miRNA patterns in male LUSC patients - the 3-way mirror: Tissue, plasma and exosomes.

Lung cancer remains one of the leading causes of cancer-related deaths worldwide. It is classified into two main histological groups: non-small cell lung cancer (NSCLC) and small cell lung cancer. Improving the outcome of cancer patients could be possible by enhancing the early diagnosis. In the current study, we evaluated the levels of three microRNAs - miR-21-5p, miR-155-5p, and miR-181a-5p in tumor (TT) vs adjacent normal tissue (NT), as well as their expression levels in plasma and extracellular vesicles (EVs) from plasma in lung squamous cell carcinoma (LUSC) male patients vs healthy individuals as means to identify a panel of miRNAs that could serve as novel biomarkers for the diagnosis of LUSC in male patients. Matched paired tissue samples from male LUSC (n=40) patients were used for miRNA expression analysis. MiR-21-5p and miR-155-5p in tumor tissue were overexpressed, while underexpression of miR-181a-5p was observed in LUSC TT vs NT. These results were further validated in the TCGA LUSC dataset, considering 279 male samples. These alterations of miR-21-5p, miR-181a-5p, and miR-155-5p in tumor tissue are also present in plasma and plasma extracellular vesicles in LUSC male patients. In addition, ROC curves were performed to assess the sensitivity and specificity of different combinations of these miRNAs, confirming a high diagnostic accuracy for LUSC of up to 88 % in male subjects. The expression levels in tissue samples and the abundance in plasma and plasma EVs of the three miRNAs combined - miR-21-5p, miR-155-5p and miR-181a-5p - could be considered for further studies on biomarkers for the early detection of LUSC in male subjects.

Arsenic Methyltransferase and Apolipoprotein E Polymorphism in Pregnant Women Exposed to Inorganic Arsenic in Drinking Water in Western Romania.

Previous studies have shown that inorganic arsenic (iAs) exposure may be associated with genotoxic and cytotoxic effects. The aim of this study was to evaluate the relationship between several polymorphisms in AS3MT and APOE genes and urinary As and the relationship between these polymorphisms and pregnancy loss. We determined urinary As concentrations and performed genotyping analysis in 50 cases of spontaneous pregnancy loss and 50 controls, matched to cases on gestational age. The most frequently identified AS3MT polymorphisms in both cases and controls were in rs10748835 (80% cases and 68% controls), rs3740400 (78% cases and 64% controls), rs7085104 (74% cases and 48% controls), and rs1046778 (62% cases and 54% controls). We identified 30 different haplotypes in AS3MT SNPs, with four predominant haplotypes (>8%). Cases with Haplotype 1 had four-fold higher urinary DMA and two-fold higher MMA concentration than those without this haplotype, the MMA levels were lower in cases and controls with Haplotype 4 compared to Haplotype 1, and the DMA levels were significantly lower in cases with Haplotype 4 compared to Haplotype 3. Cases with Haplotype 1 had higher levels of all analyzed biomarkers, suggesting that Haplotype 1 may be associated with greater exposure to iAs and tobacco smoke. Our results suggest the importance of the AS3MT gene in iAs metabolism among pregnant women with low-level drinking water iAs exposure.

Profiling canine mammary tumors: A potential model for studying human breast cancer.

Despite all clinical progress recorded in the last decades, human breast cancer (HBC) remains a major challenge worldwide both in terms of its incidence and its management. Canine mammary tumors (CMTs) share similarities with HBC and represent an alternative model for HBC. The utility of the canine model in studying HBC relies on their common features, include spontaneous development, subtype classification, mutational profile, alterations in gene expression profile, and incidence/prevalence. This review describes the similarities between CMTs and HBC regarding genomic landscape, microRNA expression alteration, methylation, and metabolomic changes occurring during mammary gland carcinogenesis. The primary purpose of this review is to highlight the advantages of using the canine model as a translational animal model for HBC research and to investigate the challenges and limitations of this approach.

Developing cancer biobanks in Romania: understanding the knowledge and recommendations in assessing a clinical dataset for biospecimens.

Background and aims: In the context of the novelty of personalized medicine and biobanking in Romania, there is an acute need to analyze the degree of knowledge of the key actors in the domain. The present study sought to investigate the understanding of 'biobanking' and 'personalized medicine' in three categories of participants in the development of a biobank - health professionals (clinicians/diagnosticians), scientific researchers, and patients, in order to identify possible faults regarding the level of information. The secondary objective of this study was to identify key elements and relevant data that should be detailed in the clinical dataset that accompanies a biological sample. Methods: A total of 120 participants were included in this study that were divided into three categories that represent key actors in the development and management of a cancer biobank - clinicians (n=40), scientific researchers (n=40), and oncology patients (n=40). Results: The survey indicated that the terms 'biobank' and 'personalized medicine' are unknown only in a proportion of patients, while for the other two groups, these terms are already known. The second questionnaire allowed the arrangement of a recommended clinical dataset to be filled when a biological sample is provided to be included in a cancer biobank. Conclusions: The trust of patients and healthcare professionals in building biobanks that adhere to ethical and operational standards in Romania is important, as the development of artificial intelligence and databases allows advanced knowledge and connection of findings from different databases and, therefore, brings the concept of personalized medicine closer to the clinical practice. The information included in this dataset will be integrated and constitutes a comprehensive biobank database. All these aspects are meant to increase the utility of the specimens in cancer research, as clearly annotated samples, along with prospective data, bring valuable knowledge that helps scientific researchers and clinicians make the clinical connection between the molecular alterations and the phenotype of particular patients or a disease.

Artificial miRNAs derived from miR-181 family members have potential in cancer therapy due to an altered spectrum of target mRNAs.

miRNAs are a class of noncoding RNAs with gene regulation properties, and they function as key factors in cell homeostasis. The interaction of miRNAs with their target mRNAs is largely considered to rely on sequence complementarity; however, some evidence indicates that mature miRNAs can adopt diverse conformations with implications for their function. Using the oncogenic miR-181 family as a study model, we suggest that a potential relationship between the primary sequence and secondary structure of miRNAs may have an impact on the number and spectrum of targeted cellular transcripts. We further emphasize that specific alterations in miR-181 primary sequences might impose certain constraints on target gene selection compared with the wild-type sequences, leading to the targeting of new transcripts with upregulated function in cancer.

Identification of a miRNA Panel with a Potential Determinant Role in Patients Suffering from Periodontitis.

In recent years, the role of microRNA (miRNA) in post-transcriptional gene regulation has advanced and supports strong evidence related to their important role in the regulation of a wide range of fundamental biological processes. Our study focuses on identifying specific alterations of miRNA patterns in periodontitis compared with healthy subjects. In the present study, we mapped the major miRNAs altered in patients with periodontitis (n = 3) compared with healthy subjects (n = 5), using microarray technology followed by a validation step by qRT-PCR and Ingenuity Pathways Analysis. Compared to healthy subjects, 159 differentially expressed miRNAs were identified among periodontitis patients, of which 89 were downregulated, and 70 were upregulated, considering a fold change of ±1.5 as the cut-off value and p ≤ 0.05. Key angiogenic miRNAs (miR-191-3p, miR-221-3p, miR-224-5p, miR-1228-3p) were further validated on a separate cohort of patients with periodontitis versus healthy controls by qRT-PCR, confirming the microarray data. Our findings indicate a periodontitis-specific miRNA expression pattern representing an essential issue for testing new potential diagnostic or prognostic biomarkers for periodontal disease. The identified miRNA profile in periodontal gingival tissue was linked to angiogenesis, with an important molecular mechanism that orchestrates cell fate.

Emerging roles and mechanisms of semaphorins activity in cancer.

Semaphorins are regulatory molecules that are linked to the modulation of several cancer processes, such as angiogenesis, cancer cell invasiveness and metastasis, tumor growth, as well as cancer cell survival. Semaphorin (SEMA) activity depends on the cancer histotypes and their particularities. In broad terms, the effects of SEMAs result from their interaction with specific receptors/co-receptors - Plexins, Neuropilins and Integrins - and the subsequent effects upon the downstream effectors (e.g. PI3K/AKT, MAPK/ERK). The present article serves as an integrative review work, discussing the broad implications of semaphorins in cancer, focusing on cell proliferation/survival, angiogenesis, invasion, metastasis, stemness, and chemo-resistance/response whilst highlighting their heterogeneity as a family. Herein, we emphasized that semaphorins are largely implicated in cancer progression, interacting with the tumor microenvironment components. Whilst some SEMAs (e.g. SEMA3A, SEMA3B) function widely as tumor suppressors, others (e.g. SEMA3C) act as pro-tumor semaphorins. The differences observed in terms of the biological structure of SEMAs and the particularities of each cancer histotypes require that each semaphorin be viewed as a unique entity, and its roles must be researched accordingly. A more in-depth and comprehensive view of the molecular mechanisms that promote and sustain the malignant behavior of cancer cells is of utmost importance.

Identification of Potential microRNA Panels for Male Non-Small Cell Lung Cancer Identification Using Microarray Datasets and Bioinformatics Methods.

Background: Non-small cell lung cancer (NSCLC) is still one of the types of cancer with the highest death rates. MicroRNAs (miRNAs) play essential roles in NSCLC development. This study evaluates miRNA expression patterns and specific mechanisms in male patients with NSCLC. Methods: We report an integrated microarray analysis of miRNAs for eight matched samples of males with NSCLC compared to the study of public datasets of males with NSCLC from TCGA, followed by qRT-PCR validation. Results: For the TCGA dataset, we identified 385 overexpressed and 75 underexpressed miRNAs. Our cohort identified 54 overexpressed and 77 underexpressed miRNAs, considering a fold-change (FC) of ±1.5 and p < 0.05 as the cutoff value. The common miRNA signature consisted of eight overexpressed and nine underexpressed miRNAs. Validation was performed using qRT-PCR on the tissue samples for miR-183-3p and miR-34c-5p and on plasma samples for miR-34c-5p. We also created mRNA-miRNA regulatory networks to identify critical molecules, revealing NSCLC signaling pathways related to underexpressed and overexpressed transcripts. The genes targeted by these transcripts were correlated with overall survival. Conclusions: miRNAs and some of their target genes could play essential roles in investigating the mechanisms involved in NSCLC evolution and provide opportunities to identify potential therapeutic targets.

Genome Editing Approaches with CRISPR/Cas9 for Cancer Treatment: Critical Appraisal of Preclinical and Clinical Utility, Challenges, and Future Research.

The increasing burden on human malignant diseases became a major concern for healthcare practitioners, that must deal with tumor relapse and the inability to efficiently treat metastasis, in addition to side effects. Throughout the decades, many therapeutic strategies have been employed to improve the clinical outcomes of cancer patients and great efforts have been made to develop more efficient and targeted medicines. The malignant cell is characterized by genetic and epigenetic modifications, therefore targeting those specific drivers of carcinogenesis is highly desirable. Among the genome editing technologies, CRISPR/Cas9 stood as a promising candidate for cancer treatment alternatives, due to its low complexity design. First described as a defense mechanism of bacteria against invading foreign DNA, later it was shown that CRISPR components can be engineered to target specific DNA sequences in a test tube, a discovery that was awarded later with the Nobel Prize in chemistry for its rapid expansion as a reliable genome editing tool in many fields of research, including medicine. The present paper aims of describing CRISPR/Cas9 potential targets for malignant disorders, and the approaches used for achieving this goal. Aside from preclinical studies, we also present the clinical trials that use CRISPR-based technology for therapeutic purposes of cancer. Finally, a summary of the presented studies adds a more focused view of the therapeutic value CRISPR/Cas9 holds and the associated shortcomings.

Cellular and Molecular Profiling of Tumor Microenvironment and Early-Stage Lung Cancer.

Lung cancers are broadly divided into two categories: non-small-cell lung carcinoma (NSCLC), which accounts for 80-85% of all cancer cases, and small-cell lung carcinoma (SCLC), which covers the remaining 10-15%. Recent advances in cancer biology and genomics research have allowed an in-depth characterization of lung cancers that have revealed new therapy targets (EGFR, ALK, ROS, and KRAS mutations) and have the potential of revealing even more biomarkers for diagnostic, prognostic, and targeted therapies. A new source of biomarkers is represented by non-coding RNAs, especially microRNAs (miRNAs). MiRNAs are short non-coding RNA sequences that have essential regulatory roles in multiple cancers. Therefore, we aim to investigate the tumor microenvironment (TME) and miRNA tumor profile in a subset of 51 early-stage lung cancer samples (T1 and T2) to better understand early tumor and TME organization and molecular dysregulation. We analyzed the immunohistochemistry expression of CD4 and CD8 as markers of the main TME immune populations, E-cadherin to evaluate early-stage epithelial-to-mesenchymal transition (EMT), and p53, the main altered tumor suppressor gene in lung cancer. Starting from these 4 markers, we identified and validated 4 miRNAs that target TP53 and regulate EMT that can be further investigated as potential early-stage lung cancer biomarkers.

Next-Generation Sequencing in Lung Cancer Patients: A Comparative Approach in NSCLC and SCLC Mutational Landscapes.

Background: Lung cancer remains one of the most diagnosed malignancies, being the second most diagnosed cancer, while still being the leading cause of cancer-related deaths. Late diagnosis remains a problem, alongside the high mutational burden encountered in lung cancer. Methods: We assessed the genetic profile of cancer genes in lung cancer using The Cancer Genome Atlas (TCGA) datasets for mutations and validated the results in a separate cohort of 32 lung cancer patients using tumor tissue and whole blood samples for next-generation sequencing (NGS) experiments. Another separate cohort of 32 patients was analyzed to validate some of the molecular alterations depicted in the NGS experiment. Results: In the TCGA analysis, we identified the most commonly mutated genes in each lung cancer dataset, with differences among the three histotypes analyzed. NGS analysis revealed TP53, CSF1R, PIK3CA, FLT3, ERBB4, and KDR as being the genes most frequently mutated. We validated the c.1621A>C mutation in KIT. The correlation analysis indicated negative correlation between adenocarcinoma and altered PIK3CA (r = −0.50918; p = 0.0029). TCGA survival analysis indicated that NRAS and IDH2 (LUAD), STK11 and TP53 (LUSC), and T53 (SCLC) alterations are correlated with the survival of patients. Conclusions: The study revealed differences in the mutational landscape of lung cancer histotypes.

New insights in gene expression alteration as effect of doxorubicin drug resistance in triple negative breast cancer cells.

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous disease with aggressive behavior and an unfavorable prognosis rate. Due to the lack of surface receptors, TNBC must be intensely investigated in order to establish a suitable treatment for patients with this pathology. Chemoresistance is an important reason for therapeutic failure in TNBC. METHOD: The aim of this study was to investigate the effect of doxorubicin in TNBC cell lines and to highlight cellular and molecular alterations after a long exposure to doxorubicin. RESULTS: The results revealed that doxorubicin significantly increased the half maximal inhibitory concentration (IC50) values at P12 and P24 compared to parenteral cells P0. Modifications in gene expression were investigated through microarray technique, and for detection of mutational pattern was used Next Generation Sequencing (NGS). 196 upregulated and 115 downregulated genes were observed as effect of multiple dose exposure, and 15 overexpressed genes were found to be involved in drug resistance. Also, the presence of some additional mutations in both cell lines was observed. CONCLUSION: The outcomes of this research may provide novel biomarkers for drug resistance in TNBC. Also, this activity can highlight the potential mechanisms associated with drug resistance, as well as the potential therapies to counteract these mechanisms.

The Clinical Utility of miR-21 and let-7 in Non-small Cell Lung Cancer (NSCLC). A Systematic Review and Meta-Analysis.

Non-small cell lung cancer (NSCLC) remains a problem worldwide due to its rapid progression and low rate of response to treatment. The heterogeneity of these tumors observed in histopathology exam but also in the mutational status and gene expression pattern makes this malignancy difficult to treat in clinic. The present study investigated the effect of miR-21 and let-7 family members as prognostic biomarkers in NSCLC patients based on the results published in different studies regarding this subject until March 2019. The analysis revealed that these two transcripts are steady biomarkers for prediction of patient outcome or survival. Upregulated expression of miR-21 is associated with poor outcome of patients with NSCLC [HR = 1.87, 95% CI = (1.41, 2.47), p < 0.001]. The analysis regarding let-7 family, specifically let-7a/b/e/f, revealed that downregulated expression of these transcripts predicts poor outcome for NSCLC patients [HR = 2.61, 95% CI = (1.58, 4.30), p < 0.001]. Besides, the reliability of these microRNAs is reflected in the fact that their prognostic significance is constant given the different sample types (tissue, FFPE tissue, serum, serum/plasma or exosomes) used in the selected studies.

Tiny Actors in the Big Cellular World: Extracellular Vesicles Playing Critical Roles in Cancer.

Communications among cells can be achieved either via direct interactions or via secretion of soluble factors. The emergence of extracellular vesicles (EVs) as entities that play key roles in cell-to-cell communication offer opportunities in exploring their features for use in therapeutics; i.e., management and treatment of various pathologies, such as those used for cancer. The potential use of EVs as therapeutic agents is attributed not only for their cell membrane-bound components, but also for their cargos, mostly bioactive molecules, wherein the former regulate interactions with a recipient cell while the latter trigger cellular functions/molecular mechanisms of a recipient cell. In this article, we highlight the involvement of EVs in hallmarks of a cancer cell, particularly focusing on those molecular processes that are influenced by EV cargos. Moreover, we explored the roles of RNA species and proteins carried by EVs in eliciting drug resistance phenotypes. Interestingly, engineered EVs have been investigated and proposed as therapeutic agents in various in vivo and in vitro studies, as well as in several clinical trials.

New Insights in Gene Expression Alteration as Effect of Paclitaxel Drug Resistance in Triple Negative Breast Cancer Cells.

BACKGROUND/AIMS: Triple negative breast cancer (TNBC) is a highly aggressive form of cancer which lacks targeted therapy options. Thus, TNBC patients have poor outcomes and a decreased survival rate than patients with other types of breast cancers. Due to the lack of surface receptors, TNBC needs a comprehensive investigation to provide more information regarding patient's therapy, as well as to understand the way how to counteract drug resistance mechanisms. Nowadays, chemotherapy remains an unsolved issue which rise a lot of questions in oncology field. METHODS: In this article, we investigated the implication of paclitaxel in TNBC cell lines after a prolong administration, after 12, respectively 24 passages followed by evaluation of morphological alteration, mutational pattern by next generation sequencing and the altered gene expression pattern by microarray technology and validation by qRT-PCR of the resistance to therapy relevant genes. RESULTS: Using functional assays, we showed that paclitaxel exhibits antiproliferative activity on Hs578T/Pax and MDA-MB-231/Pax demonstrating the activation of cell death mechanisms. Confocal microscopy revealed significant modifications which occur in the morphological structure with a disruption of the actin-filaments and also mitotic catastrophe. The presence of these nuclear alterations is due to some modifications at the cellular and molecular levels. Important alterations at the transcriptomic and genomic levels were observed from this a common drug resistance signature (IL-6, CXCL8, VEGFA, EGR1, PTGS2 and TRIB1) for both cell lines at 24 passages was discovered. Also, an important mutation (TP53) linked with drug response was identified. CONCLUSION: These results might be used to furnish novel biomarkers in TNBC, as well as to find a strategy to counteract the resistance to therapy in order to increase survival rate and to enhance the prognosis of patients with TNBC.

Prognostic Value of MiR-21: An Updated Meta-Analysis in Head and Neck Squamous Cell Carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is a group of malignancies with serious impact on patient quality of life due to a reduced rate of response to chemotherapy or radiation therapy. MiR-21 has been identified as one of the most common proto-oncogenes. It is hypothesized that upregulated miR-21 could serve as a potential biomarker for human cancer diagnosis. Considering the target genes identified for miR-21 in HNSCC, this transcript is an important player in several cellular processes that control carcinogenesis. The abnormal expression of miR-21 in this group of pathologies has been assessed in several publications, but given the heterogeneity of the published results, a meta-analysis and proper bioinformatics analysis of expression databases are needed to correctly establish the prognostic potential of this molecule. The present meta-analysis comprises the published survival data on HNSCC patients, reported as HR and 95% CI, in association with the expression levels of miR-21. Our investigation revealed that miR-21 could be used successfully as a prognostic biomarker in HNSCC patients, confirming its oncogenic potential. Specifically, the upregulation of miR-21 in these patients predicts a worse outcome in terms of survival rate.

MiR-181 family-specific behavior in different cancers: a meta-analysis view.

The involvement of microRNAs in malignant transformation and cancer progression was previously grounded. The observations made by multiple published studies led to the conclusion that some of these small sequences could be eventually used as biomarkers for diagnosis/prognosis. This meta-analysis investigated whether microRNA-181 family members could predict the outcome of patients carrying different types of cancer. We searched the PubMed and Embase databases for studies evaluating the expression levels of miR-181a/b/c/d in patients with cancer, selecting the publications that assessed the relation between low and high levels of one of these four microRNAs and patients' outcome. Hazard ratios (HRs) or risk ratios (RRs) were extracted from the studies, and random-effect model was performed to investigate the role of miR-181 in the outcome of these patients. The meta-analysis comprised 26 studies including 2653 cancer patients from 6 countries. The results showed significant association between the expression of miR-181 family members and colorectal cancer. Considering the heterogeneity of the pathologies, the analysis, including all types of cancer and the expression of all the miR-181 family members together, showed no association with distinct outcome (HR = 1.099, p = 0.435). When the analysis was performed on each microRNA separately, the expression of miR-181c was significantly associated with the outcome of patients with cancer (HR = 2.356, p = 0.011) and miR-181a expression levels significantly correlated with survival in patients with non-small-cell lung cancer (HR = 0.177, p < 0.05). This meta-analysis revealed evidence regarding the involvement of miR-181 family members in the outcome of patients with some types of cancer, according to their expression level.

The silent healer: miR-205-5p up-regulation inhibits epithelial to mesenchymal transition in colon cancer cells by indirectly up-regulating E-cadherin expression.

EMT represents the dominant program within advanced stages of colon cancer, where cells acquire migratory characteristics in order to invade secondary tissues and form metastasis. Where the majority of the therapeutic strategies are concentrated on the reduction of the tumor mass through different apoptotic mechanisms, the present study advocates an important role for miR-205-5p in impairment of colon cancer cells migration and restoration of the epithelial phenotype. Upon identification of a homogenous downregulated profile for miR-205-5p in colon adenocarcinoma patients, functional studies demonstrated that experimental upregulation of this sequence is able to significantly raise the levels of E-cadherin through direct inhibition of ZEB1. Moreover, the elevation in CDH1 expression was translated into functional parameters where cells lost their invasion and migratory characteristics and formed homogenous clusters through adhesion interactions. Survival analysis of colon adenocarcinoma patients revealed that low levels of miR-205-5p are associated with an unfavorable prognostic compared to those with increased expression, demonstrating the possible clinical utility of miR-205-5p replacement. Exogenous administration of miRNA mimics was not associated with significant changes in cell viability or inflammatory pathways. Therefore, the proposed strategy is aiming towards inhibition of metastasis and limitation of the tumor borders in advanced stages patients in order to prolong the survival time and to increase the efficiency of the current therapeutic strategies.

Understanding the Role of Non-Coding RNAs in Bladder Cancer: From Dark Matter to Valuable Therapeutic Targets.

The mortality and morbidity that characterize bladder cancer compel this malignancy into the category of hot topics in terms of biomolecular research. Therefore, a better knowledge of the specific molecular mechanisms that underlie the development and progression of bladder cancer is demanded. Tumor heterogeneity among patients with similar diagnosis, as well as intratumor heterogeneity, generates difficulties in terms of targeted therapy. Furthermore, late diagnosis represents an ongoing issue, significantly reducing the response to therapy and, inevitably, the overall survival. The role of non-coding RNAs in bladder cancer emerged in the last decade, revealing that microRNAs (miRNAs) may act as tumor suppressor genes, respectively oncogenes, but also as biomarkers for early diagnosis. Regarding other types of non-coding RNAs, especially long non-coding RNAs (lncRNAs) which are extensively reviewed in this article, their exact roles in tumorigenesis are-for the time being-not as evident as in the case of miRNAs, but, still, clearly suggested. Therefore, this review covers the non-coding RNA expression profile of bladder cancer patients and their validated target genes in bladder cancer cell lines, with repercussions on processes such as proliferation, invasiveness, apoptosis, cell cycle arrest, and other molecular pathways which are specific for the malignant transformation of cells.