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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.

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.

Simple Summary Multiple myeloma bone disease (MMBD) is one of the most important complications of multiple myeloma with a great impact on quality of life. Recent advances in the field of imaging techniques provided clinicians with a variety of imaging modalities with high sensitivity for the diagnosis of MMBD. However, no circulating biomarkers are available to support the diagnosis of MMBD in cases where the results are inconclusive. The aim of our study was to investigate the clinical utility of 19 miRNAs implicated in osteoporosis in MMBD. Our results suggest that the levels of circulating let-7b-5p, miR-143-3p, miR-17-5p, miR-335-5p, and miR-214-3p (standalone or combined in multi-miRNA models) can effectively predict the presence of MMBD in newly diagnosed MM patients. Abstract Background: Multiple myeloma bone disease (MMBD) constitutes a common and severe complication of multiple myeloma (MM), impacting the quality of life and survival. We evaluated the clinical value of a panel of 19 miRNAs associated with osteoporosis in MMBD. Methods: miRNAs were isolated from the plasma of 62 newly diagnosed MM patients with or without MMBD. First-strand cDNA was synthesized, and relative quantification was performed using qPCR. Lastly, we carried out extensive biostatistical analysis. Results: Circulating levels of let-7b-5p, miR-143-3p, miR-17-5p, miR-214-3p, and miR-335-5p were significantly higher in the blood plasma of MM patients with MMBD compared to those without. Receiver operating characteristic curve and logistic regression analyses showed that these miRNAs could accurately predict MMBD. Furthermore, a standalone multi-miRNA–based logistic regression model exhibited the best predictive potential regarding MMBD. Two of those miRNAs also have a prognostic role in MM since survival analysis indicated that lower circulating levels of both let-7b-5p and miR-335-5p were associated with significantly worse progression-free survival, independently of the established prognostic factors. Conclusions: Our study proposes a miRNA signature to facilitate MMBD diagnosis, especially in ambiguous cases. Moreover, we provide evidence of the prognostic role of let-7b-5p and miR-335-5p as non-invasive prognostic biomarkers in MM.

Background Current chemotherapy regimens for the treatment of colorectal cancer (CRC) include oxaliplatin, irinotecan, and fluorouracil along with leucovorin. Cytotoxicity involves the induction of programmed cell death. Objective The purpose of this study was to assess the molecular effects of doxorubicin (a 14-OH derivative of the natural product daunorubicin) and common chemotherapeutic drugs (used in the clinical practice to treat CRC) on the expression of the most prominent members of the BCL2 family, namely BCL2, BAX, BCLX, and MCL1. Moreover, we sought to define the role of BCL2L12, another member of the BCL2 family, the apoptotic role of which is ambiguous. Methods The MTT cell proliferation assay was used to determine the IC50 of each chemotherapeutic drug at 72 hours of treatment of Caco-2 and DLD-1 colorectal adenocarcinoma cell lines. Real-time PCR was used to quantify the antiapoptotic BCL2-α, BLCX-L, and MCL1-L transcripts, the proapoptotic BAX, BLCX-S, BLCX-ES, MCL1-S, and MCL1-ES transcripts, and BCL2L12 expression in relation to GAPDH mRNA levels. Results We constructed growth curves of Caco-2 and DLD-1 cells and determined the IC50 of each drug at 72 hours of treatment. Significant alterations in the expression levels of the studied BCL2 family genes and/or particular transcripts were observed. Conclusion The intrinsic apoptotic pathway is activated during treatment of CRC cells with common chemotherapeutic drugs. Moreover, BCL2L12 mRNA expression increases progressively during treatment, similarly to the expression of other BCL2 family genes favoring apoptosis and/or particular proapoptotic transcripts, thus suggesting a proapoptotic role for BCL2L12 in chemotherapy-treated CRC cells.

Background Treatment of cancer with natural chemotherapeutic agents induces apoptosis along with remarkable alterations in the expression of apoptosis-related genes. Deregulation of microRNA (miRNA) expression is implicated in several human malignancies. Vinca alkaloids compose a class of antimitotic drugs preventing cancer cells from dividing, leading to apoptosis. They are commonly used in clinical practice for breast cancer treatment. Objective The present study focused on the effects of vinca alkaloids (vincristine, vinblastine, and vinorelbine) on miRNA expression of treated breast cancer cells. Methods We investigated the effect of vincristine, vinblastine, and vinorelbine on the expression of oncogenic and tumor-suppressive miRNAs (miR-15a-5p, miR-16-5p, miR-21-5p, miR-25-3p, miR- 29b-3p, miR-125b-5p, miR-148a-3p, miR-214-3p, miR-221-3p, miR-222-3p, and miR-421), as well as on the expression of the apoptosis-related genes BAX, BCL2, TP53, and CDKN1B in BT-20 and SKBR- 3 breast adenocarcinoma cells. Results Treatment of BT-20 cells with vincristine, vinblastine, and/or vinorelbine resulted in upregulation of TP53 expression. However, no alterations in the mRNA levels of the pivotal BCL2 family members BAX and BCL2 were observed. On the other hand, treatment of SK-BR-3 cells with any of these vinca alkaloids led to an increase in the BAX/BCL2 mRNA ratio, implying the activation of the intrinsic apoptotic pathway. No concomitant alteration in TP53 expression was observed in treated SKBR- 3 cells. Regarding the miRNAs examined in this study, miR-222-3p expression exhibited the most remarkable modulations in both treated cell lines. Conclusion This study suggests the possible involvement of miR-222-3p expression in breast cancer cell apoptosis, triggered by vincristine, vinblastine, and vinorelbine.

Simple Summary Circular RNAs (circRNAs) are single-stranded RNA molecules that form a covalently closed loop structure. They are characterized by distinct features and multifariously implicated in the regulation of both physiological and pathological states. The aberrant expression of circRNAs has been evidenced in various malignancies. Circular transcripts can effectively modulate gene expression. The most prevalent manner through which circRNAs promote cancer development and progression is their interaction with key components of major signaling pathways. In particular, abnormally expressed circRNAs can dictate the crosstalk between signaling cascades. In recent years, there has been great progress regarding circRNA research in the context of cancer progression, and various regulatory axes have been described. As our knowledge of signaling regulation by circRNAs continuously expands, novel therapeutic approaches can be assessed and established, seeking to overcome clinical challenges, such as the treatment of cancer patients with distant metastasis and those who relapse. Abstract 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/β-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.

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.

Simple Summary The involvement of miRNAs in physiological cellular processes has been well documented. The development of B cells, which is dictated by a miRNA-transcription factor regulatory network, suggests a typical process partly orchestrated by miRNAs. Besides their contribution in normal hematopoiesis, miRNAs have been severally reported to be implicated in hematological malignancies, a typical example of which is B-cell chronic lymphocytic leukemia (B-CLL). Numerous studies have attempted to highlight the regulatory role of miRNAs in B-CLL or establish some of them as molecular biomarkers or therapeutic targets. Thus, a critical review summarizing the current knowledge concerning the multifaceted role of miRNAs in normal B-cell development and B-CLL progression, prognosis, and therapy, is urgent. Moreover, this review aims to highlight important miRNAs in both normal B-cell development and B-CLL and discuss future perspectives concerning their regulatory potential and establishment in clinical practice. Abstract 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.

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&rsquo, 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.

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 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.

The hypoxia inducible factor 1 (HIF1) is a heterodimeric transcription factor that ultimately regulates cellular responses to changes in oxygen tension. In this study, we examined the potential diagnostic and prognostic potential of the mRNA expression of HIF1 regulatory α-subunit (HIF1A) in chronic lymphocytic leukemia (CLL). For this purpose, total RNA was isolated from peripheral blood mononuclear cells collected from 88 CLL patients and 33 non-leukemic blood donors, and poly(A)-RNA was reversely transcribed. HIF1A mRNA levels were quantified using real-time PCR. Kaplan-Meier survival analysis showed that high HIF1A mRNA expression predicts inferior overall survival for CLL patients (p=0.001). Bootstrap univariate Cox regression analysis confirmed that HIF1A mRNA overexpression is a significant unfavorable prognosticator in CLL (hazard ratio=3.75, bias-corrected and accelerated 95% confidence interval=1.43-24.36, bootstrap p0.001), independent of other established prognostic factors, including CD38 expression, the mutational status of the immunoglobulin heavy chain variable region (IGHV), and the clinical stage (Binet or Rai stage) or risk group (p0.001 in all cases). Interestingly, HIF1A mRNA positivity retains its unfavorable prognostic value in distinct subgroups of patients, stratified according to established prognostic factors. Thus, HIF1A mRNA overexpression can be regarded as a promising, independent molecular biomarker of unfavorable prognosis in CLL.

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.

Background Laryngeal squamous cell carcinoma (LSCC) constitutes the third most frequent head and neck cancer. Several tissue biomarkers have been studied for their prognostic significance in LSCC. Objective To investigate the prognostic significance of BCL2L12, a new member of the BCL2 family, in primary LSCC along with well-examined biomarkers such as BCL2 and BAX. Methods Cancerous tissue specimens of patients with primary LSCC were collected during 2005 and 2012 as pretreatment tissue biopsy. The specimens were immunohistochemically evaluated for the protein expression of BCL2L12, BCL2 and BAX. Kaplan-Meier survival curves and Cox proportional hazard regression models were performed to evaluate prognosis. Results In the study cohort of 78 patients with primary LSCC, Kaplan-Meier survival curves demonstrated that advanced-stage LSCC patients with BCL2L12-positive tumors had significantly higher OS time in comparison with advanced-stage LSCC patients with BCL2L12-negative tumors (p= 0.014). Also, advanced-stage LSCC patients with BCL2L12-positive tumors had significantly lower risk of death from LSCC compared to advanced-stage LSCC patients with BCL2L12-negative tumors (HR = 0.228, 95%CI = 0.063-0.833, p= 0.025). Conclusions BCL2L12 protein expression could be used as a favorable prognostic tissue biomarker in patients with primary advanced-stage LSCC. On the contrary, BCL2 and BAX did not correlate with prognosis in patients with primary LSCC.

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.

Purpose: Azacitidine is the mainstay of high-risk myelodysplastic syndromes (MDS) therapy, but molecular predictors of response and the mechanisms of resistance to azacitidine remain largely unidentified. Deregulation of signaling via Stat3 and Stat5 in acute myeloid leukemia (AML) is associated with aggressive disease. Numerous genes involved in cell signaling are aberrantly methylated in MDS, yet the alterations and the effect of azacitidine treatment on Stat3/5 signaling in high-risk MDS have not been explored. Experimental Design: We assessed longitudinally constitutive and ligand-induced phospho-Stat3/5 signaling responses by multiparametric flow cytometry in 74 patients with MDS and low blast count AML undergoing azacitidine therapy. Pretreatment Stat3/5 signaling profiles in CD34+ cells were grouped by unsupervised clustering. The differentiation stage and the molecular properties of the CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation were performed by flow cytometry and quantitative real-time PCR in isolated MDS progenitors. Results: The pretreatment Stat3/5 signaling profiles in CD34+ cells correlated strongly with response and cytogenetics and independently predicted event-free survival. We further identified a CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation (DP subset) whose pretreatment levels were inversely associated with treatment response and cytogenetics. The kinetics of the DP subset followed the response to azacitidine and the disease course, whereas its molecular characteristics and cellular hierarchy were consistent with a leukemia propagating cell phenotype. Conclusions: Our findings provide a novel link among Stat3/5 signaling and MDS pathobiology and suggest that the Stat3/5 signaling biosignature may serve as both a response biomarker and treatment target. Clin Cancer Res; 22(8); 1958–68. ©2015 AACR.

Purpose: Programmed death-ligand 1 (PD-L1; also known as CD274 or B7-H1) expression represents a mechanism of immune escape for cancer. Our purpose was to characterize tumor PD-L1 expression and associated T-cell infiltration in primary laryngeal squamous cell carcinomas (SCC). Experimental Design: A well-annotated cohort of 260 operable primary laryngeal SCCs [formalin-fixed paraffin-embedded (FFPE) specimens] was morphologically characterized for stromal tumor-infiltrating lymphocytes (TIL), on hematoxylin/eosin-stained whole sections and for PD-L1 mRNA expression by qRT-PCR in FFPE specimens. For PD-L1 protein expression, automated quantitative protein analysis (AQUA) was applied on tissue microarrays consisting of two cores from these tumors. In addition, PD-L1 mRNA expression in fresh-frozen tumors and normal adjacent tissue specimens was assessed in a second independent cohort of 89 patients with primary laryngeal SCC. Results: PD-L1 mRNA levels were upregulated in tumors compared with surrounding normal tissue (P = 0.009). TILs density correlated with tumor PD-L1 AQUA levels (P = 0.021). Both high TILs density and high PD-L1 AQUA levels were significantly associated with superior disease-free survival (DFS; TILs: P = 0.009 and PD-L1: P = 0.044) and overall survival (OS; TILs: P = 0.015 and PD-L1: P = 0.059) of the patients and retained significance in multivariate analysis. Conclusions: Increased TILs density and PD-L1 levels are associated with better outcome in laryngeal squamous cell cancer. Assessment of TILs and PD-L1 expression could be useful to predict response to immune checkpoint inhibitors. Clin Cancer Res; 22(3); 704–13. ©2015 AACR.

Simple Summary The profiles of miRNAs, tRNA-derived fragments and tRNAs from lung cancer biopsy specimens indicate involvement of gene networks that modulate signaling and translation initiation. The current study highlights the important role of several regulatory small non-coding RNAs in aberrant signaling and translation deregulation in lung cancer. Abstract Transcriptomics profiles of miRNAs, tRNAs or tRFs are used as biomarkers, after separate examination of several cancer cell lines, blood samples or biopsies. However, the possible contribution of all three profiles on oncogenic signaling and translation as a net regulatory effect, is under investigation. The present analysis of miRNAs and tRFs from lung cancer biopsies indicated putative targets, which belong to gene networks involved in cell proliferation, transcription and translation regulation. In addition, we observed differential expression of specific tRNAs along with several tRNA-related genes with possible involvement in carcinogenesis. Transfection of lung adenocarcinoma cells with two identified tRFs and subsequent NGS analysis indicated gene targets that mediate signaling and translation regulation. Broader analysis of all major signaling and translation factors in several biopsy specimens revealed a crosstalk between the PI3K/AKT and MAPK pathways and downstream activation of eIF4E and eEF2. Subsequent polysome profile analysis and 48S pre-initiation reconstitution experiments showed increased global translation rates and indicated that aberrant expression patterns of translation initiation factors could contribute to elevated protein synthesis. Overall, our results outline the modulatory effects that possibly correlate the expression of important regulatory non-coding RNAs with aberrant signaling and translation deregulation in lung cancer.

Simple Summary Circular RNAs (circRNAs) are a relatively new and unexplored RNA type, implicated in several aspects of cell life, in normal and pathological states. This review aims to discuss the roles of circRNAs in colorectal cancer (CRC) initiation, progression, and therapy resistance, as well as their potential clinical value as CRC biomarkers. CRC is characterized by an elevated mortality rate, poor prognosis of high-grade tumors, high metastatic potential, and resistance to conventional therapies. Therefore, there is an urgent need for identification of novel molecules involved in colorectal carcinogenesis. Prompted by several studies examining circRNA involvement in CRC, in this review we seek to summarize the existing knowledge on circRNA expression in CRC and their implication in cellular pathways and molecular mechanisms underlying colorectal carcinogenesis. Lastly, we discuss the limitations and future perspectives highlighting the missing pieces of the puzzle and aspects of the circRNA research field that should be further investigated. Abstract 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.

Hypomethylating agents are widely used in chronic myelomonocytic leukemia (CMML). We analyzed the characteristics of 88 patients with CMML homogeneously treated with 5-azacytidine (Hellenic 5-Azacytidine Registry). The overall response rate was 48.9% and the median overall survival (OS) 29.7 months. Out of the seven most widely used prognostic scoring systems for CMML, the Dusseldorf score (DUSS) showed the best prognostic capability (HR, 2.27

MicroRNAs (miRNAs) are small, non-coding RNA molecules that epigenetically regulate gene expression. Recently, it has been shown that miRNAs are heavily involved in renal carcinogenesis by controlling the expression of proteins related to renal cell carcinoma (RCC) and, thus, the activity of key signaling pathways involved in pivotal cell processes. Areas covered: This review focuses on the role of miRNAs as emerging biomarkers in RCC. We summarize the oncogenic and/or tumor-suppressive functions of miRNAs as well as the strong clinical information that these RNA molecules provide. Expert commentary: miRNAs that could be either exploited as standalone biomarkers or integrated into molecular signatures with high clinical interest should be considered as ideal for diagnostic purposes and accurate molecular classification of renal tumors. Furthermore, miRNAs could serve as prognostic biomarkers, predicting disease-free and overall survival of RCC patients and, thus, reducing over- and/or under-treatment of patients. miRNA may also be used as predictors of patients' response to targeted therapy with tyrosine kinase inhibitors, contributing to the decision-making process for the selection of the appropriate treatment plan.

The demethylating factor 5-azacytidine (5-AZA) improves survival in intermediate-2 and high-risk myelodysplastic syndrome (MDS) patients [according to the International Prognostic Score System (IPSS)] responding to treatment. However, the outcome of patients achieving stable disease (SD) is unclear. This retrospective study of the Hellenic MDS Study Group included 353 intermediate-2 or high IPSS risk patients treated with 5-AZA. Forty-four out of 86 (51.6%) patients achieving SD and continuing treatment with 5-AZA showed a lower risk of transformation of MDS to acute myeloid leukemia (AML) and increased overall survival (OS), compared to SD patients who discontinued the treatment (estimated median AML-free survival = 38 months, 95% CI = 10.7-65.3 vs 15 months, 95% CI = 10.4-19.6, P < .001; estimated median OS = 20 months, 95% CI = 5.5-34.5 vs 11 months, 95% CI = 5.8-16.2, P < .001). Moreover, SD patients continuing treatment with 5-AZA had no differences in AML-free survival compared to patients showing response to 5-AZA (estimated median AML-free survival = 38 months, 95% CI = 10.7-65.3 vs 31 months, 95% CI = 23.6-38.4, P = .45; estimated median OS 20 months, 95% CI = 5.5-34.5 vs 25 months, 95% CI = 21.3-28.7, P = .50). In conclusion, MDS patients achieving SD in the first 6 months of treatment with 5-AZA as best response should continue receiving 5-AZA as they may benefit from prolonged treatment.

MicroRNA-92a-3p (miR-92a-3p) derives from the oncogenic miR-17/92 cluster and its highly homologous miR-106a/363 cluster. miR-92a-3p regulates the expression of key transcription factors such as HIF1 and inhibits SOCS1 to enhance the anti-apoptotic STAT3/IL6 signaling pathway. In this study, we assessed the putative usefulness of miR-92a-3p as a prognostic and/or diagnostic biomarker in chronic lymphocytic leukemia (CLL). For this purpose, total RNA was extracted from mononuclear cells isolated from the peripheral blood of 88 CLL patients and 36 non-leukemic blood donors, was polyadenylated and reversely transcribed. miR-92a-3p expression was quantified using an accurate qPCR method. miR-92a-3p levels were significantly lower in peripheral blood mononuclear cells of CLL patients. Overall survival (OS) analysis revealed that high miR-92a-3p expression predicts significantly prolonged OS of CLL patients. Interestingly, miR-92a-3p overexpression remains a significant prognosticator in subgroups of CLL patients with distinct prognosis. In conclusion, miR-92a-3p overexpression is a potential surrogate biomarker of favorable outcome of CLL patients.

MicroRNAs are small, non-coding RNAs which regulate post-transcriptionally hundreds of target mRNAs. Given that their expression is deregulated in several cancer types, they represent potential diagnostic, prognostic, and predictive biomarkers, as well as next-generation therapeutic targets. Nevertheless, the involvement of miRNAs in non-melanoma skin cancer, a cancer type with increasing prevalence, is not extensively studied, and their comprehensive characterization as regard to the initiation, promotion, and progression stages is missing. To this end, we exploited a well-established multistage mouse skin carcinogenesis model in order to identify miRNAs consistently implicated in different stages of skin carcinogenesis. The cell lines comprising this model were subjected to miRNA expression profiling using microarrays, followed by bioinformatics analysis and validation with Q-PCR, as well as treatment with miRNA modulators. We showed that among all deregulated miRNAs in our system, only a functionally coherent group consisting of the miR-200 family members and miR-205-5p displays a pattern of progressive co-downregulation from the early toward the most aggressive stages of carcinogenesis. Their overlapping, co-regulated putative targets are potentially inter-associated and, of these, the EMT-related Rap1a is overexpressed toward aggressive stages. Ectopic expression of miR-205-5p in spindle cancer cells reduces Rap1a, mitigates cell invasiveness, decreases proliferation, and delays tumor onset. We conclude that deregulation of this miRNA group is primarily associated with aggressive phenotypes of skin cancer cells. Restoration of the miR-205-5p member of this group in spindle cells reduces the expression of critical, co-regulated targets that favor cancer progression, thus reversing the EMT characteristics. © 2015 Wiley Periodicals, Inc.

MicroRNA-155-5p (miR-155-5p) is a proinflammatory, oncogenic miRNA, involved in various physiological processes, including hematopoiesis, immunity, inflammation, and cell lineage differentiation. It regulates important transcription factors, such as E2F2, hypoxia-inducible factor 1 (HIF1), and FOXO3. Recently, the dysregulation of miR-155-5p expression has been linked to chronic lymphocytic leukemia (CLL) pathogenesis. In this research study, we investigated the potential diagnostic and prognostic value of miR-155-5p in CLL. To achieve our goal, we isolated total RNA from peripheral blood mononuclear cells (PBMCs) collected from 88 CLL patients and 36 nonleukemic blood donors and performed polyadenylation of total RNA and reverse transcription. Next, we quantified miR-155-5p levels using an in-house-developed real-time quantitative PCR method, before proceeding to extensive biostatistical analysis. Thus, it appears that miR-155-5p is significantly overexpressed in PBMCs of CLL patients and can distinguish them from nonleukemic population. Kaplan-Meier OS analysis and bootstrap univariate Cox regression showed that high miR-155-5p expression predicts inferior OS for CLL patients (p<0.001). Interestingly, miR-155-5p overexpression retains its unfavorable prognostic role in CLL patients stratified according to established prognostic factors [CD38 expression and mutational status of the immunoglobulin heavy chain variable region (IGHV)]. Thus, miR-155-5p appears as a promising, independent molecular biomarker of unfavorable prognosis in CLL.

Objectives MicroRNA-34a (miR-34a) is regulated by TP53 and, in response, downregulates the expression of a gamut of protein-coding genes, including apoptosis regulators, transcription factors, cyclins, and cyclin-dependent kinases. Its upregulation initiates a reprogramming of gene expression and promotes apoptosis. The purpose of this study was the investigation of the potential clinical significance of miR-34a as a molecular prognostic biomarker in colorectal adenocarcinoma using an in-house real-time quantitative PCR (qPCR) methodology. Design and methods Total RNA was extracted from 113 primary colorectal adenocarcinoma specimens and 61 paired non-cancerous colorectal tissue samples. After polyadenylation and reverse transcription, miR-34a molecules were determined using qPCR based on SYBR Green chemistry. Calculations were performed using the comparative C T method. Finally, extensive biostatistical analysis was performed. Results miR-34a expression does not significantly differ between colorectal adenocarcinoma tissue specimens and adjacent non-cancerous mucosae. However, miR-34a expression increases progressively as colorectal adenocarcinoma loses its differentiation, being highest in grade III tumors ( P = 0.010). Moreover, miR-34a expression is a potential unfavorable prognostic biomarker in colorectal adenocarcinoma, predicting poor disease-free and overall survival ( P = 0.002 and P = 0.019, respectively), independently of classical clinicopathological parameters. Most importantly, miR-34a expression stratifies patients without local (N0) and/or distant metastasis (M0) at the time of diagnosis into two groups with substantially different prognosis ( P = 0.013 and P = 0.002, respectively). Conclusions High miR-34a levels in colorectal adenocarcinoma predict a rather increased risk for disease recurrence and poor overall survival, particularly in patients at an early TNM stage. The unfavorable prognostic potential of miR-34a expression is independent of established prognostic features of colorectal adenocarcinoma.

Accumulated data indicate a significant role of T cell dysfunction in the pathogenesis of chronic lymphocytic leukemia. In CLL, regulatory T cells are significantly higher and show lower apoptotic levels compared to healthy donors. We demonstrate that CLL derived CD4 + CD25 − CD127 − and CD4 + CD25 low CD127 − subpopulations share a common immunophenotypic profile with conventional Tregs and are associated with advanced stage disease. We further provide evidence that the increased number of Tregs contributes indirectly to the proliferation of the CLL clone, by suppressing the proliferation of Teffs which in turn suppress CLL cells. These data are further supported by our observations that CLL derived Tregs appear rather incapable of inducing apoptosis of both normal B cells and CLL cells, in contrast to normal Tregs, suggesting an immunoediting effect of CLL cells on Tregs which negatively affects the functionality of the latter and contributes to the failure of Tregs in CLL to efficiently eliminate the abnormal clone.

Colorectal adenocarcinoma constitutes the most frequent form of colorectal cancer and a serious cause of cancer-related deaths. The expression of multiple miRNAs, including miR-224, is deregulated in colorectal adenocar- cinoma. The aim of this study was the investigation of the prognostic value of miR-224 in colorectal adenocarcinoma. For this purpose, total RNA was isolated from 115 colorectal adenocarcinomas and 66 adjacent non-cancer mucosae. Total RNA (2 µg) was polyadenylated and reverse transcribed. A quantitative PCR method based on SYBR-Green chemistry was developed and applied for the quantification of miR-224 levels, followed by extensive biostatistical analysis. miR-224 levels in malignant colorectal adenocarcinomas ranged between 1.81 and 187.75 RQU (miR-224 copies/1,000 SNORD48 copies) with a median of 34.27, and were significantly elevated, compared to miR-224 levels in adjacent non-cancer mucosae (p

Natural products display special attributes in the treatment and prevention of a variety of human disorders including cancer. Their therapeutic capacities along with the fact that nature comprises a priceless pool of new compounds have attracted the interest of researchers worldwide. A significant number of organic compounds from terrestrial and marine organisms exhibit anticancer properties as attested by both in vitro and in vivo studies. Emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. As a result, numerous natural compounds or their derivatives have entered clinical practice and are currently in the forefront of chemotherapeutics, showing beneficial effects for cancer patients. Induction of apoptosis seems to be the major mechanism of action induced by these natural agents in the race against cancer. This is mainly achieved through modulations of the expression of B-cell CLL/lymphoma 2 (BCL2) family members. These molecules appear to be the pivotal players determining cellular fate. In the current review, we provide a comprehensive overview of the major alterations in the gene and/or protein levels of BCL2-family members evoked in cancer cells after treatment with a gamut of natural compounds. The data cited suggest the need for exploitation of newly discovered natural products that, along with the improvement of currently employed chemotherapeutics, will significantly enrich the anticancer armamentarium.

Classical chemotherapeutic agents such as mitotic inhibitors (spindle poisons), alkylating agents, antimetabolites, topoisomerase inhibitors, and anthracenediones (anthracyclines) inhibit DNA synthesis and mitosis, thereby killing or impeding the proliferation of rapidly dividing cells. During the last decade, targeted therapy has gained advantage over conventional treatment regimens, as it is more effective against cancer and also much less harmful to normal cells, thus minimizing the side-effects of chemotherapy. This type of treatment blocks the proliferation of cancer cells by inhibiting the function of specific targeted molecules needed for tumor growth and metastasis. Targeted therapy agents include monoclonal antibodies and small-molecule inhibitors, which most commonly target receptor and/or non-receptor tyrosine kinases. Most members of the BCL2 apoptosis-related family regulate cellular fate as a response to antineoplastic agents. Modulations at the mRNA and protein levels of these genes are usually associated with sensitivity or resistance of various types of cancer cells to chemotherapeutic drugs. Moreover, alterations in expression of BCL2-family members, induced by anticancer drug treatment, can trigger or simply facilitate apoptosis. In this review, we summarize information about changes in apoptosis-related gene expression caused directly or indirectly by antineoplastic agents, as well as about the impact of BCL2-family members on the chemosensitivity or chemoresistance of cancer cells.

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.

e17542 Background: We sought to analyze the transcriptional landscape of HNSCC in an attempt to identify tumor-intrinsic oncogenic pathways that appear to mediate T-cell infiltration of tumor tissue. In this direction, we employ a methodology that integrates histopathology data of the tumor microenvironment with its corresponding transcriptome. Methods: 32 frozen HNSCCs were subjected to RNA-seq and corresponding FFPE were scored for plasma cells, tertiary lymphoid structures and CD8a+ TILs (center, invasive margin). RNA-seq data were analyzed to identify differentially expressed genes (DEGs) between tumors scored by immunohistochemistry (IHC) as CD8a high and CD8a low. Gene ontology analysis (GO) was performed based on DEGs > 1.5 fold expression change between CD8a high and CD8a low groups. Candidate genes were investigated by hierarchical clustering in TCGA RNA-seq data and further validated by IHC and quantitative RT-PCR in our cohort. Results: 32 HNSCCs were either scored as CD8a high or CD8a low based on IHC detection of CD8a+ cells in invasive margin of tumors. Comparative analysis of mRNA expression data between CD8a high and CD8a low groups in our cohort revealed that Muc1/16 overexpression and glycosylation was highly enriched in T cell infiltrated group of tumors. This finding was further validated using antibodies that detect glycosylated epitopes for both mucins. Analysis of TCGA RNA-seq data indicated that Muc1/16 overexpressing tumors share signatures of early keratinocyte differentiation and stem cell identity and co-express high levels of enzymes that promote Muc1/16 glycosylation. Interestingly, loss of CDH1 and acquisition of epithelial mesenchymal transition (EMT) markers in the cluster of Muc1/16 overexpressing tumors is strongly correlated with elevated CD8a, IDO1, CD274 and CXCL10 mRNA levels (P < 0.0001). Conclusions: Muc1/16 overexpressing tumors represent a very immunogenic HNSCC cluster. Previous studies have shown that mucins 1 and 16 in cancer cells expose glycosylated-specific epitopes that are recognized by T cells as cancer antigens. To this end, MUC1/16 expression may serve as predictive biomarkers for response to immunotherapy and MUC-targeted immunotherapy may function as an attractive partner to checkpoint inhibitors in HNSCC.

Previous studies have uncovered several transcription factors that determine biological alterations in tumor cells to execute the invasion-metastasis cascade, including the epithelial-mesenchymal transition (EMT). We sought to investigate the role of miR-21 in colorectal cancer regulation. For this purpose, miR-21 expression was quantified in a panel of colorectal cancer cell lines and clinical specimens. High expression was found in cell lines with EMT properties and in the vast majority of human tumor specimens. We demonstrate in a cell-specific manner the occupancy of MIR-21 gene promoter by AP-1 and ETS1 transcription factors and, for the first time, the pattern of histone posttranslational modifications necessary for miR-21 overexpression. We also show that Integrin-β4 (ITGβ4), exclusively expressed in polarized epithelial cells, is a novel miR-21 target gene and plays a role in the regulation of EMT, since it is remarkably de-repressed after transient miR-21 silencing and downregulated after miR-21 overexpression. miR-21-dependent change of ITGβ4 expression significantly affects cell migration properties of colon cancer cells. Finally, in a subgroup of tumor specimens, ROC curve analysis performed on quantitative PCR data sets for miR-21, ITGβ4, and PDCD4 shows that the combination of high miR-21 with low ITGβ4 and PDCD4 expression is able to predict presence of metastasis. In conclusion, miR-21 is a key player in oncogenic EMT, its overexpression is controlled by the cooperation of genetic and epigenetic alterations, and its levels, along with ITGβ4 and PDCD4 expression, could be exploited as a prognostic tool for CRC metastasis.

Mir-17-5p and mir-20a, members of the mir-17-92 family, down-regulate E2F1, which is over-expressed in myelodysplastic syndromes (MDS). Moreover, let-7a down-regulates KRAS, which is aberrantly expressed in MDS. We evaluated the expression of the aforementioned microRNAs in CD34+ cells of 43 MDS patients using real-time PCR and their target proteins (E2F1, MYC, BCL2, CCND1, and KRAS) by Western blot. Mir-17-5p and mir-20a were under expressed in high risk MDS patients, compared to low risk MDS patients. Similarly, let-7a was under expressed in patients with intermediate or high-risk karyotype. Interestingly, there was an inverse correlation between microRNA and the expression levels of their targets. Importantly, mir-17-5p and mir-20a constitute favorable prognostic factors in MDS, since their expression was associated with increased overall survival of MDS patients.

Objectives Several miRNAs are aberrantly expressed in cancer. miR-24-3p is involved in cancer-related cellular processes, including cell cycle control, cell growth, proliferation, and apoptosis. In this study, we examined the potential diagnostic and prognostic significance of miR-24-3p expression in colorectal adenocarcinoma. Design and methods Total RNA was isolated from 182 colorectal adenocarcinoma specimens and 86 paired non-cancerous colorectal mucosae. After polyadenylation of 2 μg total RNA and reverse transcription into first-strand cDNA using an oligo-dT-adapter primer, miR-24-3p expression was quantified using an in-house–developed reverse-transcription real-time quantitative PCR method, based on the SYBR Green chemistry. SNORD43 ( RNU43 ) was used as a reference gene. Results miR-24-3p levels do not significantly differ between colorectal adenocarcinoma and non-cancerous colorectal mucosae. Thus, miR-24-3p expression cannot be used for diagnostic purposes. However, high miR-24-3p expression predicts poor disease-free survival (DFS) and overall survival (OS) of colorectal adenocarcinoma patients. Multivariate Cox regression analysis confirmed that miR-24-3p overexpression is a significant predictor of relapse in colorectal adenocarcinoma and that its prognostic significance is independent of other established prognostic factors and treatment of patients. Of note, miR-24-3p overexpression retains its rather unfavorable prognostic value in the subgroup of patients with advanced yet locally restricted colorectal adenocarcinoma (T3) and in those without distant metastasis (M0). Moreover, miR-24-3p overexpression is a potentially unfavorable prognosticator for patients who were not treated with radiotherapy. Conclusions Strong expression of miR-24-3p predicts poor DFS and OS of colorectal adenocarcinoma patients, independently of clinicopathological parameters that are currently used for prognosis in this human malignancy.

Diffuse large B-cell lymphoma (DLBCL) is recognized as a distinct entity by the World Health Organization (WHO) classification and constitutes the most common subtype of non-Hodgkin lymphomas, corr...

J Oral Pathol Med (2013)42: 154–161 Background: BCL2L12 is a recently identified gene belonging to the BCL2 family, members of which are implicated in head and neck squamous cell carcinoma (HNSCC). We have recently shown that BCL2L12 mRNA expression is an unfavorable prognostic indicator in nasopharyngeal carcinoma (NPC) and that BCL2L12 can be regarded as a novel, useful tissue biomarker for the prediction of NPC patients’ short-term relapse. The aim of this study was to analyze the mRNA expression of the novel apoptosis-related gene BCL2L12 in patients with HNSCC. Methods: Total RNA was isolated from 53 malignant tumors originating in larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissue specimens, resected from patients with HNSCC. A highly sensitive real-time PCR method for BCL2L12 mRNA quantification in head and neck tissues was developed using the SYBR® Green chemistry. After preparing cDNA by reverse transcription, relative quantification was performed using the comparative CT () method. Results: BCL2L12 mRNA levels were lower in laryngeal tumors of advanced tumor, node, metastasis (TNM) stage or bigger size and in well-differentiated malignant tongue neoplasms, compared with early-stage laryngeal tumors or poorly differentiated tongue tumors. Interestingly, the BCL2L12 expression showed significant discriminatory value, distinguishing efficiently patients with tongue squamous cell carcinoma (SCC) from non-cancerous population. Conclusions: This is the first study examining the BCL2L12 mRNA expression in HNSCC. Our results suggest that BCL2L12 mRNA expression may serve as a potential prognostic biomarker in tongue and/or larynx SCC, which principally constitute the great majority of HNSCC cases worldwide.

Apoptosis is a tightly regulated process that plays a critical role in many biological events. Members of the BCL2 (Bcl-2) family of apoptosis-related genes have been found to be differentially expressed in various malignancies and have been proposed as prognostic tumor biomarkers. We have recently discovered and cloned a new member of the BCL2 gene family, BCL2L12, expressed in colon tissues. Here we have investigated expression of the BCL2L12 gene in colon cancer tissues and assessed its prognostic value. Total RNA was isolated from 96 specimens of malignant colon tissue. After testing the RNA quality, cDNA was prepared by reverse transcription. A highly sensitive real-time PCR method for BCL2L12 mRNA quantification was developed using SYBR® Green chemistry. GAPDH served as a housekeeping gene. Relative quantification analysis was performed using the comparative CT method (2-ΔΔCT). High BCL2L12 expression levels were found in smaller (≤5 cm, p=0.027) and well-differentiated tumors (p=0.034), as well as in early-stage tumors (p=0.039). Survival analysis demonstrated that patients with BCL2L12-positive colon tumors have significantly longer disease-free survival and overall survival (p=0.015 and p=0.027, respectively). Our results suggest that BCL2L12 gene expression may represent a potential new biomarker for colon cancer.

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.