The aim of this review is to discuss current findings regarding the roles of miRNAs in periodontal diseases and the potential use of saliva as a diagnostic medium for corresponding miRNA investigations. For periodontal disease, investigations have been restricted to tissue samples and five miRNAs, that is, miR-142-3p, miR-146a, miR-155, miR-203, and miR-223, were repeatedly validated in vivo and in vitro by different validation methods. Particularly noticeable are the small sample sizes, different internal controls, and different case definitions of periodontitis in in vivo studies. Beside of that, the validated miRNAs are associated with inflammation and therefore with various diseases. Furthermore, several studies successfully explored the use of salivary miRNA species for the diagnosis of oral cancer. Different cancer types were investigated and heterogeneous methodology was used; moreover, no overlap of resultswas found. In conclusion, fivemiRNAs have consistently been reported for periodontitis; however, their disease specificity, detectability, and expression in saliva and their importance as noninvasive markers are questionable. In principle, a salivary miRNA diagnostic method seems feasible.However, standardized criteria and protocols for preanalytics, measurements, and analysis should be established to obtain comparable results across different studies.

Markéta Sypecká CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years. However, every stage of this disease has significantly higher level of miR-155, which is known as oncomiR. Micro RNAs represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets. We hypothesized that by gene editing of CLL B - cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Method we use for gene editing is CRISPR/Cas9, which enables to delete sequence of mature miR-155 in genome of leukemic B-cells. Methods: CRISPR/Cas9, nucleofection, qRT-PCR, FACS Results:We achieved to isolate clone that bears one allelic deletion (miR-155-/+) in sequence for mature...

The emerging potential of miRNA molecules as diagnostic biomarkers calls for the development of a new quantification method. Current approaches usually require time-consuming and costly miRNA isolation for proper sample analysis. In this thesis, a new, isolation-free, oligonucleotide- modified gold nanoparticle (AuNP/DNA) system is proposed and designed for miRNA detection and quantification in living cells. This cascade, entropy-driven, and enzyme-free amplification system provides fluorescence signal upon selective interaction with the target miRNA. For this purpose, citrate-stabilized gold nanoparticles were synthesized, and their diameters were determined by dynamic light scattering and transmission electron microscopy. The AuNP/DNA conjugates were prepared following a recently published "freezing method". Their reaction kinetics with the target miRNA and selectivity to various miRNAs were compared with those of an analogous DNA system without AuNPs in a series of fluorescence measurements. Furthermore, stability experiments in glutathione environment were conducted, as well as DNA electrophoresis, demonstrating the mechanistic aspects of the reaction. The reaction yields and selectivity to target miRNA of 42.31 ± 2.91 nm AuNP/DNA constructs, containing approximately 25 DNA complexes per AuNP,...

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Acute myeloid leukemia (AML) is a very heterogeneous disease associated with cytogenetic aberrations and genetic mutations. Many of these changes have been revealed and their detection became usual part of the diagnostic process today. However, changes of expression profiles of small, noncoding RNAs, so called microRNAs (miRNAs), are less known and not used for diagnostics yet. These RNAs, 19-24 nucleotides long, take part in the regulation of expression of different genes through complementary base pairing to the 3'non- translated region (3'UTR) of the target messenger RNA (mRNA). They can influence key processes of the cell, like differentiation, proliferation or apoptosis. The changes in expression of different miRNAs are known from different types of cancers. In solid tumors, they are usually detected from bioptic samples; but also plasma samples are now in the center of attention as so called liquid biopsies providing the information about molecular genetic events in the organism. Many studies have revealed deregulated miRNAs in the bone marrow, full blood or isolated progenitor cells (CD34+) of AML patients, only four of them have analyzed plasma samples. We focused on the plasma samples and we targeted on such miRNAs, which levels differ at AML diagnosis and after the chemotherapy. Out of...

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

Cardiac dysfunction is correlated with detrimental prognosis of sepsis and contributes to a high risk of mortality. After an initial hyperinflammatory reaction, most patients enter a protracted state of immunosuppression (late sepsis) that alters both innate and adaptive immunity. The changes of cardiac function in late sepsis are not yet known. MicroRNA-155 (miR-155) is previously found to play important roles in both regulations of immune activation and cardiac function. In this study, C57BL/6 mice were operated to develop into early and late sepsis phases, and miR-155 mimic was injected through the tail vein 48 h after cecal ligation and puncture (CLP). The effect of miR-155 on CLP-induced cardiac dysfunction was explored in late sepsis. We found that increased expression of miR-155 in the myocardium protected against cardiac dysfunction in late sepsis evidenced by attenuating sepsis-reduced cardiac output and enhancing left ventricular systolic function. We also observed that miR-155 markedly reduced the infiltration of macrophages and neutrophils into the myocardium and attenuated the inflammatory response via suppression of JNK signaling pathway. Moreover, overexpression of β-arrestin 2 (Arrb2) exacerbated the mice mortality and immunosuppression in late sepsis. Furthermore, transfection of miR-155 mimic reduced Arrb2 expression, and then restored immunocompetence and improved survival in late septic mice. We conclude that increased miR-155 expression through systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling and Arrb2 mediated immunosuppression.

Background: Myocardial apoptosis plays an important role in myocardial ischemia/reperfusion (I/R) injury. Activation of PI3K/Akt signaling protects the myocardium from I/R injury. This study investigated the role of miR-214 in hypoxia/ reoxygenation (H/R)-induced cell damage in vitro and myocardial I/R injury in vivo. Methods and Results: H9C2 cardiomyoblasts were transfected with lentivirus expressing miR-214 (LmiR-214) or lentivirus expressing scrambled miR-control (LmiR-control) respectively, to establish cell lines of LmiR-214 and LmiR-control. The cells were subjected to hypoxia for 4 h followed by reoxygenation for 24 h. Transfection of LmiR-214 suppresses PTEN expression, significantly increases the levels of Akt phosphorylation, markedly attenuates LDH release, and enhances the viability of the cells subjected to H/R. In vivo transfection of mouse hearts with LmiR-214 significantly attenuates I/R induced cardiac dysfunction and reduces I/Rinduced myocardial infarct size. LmiR-214 transfection significantly attenuates I/Rinduced myocardial apoptosis and caspase-3/7 and caspase-8 activity. Increased expression of miR-214 by transfection of LmiR-214 suppresses PTEN expression, increases the levels of phosphorylated Akt, represses Bim1 expression and induces Bad phosphorylation in the myocardium. In addition, in vitro data shows transfection of miR-214 mimics to H9C2 cells suppresses the expression and translocation of Bim1 from cytosol to mitochondria and induces Bad phosphorylation. Conclusions: Our in vitro and in vivo data suggests that miR-214 protects cells from H/R induced damage and attenuates I/R induced myocardial injury. The mechanisms involve activation of PI3K/Akt signaling by targeting PTEN expression, induction of Bad phosphorylation, and suppression of Bim1 expression, resulting in decreases in I/R-induced myocardial apoptosis.

Pancreatic cancer (PC) is a highly aggressive disease with unmet therapeutic needs. Recent advances in the use of oncolytic viruses (OVs) as cancer therapeutic agents bring new hope to fight the notorious disease that is PC. Although OVs have shown promising results in certain cancers, some tumors remain resistant to OV therapy due to their inherent residual antiviral mechanisms. We hypothesized that the use of OV-encoded artificial microRNAs (amiRNAs) could help target the cellular antiviral components associated with the observed OV resistance and could also sensitize neighboring tumor cells to OV therapy and small molecule inhibitors through the secretion of amiRNA-containing extracellular vesicles (EVs) from infected cells. To find such amiRNAs, a viral surrogate library encoding ~16,000 unique amiRNAs was passaged in pancreatic cancer cell lines to enrich for sequences that could enhance OV replication. An amiRNA that improves PC cell killing when expressed from an OV was identified. Target identification of this amiRNA (amiR-4) revealed ARID1A as a key player in resistance to OV therapy in pancreatic cancers. This target is of particular interest, since its downregulation acts in a synthetic lethal fashion with inhibition of the EZH2 methyltransferase. Combining VSV51-amiR-4 with a small molecule inhibitor of EZH2 enhances PC cell death. Moreover, amiR-4 is packaged in cancer cell-secreted EVs which can reach neighboring naïve cells to sensitize them to EZH2 inhibition-mediated cell death and to spread the OV-mediated tumor killing effect throughout the tumor. This data translates into tumor debulking and survival in animal models of highly aggressive PC. This work not only broadens our knowledge on the resistance of select tumors to oncolytic virotherapy and the EV-mediated bystander killing effect in OV-infected tumors, but it also establishes OVs as a novel tool to produce anti-cancer therapeutic EVs in situ to improve therapeutic gain. Ultimately, our work provides new hope for a cure to the grim disease that is PC.