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.

Expression of Human L-Dopa Decarboxylase (DDC) under Conditions of Oxidative Stress.

Oxidative stress is known to influence mRNA levels, translation, and proteolysis. The importance of oxidative stress has been demonstrated in several human diseases, including neurodegenerative disorders. L-Dopa decarboxylase (DDC) is the enzyme that converts L-Dopa to dopamine (DA). In spite of a large number of studies, little is known about the biological significance of the enzyme under physiological and pathological conditions. Here, we investigated the relationship between DDC expression and oxidative stress in human neural and non-neural cells. Oxidative stress was induced by treatment with H2O2. Our data indicated that mRNA and protein expression of DDC was enhanced or remained stable under conditions of ROS induction, despite degradation of total RNA and increased cytotoxicity and apoptosis. Moreover, DDC silencing caused an increase in the H2O2-induced cytotoxicity. The current study suggests that DDC is involved in the mechanisms of oxidative stress.

3'-tRF-CysGCA overexpression in HEK-293 cells alters the global expression profile and modulates cellular processes and pathways.

tRNA fragments (tRFs) are small non-coding RNAs generated through specific cleavage of tRNAs and involved in various biological processes. Among the different types of tRFs, the 3'-tRFs have attracted scientific interest due to their regulatory role in gene expression. In this study, we investigated the role of 3'-tRF-CysGCA, a tRF deriving from cleavage in the T-loop of tRNACysGCA, in the regulation of gene expression in HEK-293 cells. Previous studies have shown that 3'-tRF-CysGCA is incorporated into the RISC complex and interacts with Argonaute proteins, suggesting its involvement in the regulation of gene expression. However, the general role and effect of the deregulation of 3'-tRF-CysGCA levels in human cells have not been investigated so far. To fill this gap, we stably overexpressed 3'-tRF-CysGCA in HEK-293 cells and performed transcriptomic and proteomic analyses. Moreover, we validated the interaction of this tRF with putative targets, the levels of which were found to be affected by 3'-tRF-CysGCA overexpression. Lastly, we investigated the implication of 3'-tRF-CysGCA in various pathways using extensive bioinformatics analysis. Our results indicate that 3'-tRF-CysGCA overexpression led to changes in the global gene expression profile of HEK-293 cells and that multiple cellular pathways were affected by the deregulation of the levels of this tRF. Additionally, we demonstrated that 3'-tRF-CysGCA directly interacts with thymopoietin (TMPO) transcript variant 1 (also known as LAP2α), leading to modulation of its levels. In conclusion, our findings suggest that 3'-tRF-CysGCA plays a significant role in gene expression regulation and highlight the importance of this tRF in cellular processes.

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.

MicroRNA-675-5p Overexpression Is an Independent Prognostic Molecular Biomarker of Short-Term Relapse and Poor Overall Survival in Colorectal Cancer.

Colorectal cancer (CRC) is the main cause of cancer-related deaths globally, highlighting the importance of accurate biomarkers for early detection and accurate prognosis. MicroRNAs (miRNAs) have emerged as effective cancer biomarkers. The aim of this study was to investigate the prognostic potential of miR-675-5p as a molecular prognostic biomarker in CRC. For this reason, a quantitative PCR assay was developed and applied to determine miR-675-5p expression in cDNAs from 218 primary CRC and 90 paired normal colorectal tissue samples. To assess the significance of miR-675-5p expression and its association with patient outcome, extensive biostatistical analysis was performed. miR-675-5p expression was found to be significantly downregulated in CRC tissue samples compared to that in adjacent normal colorectal tissues. Moreover, high miR-675-5p expression was associated with shorter disease-free (DFS) and overall survival (OS) in CRC patients, while it maintained its unfavorable prognostic value independently of other established prognostic factors. Furthermore, TNM stage stratification demonstrated that higher miR-675-5p levels were associated with shorter DFS and OS intervals, particularly in patients with CRC of TNM stage II or III. In conclusion, our findings suggest that miR-675-5p overexpression constitutes a promising molecular biomarker of unfavorable prognosis in CRC, independent of other established prognostic factors, including TNM staging.

Identification of novel alternative transcripts of the human Ribonuclease κ (RNASEK) gene using 3' RACE and high-throughput sequencing approaches.

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.

Circulating miR-146a and miR-134 in predicting drug-resistant epilepsy in patients with focal impaired awareness seizures.

OBJECTIVE: Epilepsy is one of the most prevalent neurologic disorders, causing serious psychological problems and reducing quality of life. Although 20 different antiepileptic drugs (AEDs) have been approved by the US Food and Drug Administration (FDA), 30% of patients have drug-resistant epilepsy (DRE). Considering the role of miR-146a and miR-134 in neuroinflammation and dendritic functionality, respectively, the aim of this study was the clinical evaluation of circulating miR-146a and miR-134 as novel noninvasive molecular markers for the prognosis of refractory epilepsy. METHODS: The study included 162 patients with focal impaired awareness seizures. Total RNA was extracted from serum samples spiked with synthetic cel-miR-39-3p for normalization purposes. First-strand complementary DNA (cDNA) synthesis was performed using microRNA-specific stem-loop primers, and hsa-miR-134/146a levels were quantified by quantitative polymerase chain reaction (qPCR). DRE was used as clinical end point event. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit on disease prognosis. RESULTS: The circulating levels of both miR-134 and miR-146a were elevated in patients with drug-resistant seizures. The receiver-operating characteristic (ROC) curve and logistic regression analysis demonstrated that patients with increased circulating miR-134/146a levels are at significantly higher risk for developing DRE, independently of temporal lobe sclerosis, epilepsy duration, familial history, age at first seizure, age, body mass index (BMI), smoking behavior, and gender. Finally, decision curve analysis highlighted that the evaluation of circulating miR-134/146a led to superior clinical benefit for DRE prognosis and patients' risk stratification. SIGNIFICANCE: Elevated serum miR-134/146a levels are associated with a higher risk for AED-resistant epilepsy and could constitute novel noninvasive molecular markers to improve disease early prognosis and support precision medicine.

Loss of GAS5 tumour suppressor lncRNA: an independent molecular cancer biomarker for short-term relapse and progression in bladder cancer patients.

BACKGROUND: Bladder cancer (BlCa) heterogeneity and the lack of personalised prognosis lead to patients' highly variable treatment outcomes. Here, we have analysed the utility of the GAS5 tumour-suppressor lncRNA in improving BlCa prognosis. METHODS: GAS5 was quantified in a screening cohort of 176 patients. Hedegaard et al. (2016) (n = 476) and TCGA provisional (n = 413) were used as validation cohorts. Survival analysis was performed using recurrence and progression for NMIBC, or death for MIBC. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit on disease prognosis. RESULTS: GAS5 levels were significantly downregulated in BlCa and associated with invasive high-grade tumours, and high EORTC-risk NMIBC patients. GAS5 loss was strongly and independently correlated with higher risk for NMIBC early relapse (HR = 2.680, p = 0.011) and progression (HR = 6.362, p = 0.035). Hedegaard et al. and TCGA validation cohorts' analysis clearly confirmed the association of GAS5 loss with NMIBC worse prognosis. Finally, multivariate models incorporating GAS5 with disease established markers resulted in higher clinical benefit for NMIBC prognosis. CONCLUSIONS: GAS5 loss is associated with adverse outcome of NMIBC and results in improved positive prediction of NMIBC patients at higher risk for short-term relapse and progression, supporting personalised prognosis and treatment decisions.

BCL2L12: a multiply spliced gene with independent prognostic significance in breast cancer.

Background Alternative splicing is a key process in carcinogenesis and, from a clinical aspect, holds great promises, as alternatively spliced variants have emerged as an untapped source of diagnostic and prognostic markers. Our aim was to assess the prognostic value of three recently recognized splice variants of the apoptosis-related gene, BCL2L12, in breast cancer (BC). Methods Total RNA was extracted from breast samples (150 BC and 80 tumor-adjacent normal tissues) and, following cDNA synthesis, a variant-specific qPCR was performed for the expressional quantification of BCL2L12 v.1, v.2 and v.4 transcript variants. Extensive statistical analysis, including bootstrap resampling and internal validation, was conducted in order to evaluate the associations of v.1, v.2 and v.4 expression with patients' clinopathological and survival data. Results All examined BCL2L12 variants were significantly upregulated in BC specimens compared to their non-cancerous counterpart (v.1, p<0.001; v.2, p=0.009; v.4, p=0.004). Increased BCL2L12 v.4 mRNA expression was associated with markers of unfavorable prognosis namely, advanced tumor grade (p=0.002), ER- (p=0.015)/PR- (p<0.001) negativity, Ki-67-positivity (p=0.007) and high NPI (Nottingham prognostic index) score (p=0.033). Moreover, v.4 was significantly overexpressed in women with triple negative BC (TNBC) and HER2-positive tumors compared to those harboring luminal tumors (p<0.001). Survival analysis disclosed that BCL2L12 v.2 overexpression, as a continuous variable ([HR]=0.45, 95% CI=0.17-0.82, p=0.010), is a strong and independent marker of favorable prognosis for BC patients. Interestingly, v.2 retains its prognostic value in patients with Grade II/III ([HR]=0.21, 95% CI=0.05-0.57, p=0.006) or HER2-positive/TNBC tumors ([HR]=0.25, 95% CI=0.05-0.74, p=0.042). Conclusions BCL2L12 v.1, v.2, v.4 are aberrantly expressed in BC. Their expressional analysis by cost-effective molecular methods could provide a novel molecular tool for BC management.

Exploring the time-dependent regulatory potential of microRNAs in breast cancer cells treated with proteasome inhibitors.

PURPOSE: Breast cancer (BrCa) is a predominant type of cancer with a disparate molecular nature. MicroRNAs (miRNAs) have emerged as promising key players in the regulation of pathological processes in BrCa. Proteasome inhibitors (PIs) emerged as promising anticancer agents for several human malignancies, including BrCa, inhibiting the function of the proteasome. Aiming to shed light on the miRNA regulatory effect in BrCa after treatment with PIs, we used two PIs, namely bortezomib and carfilzomib. MATERIALS AND METHODS: Four BrCa cell lines of distinct molecular subtypes were treated with these PIs. Cell viability and IC50 concentrations were determined. Total RNA was extracted, polyadenylated, and reversely transcribed. Next, the levels of specific miRNAs with a significant role in BrCa were determined using relative quantification, and their regulatory effect was assessed. RESULTS: High heterogeneity was discovered in the levels of miRNAs in the four cell lines, after treatment. The miRNA levels fluctuate with distinct patterns, in 24, 48, or 72 hours. Interestingly, miR-1-3p, miR-421-3p, and miR-765-3p appear as key molecules, as they were found deregulated, in almost all combinations of cell lines and PIs. In the SK-BR-3 cell line, the majority of the miRNAs were significantly downregulated in treated compared to untreated cells, with miR-21-5p being the only one upregulated. Finally, various significant biological processes, molecular functions, and pathways were predicted to be affected. CONCLUSIONS: The diversity of pathways predicted to be affected by the diversity in miRNA expression after treatment with PIs paves the way for the recognition of new regulatory axes in BrCa.

Spike-Seq: An amplicon-based high-throughput sequencing approach for the sensitive detection and characterization of SARS-CoV-2 genetic variations in environmental samples.

Ever since the outbreak of COVID-19 disease in Wuhan, China, different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been identified. Wastewater-based epidemiology (WBE), an approach that has been successfully applied in numerous case studies worldwide, offers a cost-effective and rapid way for monitoring trends of SARS-Cov-2 in the community level without selection bias. Despite being a gold-standard procedure, WBE is a challenging approach due to the sample instability and the moderate efficiency of SARS-CoV-2 concentration in wastewater. In the present study, we introduce Spike-Seq, a custom amplicon-based approach for the S gene sequencing of SARS-CoV-2 in wastewater samples, which enables not only the accurate identification of the existing Spike-related genetic markers, but also the estimation of their frequency in the investigated samples. The implementation of Spike-Seq involves the combination of nested PCR-based assays that efficiently amplify the entire nucleotide sequence of the S gene and next-generation sequencing, which enables the variant detection and the estimation of their frequency. In the framework of the current work, Spike-Seq was performed to investigate the mutational profile of SARS-CoV-2 in samples from the Wastewater Treatment Plant (WWTP) of Athens, Greece, which originated from multiple timepoints, ranging from March 2021 until July 2022. Our findings demonstrate that Spike-Seq efficiently detected major genetic markers of B.1.1.7 (Alpha), B.1.617.2 (Delta) as well as B.1.1.529 (Omicron) variants in wastewater samples and provided their frequency levels, showing similar variant distributions with the published clinical data from the National Public Health organization. The presented approach can prove to be a useful tool for the detection of SARS-CoV-2 in challenging wastewater samples and the identification of the existing genetic variants of S gene.

Exploring the molecular biomarker utility of circCCT3 in multiple myeloma: A favorable prognostic indicator, particularly for R-ISS II patients.

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

CDKN2A copy number alteration in bladder cancer: Integrative analysis in patient-derived xenografts and cancer patients.

Bladder cancer (BlCa) is an extensively heterogeneous disease that leads to great variability in tumor evolution scenarios and lifelong patient surveillance, emphasizing the need for modern, minimally invasive precision medicine. Here, we explored the clinical significance of copy number alterations (CNAs) in BlCa. CNA profiling was performed in 15 patient-derived xenografts (PDXs) and validated in The Cancer Genome Atlas BlCa (TCGA-BLCA; n = 408) and Lindgren et al. (n = 143) cohorts. CDKN2A copy number loss was identified as the most frequent CNA in bladder tumors, associated with reduced CDKN2A expression, tumors of a papillary phenotype, and prolonged PDX survival. The study's screening cohort consisted of 243 BlCa patients, and CDKN2A copy number was assessed in genomic DNA and cell-free DNA (cfDNA) from 217 tumors and 189 pre-treatment serum samples, respectively. CDKN2A copy number loss was correlated with superior disease-free and progression-free survival of non-muscle-invasive BlCa (NMIBC) patients. Moreover, a higher CDKN2A index (CDKN2A/LEP ratio) in pre-treatment cfDNA was associated with advanced tumor stage and grade and short-term NMIBC progression to invasive disease, while multivariate models fitted for CDKN2A index in pre-treatment cfDNA offered superior risk stratification of T1/high-grade and EORTC high-risk patients, enhancing prediction of treatment outcome. CDKN2A copy number status could serve as a minimally invasive tool to improve risk stratification and support personalized prognosis in BlCa.

L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.

The enzyme L-Dopa Decarboxylase (DDC) synthesizes the catecholamine dopamine and the indolamine serotonin. Apart from its role in the brain as a neurotransmitter biosynthetic enzyme, DDC has been detected also in the liver and other peripheral organs, where it is implicated in cell proliferation, apoptosis, and host-virus interactions. Dengue virus (DENV) suppresses DDC expression at the later stages of infection, during which DENV also inhibits autophagosome-lysosome fusion. As dopamine affects autophagy in neuronal cells, we investigated the possible association of DDC with autophagy in human hepatocytes and examined whether DDC mediates the relationship between DENV infection and autophagy. We performed DDC silencing/overexpression and evaluated autophagic markers upon induction of autophagy, or suppression of autophagosome-lysosome fusion. Our results showed that DDC favored the autophagic process, at least in part, through its biosynthetic function, while knockdown of DDC or inhibition of DDC enzymatic activity prevented autophagy completion. In turn, autophagy induction upregulated DDC, while autophagy reduction by chemical or genetic (ATG14L knockout) ways caused the opposite effect. This study also implicated DDC with the cellular energetic status, as DDC silencing reduced the oxidative phosphorylation activity of the cell. We also report that upon DDC silencing, the repressive effect of DENV on the completion of autophagy was enhanced, and the inhibition of autolysosome formation did not exert an additive effect on viral proliferation. These data unravel a novel role of DDC in the autophagic process and suggest that DENV downregulates DDC expression to inhibit the completion of autophagy, reinforcing the importance of this protein in viral infections.

Recent Advances in Genome-Engineering Strategies.

In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice. Due to their advantageous features, the CRISPR-Cas systems are already employed in several biological and medical research fields as the most suitable technique for genome engineering. In this review, we aim to describe the CRISPR-Cas systems that have been identified among prokaryotic organisms and engineered for genome manipulation studies. Furthermore, a comprehensive comparison between the innovative CRISPR-Cas methodology and the previously utilized ZFN and TALEN editing nucleases is also discussed. Ultimately, we highlight the contribution of CRISPR-Cas methodology in modern biomedicine and the current plethora of available applications for gene KO, repression and/or overexpression, as well as their potential implementation in therapeutical strategies that aim to improve patients' quality of life.

Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

miRNA-Based Technologies in Cancer Therapy.

The discovery of therapeutic miRNAs is one of the most exciting challenges for pharmaceutical companies. Since the first miRNA was discovered in 1993, our knowledge of miRNA biology has grown considerably. Many studies have demonstrated that miRNA expression is dysregulated in many diseases, making them appealing tools for novel therapeutic approaches. This review aims to discuss miRNA biogenesis and function, as well as highlight strategies for delivering miRNA agents, presenting viral, non-viral, and exosomic delivery as therapeutic approaches for different cancer types. We also consider the therapeutic role of microRNA-mediated drug repurposing in cancer therapy.

High Intratumoral i-tRF-GlyGCC Expression Predicts Short-Term Relapse and Poor Overall Survival of Colorectal Cancer Patients, Independent of the TNM Stage.

Colorectal cancer (CRC), one of the most prevalent types of cancer, requires the discovery of new tumor biomarkers for accurate patient prognosis. In this work, the prognostic value of the tRNA fragment i-tRF-GlyGCC in CRC was examined. Total RNA extraction from 211 CRC patient cancer tissue specimens and 83 adjacent normal tissues was conducted. Each RNA extract was subjected to in vitro polyadenylation and reverse transcription. A real-time quantitative PCR assay was used to quantify i-tRF-GlyGCC in all samples. Extensive biostatics analysis showed that i-tRF-GlyGCC levels in CRC tissues were significantly lower than in matched normal colorectal tissues. Additionally, the disease-free survival (DFS) and overall survival (OS) time intervals were considerably shorter in CRC patients with high i-tRF-GlyGCC expression. i-tRF-GlyGCC expression maintained its prognostic value independently of other established prognostic factors, as shown by the multivariate Cox regression analysis. Additionally, survival analysis after TNM stage stratification revealed that higher i-tRF-GlyGCC levels were linked to shorter DFS time intervals in patients with TNM stage II tumors, as well as an increased probability of having a worse OS for patients in TNM stage II. In conclusion, i-tRF-GlyGCC has the potential to be a useful molecular tissue biomarker in CRC, independent of other clinicopathological variables.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

Efficient cloning of alternatively polyadenylated transcripts via hybridization capture PCR.

Cloning of alternatively polyadenylated transcripts is crucial for studying gene expression and function. Recent transcriptome analysis has mainly focused on large EST clone collections. However, EST sequencing techniques in many cases are incapable of isolating rare transcripts or address transcript variability. In most cases, 3' RACE is applied for the experimental identification of alternatively polyadenylated transcripts. However, its application may result in nonspecific amplification and false positive products due to the usage of a single gene specific primer. Additionally, internal poly(A) stretches primed by oligo(dT) primer in mRNAs with AU-rich 3'UTR may generate truncated cDNAs. To overcome these limitations, we have developed a simple and rapid approach combining SMART technology for the construction of a full length cDNA library and hybrid capture PCR for the selection and amplification of target cDNAs. Our strategy is characterized by enhanced specificity compared to other conventional RT-PCR and 3' RACE procedures.