Non-coding RNAs in disease: from mechanisms to therapeutics.

Non-coding RNAs (ncRNAs) are a heterogeneous group of transcripts that, by definition, are not translated into proteins. Since their discovery, ncRNAs have emerged as important regulators of multiple biological functions across a range of cell types and tissues, and their dysregulation has been implicated in disease. Notably, much research has focused on the link between microRNAs (miRNAs) and human cancers, although other ncRNAs, such as long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are also emerging as relevant contributors to human disease. In this Review, we summarize our current understanding of the roles of miRNAs, lncRNAs and circRNAs in cancer and other major human diseases, notably cardiovascular, neurological and infectious diseases. Further, we discuss the potential use of ncRNAs as biomarkers of disease and as therapeutic targets.

Comparative Evaluation of the Repair Bond Strength of Dental Resin Composite after Sodium Bicarbonate or Aluminum Oxide Air-Abrasion.

The dental prophylactic cleaning of a damaged resin-based composite (RBC) restoration with sodium bicarbonate can change the surface characteristics and influence the repair bond strength. The purpose of this study was to compare the effect of sodium bicarbonate (SB) and aluminum oxide (AO) surface treatments on the microtensile bond strength (µTBS) of repaired, aged RBC. Bar specimens were prepared from microhybrid RBC and aged in deionized water for 8 weeks. Different surface treatments (AO air-abrasion; SB air-polishing), as well as cleaning (phosphoric acid, PA; ethylene-diamine-tetraacetic-acid, EDTA) and adhesive applications (single bottle etch-and-rinse, ER; universal adhesive, UA), were used prior to the application of the repair RBC. Not aged and aged but not surface treated RBCs were used as positive and negative controls, respectively. The repaired blocks were cut into sticks using a precision grinding machine. The specimens were tested for tensile fracture and the µTBS values were calculated. Surface characteristics were assessed using scanning electron microscopy. AO-PA-UA (62.6 MPa) showed a 20% increase in µTBS compared to the NC (50.2 MPa), which proved to be the most significant. This was followed by SB-EDTA-UA (58.9 MPa) with an increase of 15%. In addition to AO-PA-UA, SB-EDTA-UA could also be a viable alternative in the RBC repair protocol.

Standardisation of protocols can be crucial in long non-coding RNA research.

In this issue, Traversa et al. [1] reviewed our current knowledge about the role of circular and linear forms of PVT1 non-coding RNA in cancer and human diseases. They highlighted the technical challenges of these studies and raised a potential bias in the publications, which require more attention from researchers.

The effect of dual inhibition of Ras-MEK-ERK and GSK3ß pathways on development of in vitro cultured rabbit embryos.

Dual inhibition (2i) of Ras-MEK-ERK and GSK3ß pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFß/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFß/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

[The role of miRNAs in the pathogenesis of pituitary adenomas]. / A mikro-RNS-ek szerepe az agyalapimirigy-daganatok tumorbiológiájában.

MicroRNAs (miRNAs) are short, single stranded RNA molecules which play regulatory roles through posttranscriptional regulation of their target genes. Based on our current knowledge, more than 30% of the human protein-coding genes are regulated by miRNAs, hence influencing basic cellular mechanisms including cell proliferation, differentiation and cell death. Differential miRNA expression pattern has been detected in many different types of tumors and, recently, several publications have referred to miRNAs as potential therapeutic targets. Through adjustment of miRNA levels by artificial miRNAs administration or miRNA inhibition, we can influence not only one target gene but also complex biological pathways. Pituitary adenoma is the second most frequent intracranial tumor. In spite of this, the molecular mechanism of the pituitary adenoma formation is not yet entirely revealed. Recently, more and more evidences have been found suggesting that miRNAs have an important role in pituitary adenoma pathogenesis. Here, we summarize the recent results related to this role and highlight the therapeutic potentials in pituitary adenomas. Orv Hetil. 2018; 159(7): 252-259.

miRNA Biology in Chronic Lymphocytic Leukemia.

microRNAs (miRNAs) are a class of small non-coding RNAs that play a crucial regulatory role in fundamental biological processes and have been implicated in various diseases, including cancer. The first evidence of the cancer-related function of miRNAs was discovered in chronic lymphocytic leukemia (CLL) in the early 2000s. Alterations in miRNA expression have since been shown to strongly influence the clinical course, prognosis, and response to treatment in patients with CLL. Therefore, the identification of specific miRNA alterations not only enhances our understanding of the molecular mechanisms underlying CLL but also holds promise for the development of novel diagnostic and therapeutic strategies. This review aims to provide a comprehensive summary of the current knowledge and recent insights into miRNA dysregulation in CLL, emphasizing its pivotal roles in disease progression, including the development of the lethal Richter syndrome, and to provide an update on the latest translational research in this field.

Histological diagnosis determines complications of percutaneous renal biopsy: a single-center experience in 353 patients.

BACKGROUND: We studied the connection between complication occurrence related to renal biopsies and histological diagnoses of the biopsy specimen. We also analyzed the distribution of diagnoses in our population. METHODS: We retrospectively studied 353 patients undergoing renal biopsy at the same center. Biopsies were performed after marking the site of puncture by ultrasound imaging. Connection of complications with diagnoses and clinical parameters was evaluated. RESULTS: Complication rate was 44.5% in our study. There was a significantly lower rate of complications in patients with diabetic nephropathy (likelihood ratio, LR = 0.44) or acute tubular necrosis (LR = 0.38), while patients with thin basement membrane syndrome had a more than 6-fold higher risk for development of intrarenal hemorrhage than others. Patients with vasculitis (LR = 2.88) and acute interstitial nephritis (LR = 3.18) have a more than doubled risk for arteriovenous shunts, while in patients with severe arteriosclerosis the prevalence of this complication was lower (LR = 0.46). Arteriovenous shunts developed also at a significantly higher rate in patients with rapidly progressive glomerulonephritis. CONCLUSION: Patients with thin basement membrane syndrome, vasculitis, rapidly progressive glomerulonephritis or acute interstitial nephritis should be observed more carefully after renal biopsy due to the significantly higher risk for certain complications.

Effect of Air-Polishing and Different Post-Polishing Methods on Surface Roughness of Nanofill and Microhybrid Resin Composites.

Air-abrasion is a popular prophylactic procedure to maintain oral hygiene. However, depending on the applied air-abrasive powder, it can damage the surface of the tooth and restorations, making it susceptible to plaque accumulation. The purpose of this study was to investigate the effect of 5 s and 10 s air-abrasion of calcium carbonate on surface roughness (Ra) of enamel, nanofill, and microhybrid resin-composites and the effect of post-polishing with two-step rubber- (RP) or one-step brush polisher (BP) to re-establish the surface smoothness. Surface topography was visualized by scanning-electron-microscopy. The quantitative measurement of the Ra was carried out with atomic-force-microscopy. Air-abrasion for 10 s decreased the Ra of enamel as a result of abrasion of the natural surface texture. Post-polishing with RP after 10 s air-abrasion did not change the Ra or BP; however, Ra was increased significantly by scratching the surface. Air-abrasion increased the Ra of resin composites significantly, irrespective of the application time. While RP provided a similarly smooth surface to the control in the case of microhybrid resin composite, BP increased the Ra significantly. The Ra for the control group of the nanofill-resin composite was initially high, which was further increased by air-abrasion. RP and BP re-established the initial Ra with deeper and shallower scratches after BP. Both the material and treatment type showed a large effect on Ra.

Aspirin Mediates Its Antitumoral Effect Through Inhibiting PTTG1 in Pituitary Adenoma.

CONTEXT: DNA demethylation and inhibitory effects of aspirin on pituitary cell proliferation have been demonstrated. OBJECTIVE: Our aim was to clarify the molecular mechanisms behind the aspirin-related effects in pituitary cells. METHODS: DNA methylome and whole transcriptome profile were investigated in RC-4B/C and GH3 pituitary cell lines upon aspirin treatment. Effects of aspirin and a demethylation agent, decitabine, were further tested in vitro. PTTG1 expression in 41 human PitNET samples and whole genome gene and protein expression data of 76 PitNET and 34 control samples (available in Gene Expression Omnibus) were evaluated. RESULTS: Aspirin induced global DNA demethylation and consequential transcriptome changes. Overexpression of Tet enzymes and their cofactor Uhrf2 were identified behind the increase of 5-hydroxymethylcytosine (5hmC). Besides cell cycle, proliferation, and migration effects that were validated by functional experiments, aspirin increased Tp53 activity through p53 acetylation and decreased E2f1 activity. Among the p53 controlled genes, Pttg1 and its interacting partners were downregulated upon aspirin treatment by inhibiting Pttg1 promoter activity. 5hmC positively correlated with Tet1-3 and Tp53 expression, and negatively correlated with Pttg1 expression, which was reinforced by the effect of decitabine. Additionally, high overlap (20.15%) was found between aspirin-regulated genes and dysregulated genes in PitNET tissue samples. CONCLUSION: A novel regulatory network has been revealed, in which aspirin regulated global demethylation, Tp53 activity, and Pttg1 expression along with decreased cell proliferation and migration. 5hmC, a novel tissue biomarker in PitNET, indicated aspirin antitumoral effect in vitro as well. Our findings suggest the potential beneficial effect of aspirin in PitNET.

Effects of mono- and dual blockade of the renin-angiotensin system on markers of cardiovascular status in hypertensive patients with mild and moderate renal failure.

BACKGROUND/AIMS: Dual renin-angiotensin system (RAS) blockade has no more efficiency to decrease cardiovascular mortality than mono-blockade. Our goal was to explore differences between other cardiovascular markers in patients with RAS blockade. METHODS: We analyzed two groups of patients treated with a long-term ACE inhibitor (MONO-group, n = 20) and an ACE inhibitor and angiotensin II receptor blocker (DUAL-group, n = 15). Ambulatory blood pressure monitoring, echocardiography, arterial stiffness and levels of catecholamine, endogenous ouabain (EO), pro-brain natriuretic peptide and more types of urinary albumin measurements were performed. RESULTS: In the DUAL-group, we found significantly better cardiac parameters, but the levels of EO and urinary albumins were similar in both groups. The level of EO correlates with nighttime mean arterial blood pressure (R = 0.556, p = 0.032) and arterial ß-stiffness (R = 0.512, p = 0.042). Urinary immuno-unreactive albumin showed a relationship with diastolic dysfunction of the heart (R = -0.508, p = 0.045) diurnal index of diastolic blood pressure (R = -0.569, p = 0.021) in the MONO-group. CONCLUSION: Cardiac parameters were more prosperous in the DUAL-group, but the levels of EO did not differ between groups. The level of EO correlated with blood pressure and arterial stiffness markers in the MONO-group only. The urinary immuno-unreactive albumin may be a new marker of cardiovascular conditions.

Three Dimensional Cell Culturing for Modeling Adrenal and Pituitary Tumors.

In vitro monolayer conditions are not able to reproduce the complexity of solid tumors, still, there is scarce information about the 3D cell culture models of endocrine tumor types. Therefore, our aim was to develop in vitro 3D tumor models by different methodologies for adrenocortical carcinoma (H295R), pituitary neuroendocrine tumor (RC-4B/C and GH3) and pheochromocytoma (PC-12). Various methodologies were tested. Cell biological assays (cell viability, proliferation and live cell ratio) and steroid hormone production by HPLC-MS/MS method were applied to monitor cellular well-being. Cells in hanging drops and embedded in matrigel formed multicellular aggregates but they were difficult to handle and propagate for further experiments. The most widely used methods: ultra-low attachment plate (ULA) and spheroid inducing media (SFDM) were not the most viable 3D model of RC-4B/C and GH3 cells that would be suitable for further experiments. Combining spheroid generation with matrigel scaffold H295R 3D models were viable for 7 days, RC-4B/C and GH3 3D models for 7-10 days. ULA and SFDM 3D models of PC-12 cells could be used for further experiments up to 4 days. Higher steroid production in 3D models compared to conventional monolayer culture was detected. Endocrine tumor cells require extracellular matrix as scaffold for viable 3D models that can be one reason behind the lack of the usage of endocrine 3D cultures. Our models help understanding the pathogenesis of endocrine tumors and revealing potential biomarkers and therapeutic targets. They could also serve as an excellent platform for preclinical drug test screening.

Demethylation Status of Somatic DNA Extracted From Pituitary Neuroendocrine Tumors Indicates Proliferative Behavior.

BACKGROUND: Cytosine intermediaries 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), epigenetic hallmarks, have never been investigated in pituitary neuroendocrine tumors (PitNET). OBJECTIVE: To examine methylation-demethylation status of global deoxyribonucleic acid (DNA) in PitNET tissues and to assess its correlation with clinical and biological parameters. MATERIALS AND METHODS: Altogether, 57 PitNET and 25 corresponding plasma samples were collected. 5mC and 5hmC were investigated using liquid chromatography-tandem mass spectrometry. Expression of DNA methyltransferase 1 (DNMT1); tet methylcytosine dioxygenase 1 through 3 (TET1-3); and ubiquitin-like, containing PHD and RING finger domains 1 and 2 (UHRF1-2) were measured by reverse transcription-polymerase chain reaction. Levels of 5hmC and UHRF1-2 were explored by immunohistochemistry. Effect of demethylating agent decitabine was tested on pituitary cell lines. RESULTS: 5hmC/5mC ratio was higher in less differentiated PitNET samples. A negative correlation between Ki-67 proliferation index and 5hmC, 5hmC to 5mC ratio were revealed. Higher 5mC was observed in SF-1 + gonadotroph adenomas with a higher Ki-67 index. Expressions of TET2 and TET3 were significantly higher in adenomas with higher proliferation rate. UHRF1 showed gradually increased expression in higher proliferative adenoma samples, and a significant positive correlation was detected between UHRF2 expression and 5hmC level. Decitabine treatment significantly decreased 5mC and increased 5hmC levels in both cell lines, accompanied with decreased cell viability and proliferation. CONCLUSION: The demethylation process negatively correlated with proliferation rate and the ratio of 5hmC to 5mC was higher in less differentiated adenomas. Therefore, epigenetic markers can be potential biomarkers for PitNET behavior. Altering the epigenome in adenoma cells by decitabine decreased proliferation, suggesting that this treatment might be a novel medical treatment for PitNET.

Comprehensive Analysis of Circulating miRNAs in the Plasma of Patients With Pituitary Adenomas.

BACKGROUND: Circulating miRNAs in pituitary adenomas would improve patient care, especially as minimally invasive biomarkers of tumor recurrence and progression in nonfunctioning adenoma cases. AIM: Our aim was to investigate plasma miRNA profiles in patients with pituitary adenomas. MATERIALS AND METHODS: A total of 149 plasma and extracellular vesicle (preoperative, early postoperative, and late postoperative) samples were collected from 45 patients with pituitary adenomas. Adenomas were characterized on the basis of anterior pituitary hormones and transcription factors by immunostaining. miRNA next-generation sequencing was performed on 36 samples (discovery set). Individual TaqMan assays were used for validation on an extended sample set. Pituitary adenoma tissue miRNAs were evaluated by TaqMan array and data in the literature. RESULTS: Global downregulation of miRNA expression was observed in plasma samples of pituitary adenomas compared with normal samples. Expression of 29 miRNAs and isomiR variants were able to distinguish preoperative plasma samples from normal controls. miRNAs with altered expression in both plasma and different adenoma tissues were identified. Three, seven, and 66 miRNAs expressed differentially between preoperative and postoperative plasma samples in GH-secreting, FSH/LH+, and hormone-immunonegative groups, respectively. miR‒143-3p was downregulated in late postoperative but not in early postoperative plasma samples compared with preoperative ones exclusively in FSH/LH+ adenomas. The plasma level of miR‒143-3p discriminated these samples with 81.8% sensitivity and 72.3% specificity (area under the curve = 0.79; P = 0.02). CONCLUSIONS: Differentially expressed miRNAs in pituitary adenoma tissues have low abundance in plasma, minimizing their role as biomarkers. Plasma miR‒143-3p level decreased in patients with FSH/LH+ adenomas, indicating successful surgery, but its application for evaluating tumor recurrence needs further investigation.

Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas.

Acetylsalicylic acid (ASA) is known as a cancer preventing agent, but there is no data available regarding the effect of ASA on pituitary cells. We investigated 66 nonfunctioning (NFPA) and growth hormone (GH)-producing adenomas and 15 normal pituitary samples. Functional assays (cell viability, proliferation, flow cytometry cell cycle analysis, caspase-3 activation and DNA degradation) were applied to explore the effect of ASA, YM155 (survivin inhibitor), survivin-targeting siRNA and TNF-related apoptosis-inducing ligand (TRAIL) in RC-4B/C and GH3 cells. Pituitary adenoma xenografts were generated in immunocompromised mice. We found that survivin was overexpressed and TRAIL was downregulated in NFPAs compared to normal pituitary tissue. ASA decreased proliferation but did not induce apoptosis in pituitary cells. Additionally, ASA treatment decreased cells in S phase and increased cells in G2/M phase of the cell cycle. Inhibition of survivin using an inhibitor or siRNA-mediated silencing reversed the ASA-induced growth inhibition partially. In addition, we also found survivin-independent effects of ASA on the cell cycle that were mediated through inhibition of cyclin A, cyclin dependent kinase 2 (CDK2) and phospho-CDK2. We also aimed to test the effect of acetylsalicylic acid in an animal model using RC-4 B/C cells, but in contrast to GH3 cells, RC-4 B/C cells failed to adhere and grow a xenograft. We concluded that ASA inhibited the growth of pituitary adenoma cells. Survivin inhibition is a key mechanism explaining its antineoplastic effects. Our results suggest that inhibition of survivin with small molecules or ASA could serve as potential therapeutic agents in NFPA.

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas.

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially regulated by miRNAs.

MEN1 mutations and potentially MEN1-targeting miRNAs are responsible for menin deficiency in sporadic and MEN1 syndrome-associated primary hyperparathyroidism.

Inherited, germline mutations of menin-coding MEN1 gene cause multiple endocrine neoplasia type 1 (MEN1), while somatic MEN1 mutations are the sole main driver mutations in sporadic primary hyperparathyroidism (PHPT), suggesting that menin deficiency has a central role in the pathogenesis of PHPT. MiRNAs are small, noncoding RNAs posttranscriptionally regulating gene expression. Our aim was to investigate both the role of MEN1 mutations and potentially MEN1-targeting miRNAs as the underlying cause of menin deficiency in MEN1-associated and sporadic PHPT tissues. Fifty six PHPT tissues, including 16 MEN1-associated tissues, were evaluated. Diagnosis of MEN1 syndrome was based on identification of germline MEN1 mutations. In silico target prediction was used to identify miRNAs potentially targeting MEN1. Menin expression was determined by immunohistochemistry while expression of miRNAs was analyzed by quantitative real-time PCR. Sporadic PHPT tissues were subjected to somatic MEN1 mutation analysis as well. Lack of nuclear menin was identified in all MEN1-associated and in 28% of sporadic PHPT tissues. Somatic MEN1 mutations were found in 25% of sporadic PHPTs. The sensitivity and specificity of menin immunohistochemistry to detect a MEN1 mutation were 86 and 87%, respectively. Expression levels of hsa-miR-24 and hsa-miR-28 were higher in sporadic compared to MEN1-associated PHPT tissues; however, no difference in miRNA levels occurred between menin-positive and menin-negative PHPT tissues. Menin deficiency is the consequence of a MEN1 mutation in most menin-negative PHPT tissues. Elevated expression of hsa-miR-24 and hsa-miR-28 mark the first epigenetic changes observed between sporadic and MEN1-associated PHPT.