given that some splice variants of KLK family members possess clinical value, novel alternatively spliced transcripts appear as new candidate biomarkers for diagnostic and/or prognostic purposes and as targets for therapeutic strategies.

The next-generation sequencing (NGS) technology has enabled genome-wide studies, providing massively parallel DNA sequencing. NGS applications constitute a revolution in molecular biology and genetics and have already paved new ways in cancer research. BCL2L12 is an apoptosis-related gene, previously cloned from members of our research group. Like most members of the BCL2 gene family, it is highly implicated in various types of cancer and hematological malignancies. In the present study, we used NGS to discover novel alternatively spliced variants of the apoptosis-related BCL2L12 gene in many human cancer cell lines, after 3'-RACE nested PCR. Extensive computational analysis uncovered new alternative splicing events and patterns, resulting in novel alternative transcripts of the BCL2L12 gene. PCR was then performed to validate NGS data and identify the derived novel transcripts of the BCL2L12 gene. Therefore, 50 novel BCL2L12 splice variants were discovered. Since BCL2L12 is involved in the apoptotic machinery, the quantification of distinct BCL2L12 transcripts in human samples may have clinical applications in different types of cancer., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In the past, we identified and cloned the BCL2-like 12 (BCL2L12) gene, a novel member of the BCL2 family, which is implicated in various malignancies. The classical BCL2L12 protein isoform contains a highly conserved BH2 domain, a BH3-like motif, and a proline-rich region, and is involved in apoptosis. Most members of this apoptosis-related family are subjected to alternative splicing, thus generating multiple protein isoforms with distinct properties, and sometimes even with opposite function (pro- vs. anti-apoptotic). In the current study, we report the identification, molecular cloning, and expression pattern of novel splice variants of the human BCL2L12 gene in cancer cell lines. EST clones displaying high sequence identity (≥90%) with the classical BCL2L12 transcript were aligned, in order to identify those containing at least one novel splice junction. EST database mining led to the identification of three previously unknown splice variants of this apoptotic gene. In our effort to experimentally validate these novel transcripts, we also cloned seven more, previously unidentified, BCL2L12 alternatively spliced variants. Expression analysis of all BCL2L12 splice variants in human cancer cell lines and embryonic kidney cells revealed remarkable differences between their BCL2L12 expression profiles. Interestingly, 7 out of 10 novel splice variants of BCL2L12 are predicted to encode new protein isoforms, some of which are BH3-only proteins, in contrast to the classical BCL2L12 isoform, which also contains a functional BH2 domain. The remaining three novel splice variants of BCL2L12 are nonsense-mediated mRNA decay (NMD) candidates., (Copyright © 2012 Elsevier B.V. All rights reserved.)