Interplay between LncRNAs and microRNAs in Breast Cancer.

(1) Although long noncoding RNAs (lncRNAs) are known to be precursors of microRNAs (miRNAs), they frequently act as competing endogoneous RNAs (ceRNAs), yet still their interplay with miRNA is not well known. However, their interaction with miRNAs may result in the modulation of miRNA action. (2) To determine the contribution of these RNA molecules in tumor resistance to chemotherapeutic drugs, it is essential to consider not only the oncogenic and tumor suppressive function of miRNAs but also the impact of lncRNAs on miRNAs. Therefore, we performed an extensive search in different databases including PubMed. (3) The present study concerns the interplay between lncRNAs and miRNAs in the regulatory post-transcriptional network and their impact on drugs used in the treatment of breast cancer. (4) Consideration of this interplay may improve the search for new drugs to circumvent chemoresistance.

MicroRNAs and their Implications in CD4+ T-cells, Oligodendrocytes and Dendritic Cells in Multiple Sclerosis Pathogenesis.

MicroRNAs (miRNAs) have been established as key players in various biological processes regulating differentiation, proliferation, inflammation, and autoimmune disorders. Emerging evidence suggests the critical role of miRNAs in the pathogenesis of multiple sclerosis (MS). Here, we provide a comprehensive overview of miRNAs, which are differentially expressed in MS patients or experimental autoimmune encephalomyelitis (EAE) mice and contribute to MS pathogenesis through regulating diverse pathways, including CD4+ T cells proliferation, differentiation, and activation in three subtypes of CD4+ T cells, including Th1, Th17 and regulatory T cells (Tregs). Moreover, the regulation of oligodendrocyte precursor cells (OPC) differentiation as a crucial player in MS pathogenesis is also described. Our literature research showed that miR-223 could affect different pathways involved in MS pathogenesis, such as promoting Th1 differentiation, activating the M2 phenotype of myeloid cells, and clearing myelin debris. MiR-223 was also identified as a potential biomarker, distinguishing relapsing-remitting multiple sclerosis (RRMS) from progressive multiple sclerosis (PMS), and thus, it may serve as an attractive target for further investigations. Our overview provides novel potential therapeutic targets for the treatment and new insights into miRNAs' role in MS pathogenesis.

Characterization of immunologically detectable T-cell sensitization, Immunohistochemical detection of pro-inflammatory cytokines, and clinical parameters of patients after allogeneic intraoral bone grafting procedures: a prospective randomized controlled clinical trial in humans.

BACKGROUND: The null hypotheses were tested that intraoral bone augmentation using two different allogeneic materials has no impact on the patient's blood levels of material-specific lymphocytes and on the immunohistochemical detection of pro-inflammatory cytokines IL-1α, IL1ß and TNF-α and T-cell markers CD4, CD8 in biopsies of the test groups. METHODS: In this prospective RCT, 60 systemically healthy participants were randomly assigned to two allogeneic test groups (1: Maxgraft®, freeze-dried, multiple donors, and 2: Puros®, solvent-dehydrated, single donor) and an autologous control group (10 patients). Plasma samples were collected pre-(T1) and postoperatively (2 weeks (T2) and 4 months (T3)). The Lymphocyte Transformation Test (LTT) was used for analyzing levels of transformed lymphocytes for type IV immune reactions by 3H-thymidine activity. Bone biopsies were harvested at T3 and immunohistochemically analyzed for IL-1α, IL1ß, TNF-α, CD4, CD8 and correlated with the immunological and clinical findings. RESULTS: A statistically significant difference between the tested materials was observed for LTT measurements at T3 (p = 0.033). Furthermore, three groups were identified: Group A (LTT negative T1-T3, n = 48), group B (LTT positive T1-T3, n = 7), group C (developing positive LTT at T2, n = 5). A highly significant elevation of IL-1α, IL1ß, TNF-α in patients of group C (p = 0.0001) and a significant elevation of CD4+ cells in patients of group B (p = 0.005) was shown. CONCLUSION: Our data show that following allogeneic bone grafting, local and systemic immunological reactions can be detected in some patients. These findings were statistically significant for the timepoint T3 between the tested materials as well as for the groups B and C correlated with group A for both tested materials. Therefore, the null hypotheses were rejected. A preoperative compatibility test for allogeneic materials in order to improve patient safety and the predictability of these materials would be desirable. TRIAL REGISTRATION: Ethical commission of the Ärztekammer Hamburg, Germany (PV5211) as well as by the German Registry of Clinical Studies (DRKS00013010) on 30/07/2018 ( http://apps.who.int/trialsearch/ ).

The History and Future of Basic and Translational Cell-Free DNA Research at a Glance.

We discuss the early history of the structure of DNA and its involvement in gene structure as well as its mobility in and between cells and between tissues in the form of circulating cell-free DNA (cfDNA). This is followed by a view of the present status of the studies on cfDNA and clinical applications of circulating cell-free tumor DNA (ctDNA). The future developments and roles of ctDNA are also considered.

A 3-year prospective randomized clinical trial of alveolar bone crest response and clinical parameters through 1, 2, and 3 years of clinical function of implants placed 4 months after alveolar ridge preservation using two different allogeneic bone-grafting materials.

PURPOSE: The aim of this study was to longitudinally evaluate changes in alveolar bone crest (ABC) levels and differences in resorption rates (RR) between the tested grafting materials following alveolar ridge preservation (ARP) after tooth extraction after 1, 2, and 3 years (T1-T8) of clinical function. METHODS: Patients were randomly assigned to two different bone allografts (group 1 maxgraft®, group 2 Puros®) for ARP. Non-restorable teeth were minimal traumatically extracted. Sockets were augmented with the tested materials and covered with a pericardium membrane. After 4 months of healing, 36 implants were placed and sites were clinically and radiographically monitored in the mesial (ABC-M), the distal (ABC-D, T1-T8), the bucco-lingual (ABC-BL), buccal (ABC-B) and oral (ABC-O) aspect (T1-T4). RESULTS: Changes in (ABC-M), (ABC-D), (ABC-BL), (ABC-B), and (ABC-O) levels showed statistically highly significant differences between T1 and T2 for both bone allografts (p < 0.001). Changes at the ABC-M and ABC-BL levels between T2 and T3 of group 1 showed a statistically significant difference (p < 0.001). Both groups achieved and maintained increased ABC levels without statistically significant differences throughout the monitoring periods of 1-3 years (T6-T8) of clinical function. No failures or adverse events were observed. CONCLUSIONS: To the best of our knowledge, this study is within its limitations the first study to directly compare ABC-changes and differences in RR of two different allogeneic grafting materials for a period of 3 years after ARP. It was demonstrated to be, despite significant differences in RR, a successful method of preserving increased ABC levels through 1, 2, and 3 years of clinical function. Trial registration DRKS00013010, registered 07/30/2018, http://apps.who.int/trialsearch.

Characterization of circulating molecules and activities in plasma of patients after allogeneic and autologous intraoral bone grafting procedures: a prospective randomized controlled clinical trial in humans.

BACKGROUND: The objective was to assess whether intraoral bone augmentation procedures have an impact on the patient's plasma levels of circulating nucleic acids, exosomes, miRNA levels and caspase activities. The null hypothesis was tested, that no significant differences between the two groups will be found. METHODS: In this prospective randomized controlled clinical trial 35 systemically healthy non-smoking participants were randomly allocated using sealed envelopes by a blinded clinician not involved in the clinical setting. Plasma samples were collected preoperatively and 3 times postoperatively (immediately, 5 weeks and 4 months postoperatively). The test group consisted of twenty-five patients who received allogeneic bone grafting material and the control group of ten patients who received autologous bone grafts. Levels of cell-free DNA (cfDNA) and microRNAs (miR-21, miR-27a, miR-218) were quantified by real-time PCR, caspase activities and exosome concentrations were determined by ELISA. RESULTS: Statistical evaluation reveled a significantly higher exosome level before surgery (p = 0.013) and the first postsurgical sample (p = 0.017) in the control group compared to the test group. The levels of miR-27a and miR-218 significantly differed between the plasma samples before surgery and after surgery in both groups. The levels of miR-21 only significantly differed between the pre- and postsurgical plasma samples in the test group, but not in the control group. All patients completed the study, no adverse events were recorded. CONCLUSIONS: Our data show the diagnostic potential of the plasma levels of miR-27a, miR-218 and miR-21 in detecting changes in bone metabolism after alveolar bone augmentation. Our very promising results indicate that there might be a high diagnostic potential in evaluating the plasma levels of the before mentioned miRNAs in order to detect bone resorption activities before they become clinically relevant. Trial registration Ethical commission of the Ärztekammer Hamburg, Germany (PV5211) on 11/03/2016 as well as by the German Registry of Clinical Studies (DRKS 00,013,010) on 30/07/2018 ( http://apps.who.int/trialsearch/ ).

Diagnostic and Prognostic Value of miR-16, miR-146a, miR-192 and miR-221 in Exosomes of Hepatocellular Carcinoma and Liver Cirrhosis Patients.

We aimed to identify a specific microRNA (miRNA) pattern to determine diagnostic and prognostic value in plasma exosomes of hepatocellular carcinoma (HCC) patients. A two-stage study was carried out: exosomal miRNAs were quantified in plasma of HCC patients and healthy individuals by PCR-based microarray cards containing 45 different miRNAs (training cohort). Then, four deregulated miRNAs (miR-16, miR-146a, miR-192, and miR-221) were quantified in the validation analysis using exosomes derived from 85 HCC patients, 50 liver cirrhosis patients, and 20 healthy individuals. Exosomal miR-146a (p = 0.0001), miR-192 (p = 0.002) and miR-221 (p = 0.032) were upregulated only in HCC patients. Repeated 10-fold cross validation showed that miR-146a differentiated HCC from liver cirrhosis patients with AUC of 0.80 ± 0.14 (sensitivity: 81 ± 13%, specificity: 58 ± 22%) in a logistic regression model. High miR-192 presence is associated with poor overall survival (OS) in all HCC patients (p = 0.027) and was predictor of OS in HCC patients in an uni- and multivariate Cox regression model. Moreover, decreased miR-16 levels correlated with OS in liver cirrhosis patients (p = 0.034). Our results emphasized that exosomes secreted into the plasma carry differentially expressed miRNAs of which in particular, miR-192, miR-146, and miR-16 are promising diagnostic and prognostic markers for both HCC and liver cirrhosis patients.

Exosomes in Immune Regulation.

Exosomes, small extracellular vesicles mediate intercellular communication by transferring their cargo including DNA, RNA, proteins and lipids from cell to cell. Notably, in the immune system, they have protective functions. However in cancer, exosomes acquire new, immunosuppressive properties that cause the dysregulation of immune cells and immune escape of tumor cells supporting cancer progression and metastasis. Therefore, current investigations focus on the regulation of exosome levels for immunotherapeutic interventions. In this review, we discuss the role of exosomes in immunomodulation of lymphoid and myeloid cells, and their use as immune stimulatory agents to elicit specific cytotoxic responses against the tumor.

Copy number variations in primary tumor, serum and lymph node metastasis of bladder cancer patients treated with radical cystectomy.

The aim of the present study was to analyze copy number variations (CNV) of multiple oncogenes and tumor suppressor genes in genomic DNA from primary tumor tissue, lymph node metastasis and cell-free DNA (cfDNA) from serum of 72 urothelial carcinoma of bladder (UCB) patients treated with radical cystectomy (RC), using multiplex ligation-dependent probe amplification (MLPA). We hypothesized that primary tumor and lymph node metastasis show similar CNV profiles, and CNV are more present in lymph node metastasis compared to primary tumor tissue. Samples from 43 (59.7%) patients could be analyzed. In total, 35 (83%), 26 (68%) and 8 (42%) patients had CNV in primary tumor, serum and lymph node metastasis, respectively. MYC, CCND1, ERBB2 and CCNE1 displayed the most frequent amplifications. In particular, CNV in ERBB2 was associated with aggressive tumor characteristics. CNV in both ERBB2 and TOP2A were risk factors for disease recurrence. The current findings show that CNV are present in various oncogenes and tumor suppressor genes in genomic DNA from primary tumor, lymph node metastasis and cfDNA from serum. CNV were more present in genomic DNA from primary tumor tissue compared to cfDNA from serum and genomic DNA from lymph node metastasis. Patients with CNV in ERBB2 and TOP2A are at increased risk for disease recurrence following RC. Further studies are necessary to validate, whether these genes may represent promising candidates for targeted-therapy.

A novel assay for exosomal and cell-free miRNA isolation and quantification.

The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.

Predictive value of exosomes and their cargo in drug response/resistance of breast cancer patients.

Exosomes are small extracellular vesicles engaged in intercellular communication in both healthy and tumor cells. When released by the primary tumor, they transfer their cargo including nucleic acids, proteins, and lipids to target cells, thus modulating the character and fate of the recipient cells. By propagating their oncogenic content, exosomes are able to promote tumor progression, angiogenesis, metastases, and drug resistance. Their functions as delivery vehicles of biological material make exosomes promising biomarkers for the early prediction of disease progression and drug resistance in breast cancer, as well as for therapeutic targeting of molecules to treat this deadly disease. In the present review, we accentuate the relevance of exosomes as vehicles of prognostic and predictive markers and target molecules, and describe their potential therapeutic applications as drug cargo suppliers. We made an extensive literature research to clarify the association of their cargo, including exosomal DNA and RNA molecules, with the propagation of drug resistance.

Author Correction: Different signatures of miR-16, miR-30b and miR-93 in exosomes from breast cancer and DCIS patients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Potential microRNA-related targets in clearance pathways of amyloid-ß: novel therapeutic approach for the treatment of Alzheimer's disease.

Imbalance between amyloid-beta (Aß) peptide synthesis and clearance results in Aß deregulation. Failure to clear these peptides appears to cause the development of Alzheimer's disease (AD). In recent years, microRNAs have become established key regulators of biological processes that relate among others to the development and progression of neurodegenerative diseases, such as AD. This review article gives an overview on microRNAs that are involved in the Aß cascade and discusses their inhibitory impact on their target mRNAs whose products participate in Aß clearance. Understanding of the mechanism of microRNA in the associated signal pathways could identify novel therapeutic targets for the treatment of AD.

Histological and immunohistochemical comparison of two different allogeneic bone grafting materials for alveolar ridge reconstruction: A prospective randomized trial in humans.

BACKGROUND: Preclinical studies have hypothesized a possible immunological reponse to allogeneic materials due to detection of remnants of potential immunogenic molecules. However, their impact on integration, bone remodeling and immunological reaction after the augmentation procedure is largely unknown and a direct correlation of analytical data and evaluation of human biopsies is missing. PURPOSE: The present study aimed to compare two commercially available allogeneic materials regarding their content of cellular remnants as well as the bone remodeling, and integration and potential immunologic reactions on a histological and immunohistochemical level, integrating also in vitro analytical evaluation of the specific batches that were used clinically. MATERIALS AND METHODS: Twenty patients were randomly assigned to treatment with Maxgraft or Puros for lateral ridge augmentation in a two-stage surgery. After a mean healing period of 5 months, implants were placed and biopsies were taken for histological, immunhistochemical, and histomorphometrical evaluation regarding bone remodeling and inflammation, protein concentrations in vitro and the presence of MHC molecules of the same batches used clinically. RESULTS: No differences in clinical outcome, histological, immunohistochemical, and in vitro protein analysis between the two bone grafting materials were observed. Active bone remodeling, amount of newly formed bone, and residual grafting material was independent of the materials used, but varied between subjects. MHC1 residues were not detected in any sample. CONCLUSIONS: Within the limitations of this study, both tested materials yielded equivalent results in terms of clinical outcome, new bone formation, and lack of immunological potential on a histological and immunohistochemical level.

Circulating Mitochondrial DNA is Linked to Progression and Prognosis of Epithelial Ovarian Cancer.

As peripheral blood contains fluctuated levels of circulating cell-free mitochondrial DNA (ccf mtDNA), we aimed to evaluate ccf mtDNA as a biomarker for diagnosis and prognosis of epithelial ovarian cancer (EOC). In the present study, we recruited 165 EOC patients and 60 healthy women. Quantitative RT-PCR was applied to amplify 79-bp and 230-bp fragments of the mitochondrial 16 s RNA gene in sera of these participants. MtDNA integrity was defined as the ratio of long to short mtDNA fragments. We observed that the levels of mtDNA79 and mtDNA230 were significantly increased (P = .0001), whereas the mtDNA integrity (P = .0001) was decreased in EOC patients compared with those in healthy controls. MtDNA79 showed a sensitivity of 90.3% and a specificity of 81.7% (AUC = 0.900) to discriminate EOC from healthy controls. Moreover, the amounts of mtDNA79 (P = .0001, P = .012, P = .039) and mtDNA230 (P = .0001, P = .042) continuously raised from healthy controls over FIGO I-II to FIGO III and IV, with highest levels of mtDNA79 (P = .0001) and mtDNA230 (P = .0001) in FIGO III and IV. Increasing levels of mtDNA79 (P = .003, P = .0001) and mtDNA230 (P = .041, P = .0001) were also associated with lymph node metastasis and CA125 values. The higher levels of mtDNA79 (P = .0001; HR 3.2, 95%CI:1.6-6.3) and mtDNA230 (borderline P = .048, HR 0.9, 95%CI:0.9-1.0) also correlated with poor patients' overall survival, of which mtDNA79 could act as an independent factor for overall survival. Our data show a significant association of increasing levels of ccf mtDNA with EOC progress and poor prognosis.

MicroRNA expression studies: challenge of selecting reliable reference controls for data normalization.

Accurate determination of microRNA expression levels is a prerequisite in using these small non-coding RNA molecules as novel biomarkers in disease diagnosis and prognosis. Quantitative PCR is the method of choice for measuring the expression levels of microRNAs. However, a major obstacle that affects the reliability of results is the lack of validated reference controls for data normalization. Various non-coding RNAs have previously been used as reference controls, but their use may lead to variations and lack of comparability of microRNA data among the studies. Despite the growing number of studies investigating microRNA profiles to discriminate between healthy and disease stages, robust reference controls for data normalization have so far not been established. In the present article, we provide an overview of different reference controls used in various diseases, and highlight the urgent need for the identification of suitable reference controls to produce reliable data. Our analysis shows, among others, that RNU6 is not an ideal normalizer in studies using patient material from different diseases. Finally, our article tries to disclose the challenges to find a reference control which is uniformly and stably expressed across all body tissues, fluids, and diseases.

The current role of circulating biomarkers in non-muscle invasive bladder cancer.

Non-muscle invasive bladder cancer (NMIBC) is characterized by its high rate of disease recurrence and relevant disease progression rates. Up to today clinical models are insufficiently predicting outcomes for reliable patient counseling and treatment decision-making. This particularly is a serious problem in patients with high-risk NMIBC who are at high risk for failure of local treatment and thus candidates for early radical cystectomy or even systemic (neoadjuvant) chemotherapy. Next to its clinical variability, bladder cancer is genetically a highly heterogeneous disease. There is an essential need of biomarkers for improving clinical staging, real-time monitoring of disease with or without active treatment, as well as improved outcome prognostication. Liquid biopsies of circulating biomarkers in the blood and urine are promising non-invasive diagnostics that hold the potential facilitating these needs. In this review we report the latest data and evidence on cell-free circulating tumor desoxyribonucleic acid (ctDNA) and circulating tumor cells (CTC) in NMIBC. We summarize their current status in clinical diagnostics, discuss limitations and address future needs.

MicroRNA Shuttle from Cell-To-Cell by Exosomes and Its Impact in Cancer.

The identification of exosomes, their link to multivesicular bodies and their potential role as a messenger vehicle between cancer and healthy cells opens up a new approach to the study of intercellular signaling. Furthermore, the fact that their main cargo is likely to be microRNAs (miRNAs) provides the possibility of the transfer of such molecules to control activities in the recipient cells. This review concerns a brief overview of the biogenesis of both exosomes and miRNAs together with the movement of such structures between cells. The possible roles of miRNAs in the development and progression of breast, ovarian and prostate cancers are discussed.

Characterization of circulating DNA in plasma of patients after allogeneic bone grafting.

OBJECTIVES: Cell-free DNA (cfDNA) harboring mutations has been found in patients with diseases. Experimental studies have shown that cfDNA can be transmitted, leading to transformations in the host. In the present study, we evaluated whether bone allograft material contains cfDNA and whether this foreign cfDNA can be released into the patient's blood circulation. MATERIALS AND METHODS: Plasma samples were collected preoperatively and postoperatively on the same day, at 5 weeks, and 4 months from 25 women who received bone allograft material (test group) from male donors and from 10 women who were treated with autologous graft (control group, only pre- and postoperative samples were collected). DNA was quantified and characterized in bone material and plasma samples by quantitative PCR with primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Y chromosome and gel electrophoresis. DNA in bone material was digested by different concentrations of DNase I. RESULTS: We detected between 1 and 1.8 µg cfDNA fragments at a length around 601 base pairs (bp) and smaller in each 100 mg allograft. Treatment of the allograft with DNase I completely degraded the longer but not the shorter DNA 90-bp fragments. Y-DNA was not detected in the patients' bloodstream at any time during the treatment and follow-up, but elevated levels of circulating cfDNA could be measured immediately postoperatively. CONCLUSIONS: Our results suggest that a transmission of DNA from allografts used for alveolar ridge reconstruction in humans is unlikely. The observed increase in circulating cfDNA in allograft and autograft patients immediately postoperatively may be elicited by the surgical procedure. CLINICAL RELEVANCE: The results support the safety of allograft materials. The results suggest that human allograft materials seem not to release DNA into the blood since we did not measure Y-DNA with our technique.