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Background and objectives: Sex determination of human or human skeletal remains is considered an initial step in its identification. In cases of fragmented body, it is difficult to identify the body. The mastoid process characteristics are of great utility in the diagnosis of sex. The purpose of this study was to evaluate the significance for sex determination by the measurement of the area formed by 3 craniometric points related to the mastoid process. Method: 100 skulls, 50 male and 50 female, were analyzed. The three craniometric points were marked to demarcate a triangle on the mastoid process. The area (mm2) of the mastoid triangle for each side of the skull was obtained, and the total value of these measures (T) was calculated. Results: The mean total area of mastoid triangle in males was 1393.88 ± 121.51 mm2 and in females 1095.88 ± 90.11 mm2. The analysis of the differences between males and females in right (R), left (L) and total areas (T) was statistically significant (p value < 0.001). The overlapping of the values for the total area between male and female skull was 14%. Conclusion and interpretation: The total areas show less overlapping of the values between the sexes and significant results were obtained in 3 studied areas R, L and T. Hence, this can be used for sexing human skulls. The values of the total area that were greater than or equal to 1360.48 mm2 belonged to male skull and that were less than or equal to 1120.85 mm2 belonged to female skull. [ABSTRACT FROM AUTHOR]

Purpose: Changes to the cellular expression of numerous microRNAs (miRs) have been implicated in disease development. MiR-1 expression is down-regulated in human heart failure (HF) and our in vivo rat HF model, we aimed to further understand its significance in HF. We previously showed that miR-1 expression was restored by in vitro adenovirus-mediated restoration of SERCA2a in adult rat HF cardiomyocytes, in response to diastolic calcium changes.Methods: Adult rats that underwent surgical ligation of the left anterior descending coronary artery to induce myocardial infarction (>30%) were maintained 16-20 weeks until HF developed. Cardiomyocytes were isolated for in vitro transfection by lipofection with miR-1 constructs followed by 48 hours culture. We examined cardiomyocyte calcium handling, contractile function, and gene expression analysis by quantitative RT-PCR.Results: The contractile beta-adrenoceptor (βAR) response to isoprenaline was depressed in cardiomyocytes from HF rats, but was restored in HF cardiomyocytes by increasing miR-1 expression (Figure 1) with a mature miR-1 construct. Selective pharmacological inhibition revealed that SERCA2a function was unchanged, and that it was an elevated β2AR response that had improved cell shortening in the HF cardiomyocytes to levels comparable to healthy. β2AR expression was unchanged after pre miR-1 treatment, implying it is the functional signalling of the receptor that is influenced by miR-1.Conclusion: Restoration of miR-1 reverses HF-induced βAR desensitisation. We suggest that the SERCA2a down-regulation and consequent increase of diastolic calcium may signal through miR-1 reduction to produce βAR signalling alterations in HF.miR-1 Restores Beta-AR Response in HF [ABSTRACT FROM AUTHOR]

Understanding RNA-protein interactions is crucial for deciphering the cellular functions and molecular mechanisms of regulatory RNAs. Consequently, there is a constant need to develop innovative and cost-effective methods to uncover such interactions. We developed a simple and cost-effective technique called Multiple Oligo assisted RNA Pulldown via Hybridization (MORPH) to identify proteins interacting with a specific RNA. MORPH employs a tiling array of antisense oligos (ASOs) to efficiently capture the RNA of interest along with proteins associated with it. Unlike existing techniques that rely on multiple individually biotinylated oligos spanning the entire RNA length, MORPH stands out by utilizing a single biotinylated oligo to capture all the ASOs. To evaluate MORPH's efficacy, we applied this technique combined with mass spectrometry to identify proteins interacting with lncRNA NEAT1, which has previously been studied using various methods. Our results demonstrate that despite being a simple and inexpensive procedure, MORPH performs on par with existing methods. Abbreviations : ASO, Antisense oligo; lncRNA, long non-coding RNA; MORPH, Multiple Oligo assisted RNA Pulldown via Hybridization.

Introduction: β-adrenergic stimulation using β-agonists such as isoproterenol has been routinely used to induce cardiac fibrosis in experimental animal models. Although transcriptome changes in surgical models of cardiac fibrosis such as transverse aortic constriction (TAC) and coronary artery ligation (CAL) are well-studied, transcriptional changes during isoproterenol-induced cardiac fibrosis are not well-explored. Methods: Cardiac fibrosis was induced in male C57BL6 mice by administration of isoproterenol for 4, 8, or 11 days at 50 mg/kg/day dose. Temporal changes in gene expression were studied by RNA sequencing. Results and discussion: We observed a significant alteration in the transcriptome profile across the different experimental groups compared to the saline group. Isoproterenol treatment caused upregulation of genes associated with ECM organization, cell-cell contact, three-dimensional structure, and cell growth, while genes associated with fatty acid oxidation, sarcoplasmic reticulum calcium ion transport, and cardiac muscle contraction are downregulated. A number of known long non-coding RNAs (lncRNAs) and putative novel lncRNAs exhibited differential regulation. In conclusion, our study shows that isoproterenol administration leads to the dysregulation of genes relevant to ECM deposition and cardiac contraction, and serves as an excellent alternate model to the surgical models of heart failure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Nanda, Pant, Machha, Sowpati and Kumarswamy.)

Abdominal aortic aneurysms (AAAs) are asymptomatic vascular diseases that have life-threatening outcomes. Smooth muscle cell (SMC) dysfunction plays an important role in AAA development. The contribution of non-coding genome, specifically the role of long non-coding RNAs (lncRNAs) in SMC dysfunction, is relatively unexplored. We investigated the role of lncRNA TUG1 in SMC dysfunction. To identify potential lncRNAs relevant to SMC functionality, lncRNA profiling was performed in angiotensin-II-treated SMCs. AAA was induced by angiotensin-II treatment in mice. Transcriptional regulation of TUG1 was studied using promoter luciferase and chromatin-immuno-precipitation experiments. Gain-or-loss-of-function experiments were performed in vitro to investigate TUG1-mediated regulation of SMC function. Immunoprecipitation experiments were conducted to elucidate the mechanism underlying TUG1-mediated SMC dysfunction. TUG1 was upregulated in SMCs following angiotensin-II treatment. Similarly, TUG1 levels were elevated in abdominal aorta in a mouse model of angiotensin-II-induced AAA. Further investigations showed that angiotensin-II-induced TUG1 expression could be suppressed by inhibiting Notch-signaling pathway, both in vitro and in mouse AAA model and that TUG1 is a direct transcriptional target of the Notch pathway. In aneurysmal tissues, TUG1 expression was inversely correlated with the expression of SMC contractile genes. Overexpression of TUG1 repressed SMC differentiation in vitro, whereas siRNA/shRNA-mediated TUG1 knockdown showed an opposite effect. Mechanistically, TUG1 interacts with transcriptional repressor KLF4 and facilitates its recruitment to myocardin promoter ultimately leading to the repression of SMC differentiation. In summary, our study uncovers a novel role for the lncRNA TUG1 wherein it modulates SMC differentiation via the KLF4-myocardin axis, which may have potential implications in AAA development. NEW & NOTEWORTHY TUG1 is an angiotensin-II-induced long noncoding RNA that mediates smooth muscle cell (SMC) dysfunction through interaction with transcriptional repressor KLF4.

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR&#95;015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach.

Chronic cardiac stress induces pathologic hypertrophy and fibrosis of the myocardium. The microRNA-29 (miR-29) family has been found to prevent excess collagen expression in various organs, particularly through its function in fibroblasts. Here, we show that miR-29 promotes pathologic hypertrophy of cardiac myocytes and overall cardiac dysfunction. In a mouse model of cardiac pressure overload, global genetic deletion of miR-29 or antimiR-29 infusion prevents cardiac hypertrophy and fibrosis and improves cardiac function. Targeted deletion of miR-29 in cardiac myocytes in vivo also prevents cardiac hypertrophy and fibrosis, indicating that the function of miR-29 in cardiac myocytes dominates over that in non-myocyte cell types. Mechanistically, we found cardiac myocyte miR-29 to de-repress Wnt signaling by directly targeting four pathway factors. Our data suggests that, cell- or tissue-specific antimiR-29 delivery may have therapeutic value for pathological cardiac remodeling and fibrosis.

Recent studies highlighted long noncoding RNAs (lncRNAs) to play an important role in cardiac development. However, understanding of lncRNAs in cardiac diseases is still limited. Global lncRNA expression profiling indicated that several lncRNA transcripts are deregulated during pressure overload-induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy-associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)-operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell-derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain-containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases., (Copyright © 2016, American Association for the Advancement of Science.)

Aims: Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process., Methods and Results: OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo., Conclusion: In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objective: MicroRNAs (miRNA/miR) are stably present in body fluids and are increasingly explored as disease biomarkers. Here, we investigated influence of impaired wound healing on the plasma miRNA signature and their functional importance in patients with type 2 diabetes mellitus., Approach and Results: miRNA array profiling identified 41 miRNAs significantly deregulated in diabetic controls when compared with patients with diabetes mellitus-associated peripheral arterial disease and chronic wounds. Quantitative real-time polymerase chain reaction validation confirmed decrease in circulating miR-191 and miR-200b levels in type 2 diabetic versus healthy controls. This was reverted in diabetic subjects with associated peripheral arterial disease and chronic wounds, who also exhibited higher circulating C-reactive protein and proinflammatory cytokine levels compared with diabetic controls. miR-191 and miR-200b were significantly correlated with C-reactive protein or cytokine levels in patients with diabetes mellitus. Indeed, proinflammatory stress increased endothelial- or platelet-derived secretion of miR-191 or miR-200b. In addition, dermal cells took up endothelial-derived miR-191 leading to downregulation of the miR-191 target zonula occludens-1. Altered miR-191 expression influenced angiogenesis and migratory capacities of diabetic dermal endothelial cells or fibroblasts, respectively, partly via its target zonula occludens-1., Conclusions: This study reports that (1) inflammation underlying nonhealing wounds in patients with type 2 diabetes mellitus influences plasma miRNA concentrations and (2) miR-191 modulates cellular migration and angiogenesis via paracrine regulation of zonula occludens-1 to delay the tissue repair process., (© 2015 American Heart Association, Inc.)

Background: Long noncoding RNAs (lncRNAs) are novel intracellular noncoding ribonucleotides regulating gene expression. Intriguingly, these RNA transcripts are detectable and stable in the blood of patients with cancer and cardiovascular disease. We tested whether circulating lncRNAs in plasma of critically ill patients with acute kidney injury (AKI) at inception of renal replacement therapy were deregulated and might predict survival., Methods: We performed a global lncRNA expression analysis using RNA isolated from plasma of patients with AKI, healthy controls, and ischemic disease controls. This global screen revealed several deregulated lncRNAs in plasma samples of patients with AKI. lncRNA-array-based alterations were confirmed in kidney biopsies of patients as well as in plasma of 109 patients with AKI, 30 age-matched healthy controls, and 30 disease controls by quantitative real-time PCR., Results: Circulating concentrations of the novel intronic antisense lncRNA TrAnscript Predicting Survival in AKI (TapSAKI) (P < 0.0001) were detectable in kidney biopsies and upregulated in plasma of patients with AKI. Cox regression and Kaplan-Meier curve analysis revealed TapSAKI as an independent predictor of 28-day survival (P < 0.01). TapSAKI was enriched in tubular epithelial cells subjected to ATP depletion (P = 0.03)., Conclusions: The alteration of circulating concentrations of lncRNAs in patients with AKI supports TapSAKI as a predictor of mortality in this patient cohort., (© 2014 American Association for Clinical Chemistry.)

Rationale: Many processes in endothelial cells including angiogenic responses are regulated by microRNAs. However, there is limited information available about their complex cross-talk in regulating certain endothelial functions., Aim: The objective of this study is to identify endothelial functions of the pro-hypertrophic miR-212/132 cluster and its cross-talk with other microRNAs during development and disease., Methods and Results: We here show that anti-angiogenic stimulation by transforming growth factor-beta activates the microRNA-212/132 cluster by derepression of their transcriptional co-activator cAMP response element-binding protein (CREB)-binding protein (CBP) which is a novel target of a previously identified pro-angiogenic miRNA miR-30a-3p in endothelial cells. Surprisingly, despite having the same seed-sequence, miR-212 and miR-132 exerted differential effects on endothelial transcriptome regulation and cellular functions with stronger endothelial inhibitory effects caused by miR-212. These differences could be attributed to additional auxiliary binding of miR-212 to its targets. In vivo, deletion of the miR-212/132 cluster increased endothelial vasodilatory function, improved angiogenic responses during postnatal development and in adult mice., Conclusion: Our results identify (i) a novel miRNA-cross-talk involving miR-30a-3p and miR-212, which led to suppression of important endothelial genes such as GAB1 and SIRT1 finally culminating in impaired endothelial function; and (ii) microRNAs may have different biological roles despite having the same seed sequence., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Ischemia-reperfusion (I/R) injury of the kidney is a major cause of AKI. MicroRNAs (miRs) are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R injury. miR-24 was upregulated in the kidney after I/R injury of mice and in patients after kidney transplantation. Cell-sorting experiments revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after I/R induction. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Transient overexpression of miR-24 alone induced apoptosis and altered functional parameters in these cells, whereas silencing of miR-24 ameliorated apoptotic responses and rescued functional parameters in hypoxic conditions. miR-24 effects were mediated through regulation of H2A histone family, member X, and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites along with miR-24 precursors rescued various functional parameters in endothelial and tubular epithelial cells. In vivo, silencing of miR-24 in mice before I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histologic tubular epithelial injury, and less infiltration of inflammatory cells. miR-24 also regulated heme oxygenase 1 and H2A histone family, member X, in vivo. Overall, these results indicate miR-24 promotes renal ischemic injury by stimulating apoptosis in endothelial and tubular epithelial cell. Therefore, miR-24 inhibition may be a promising future therapeutic option in the treatment of patients with ischemic AKI., (Copyright © 2014 by the American Society of Nephrology.)

Rationale: Long noncoding RNAs represent a novel class of molecules regulating gene expression. Long noncoding RNAs are present in body fluids, but their potential as biomarkers was never investigated in cardiovascular disease., Objective: To study the role of long noncoding RNAs as potential biomarkers in heart disease., Methods and Results: Global transcriptomic analyses were done in plasma RNA from patients with or without left ventricular remodeling after myocardial infarction. Regulated candidates were validated in 3 independent patient cohorts developing cardiac remodeling and heart failure (788 patients). The mitochondrial long noncoding RNA uc022bqs.1 (LIPCAR) was downregulated early after myocardial infarction but upregulated during later stages. LIPCAR levels identified patients developing cardiac remodeling and were independently to other risk markers associated with future cardiovascular deaths., Conclusions: LIPCAR is a novel biomarker of cardiac remodeling and predicts future death in patients with heart failure.

Background: Recent studies have suggested that the microRNAs miR-133a and miR-423-5p may serve as useful biomarkers in patients with left ventricular (LV) heart failure or with LV remodeling after myocardial infarction (MI). These results were however obtained in small series of patients and control subjects were used as reference groups. Whether these microRNAs may be indicators of the degree of LV remodeling after MI is unknown., Methods: 246 patients with a first anterior Q-wave MI were included. Serial echocardiographic studies were performed at hospital discharge, 3 months, and 1 year after MI and analyzed at a core laboratory. We investigated the temporal profile (baseline, 1, 3 and 12 months) of circulating miR-133a and miR-423-5p and their relations with cardiac biomarkers (B-type natriuretic peptide, C-reactive protein, and cardiac troponin I) and LV remodeling during the 1 year follow-up., Results: There were time-dependent changes in the levels of circulating miR-133a and miR-423-5p with significant increase of miR-133a at 12 months compared to 3 months and significant increase of miR-423-5p at 1, 3, and 12 months compared to baseline. However, miR-133a and miR-423-5p were not associated with indices of LV function and LV remodeling serially assessed during a 1 year period after an acute anterior MI, nor with B-type natriuretic peptide., Conclusions: Circulating levels of miR-133a and miR-423-5p are not useful biomarkers of LV remodeling after MI., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Rationale: Transforming growth factor (TGF)-β was linked to abnormal vessel function and can mediate impairment of endothelial angiogenic responses. Its effect on microRNAs and downstream targets in this context is not known., Objective: To study the role of microRNAs in TGF-β-mediated angiogenic activity., Methods and Results: MicroRNA profiling after TGF-β treatment of endothelial cells identified miR-30a-3p, along with other members of the miR-30 family, to be strongly silenced. Supplementation of miR-30a-3p restored function in TGF-β-treated endothelial cells. We identified the epigenetic factor methyl-CpG-binding protein 2 (MeCP2) to be a direct and functional target of miR-30a-3p. Viral overexpression of MeCP2 mimicked the effects of TGF-β, suggesting that derepression of MeCP2 after TGF-β treatment may be responsible for impaired angiogenic responses. Silencing of MeCP2 rescued detrimental TGF-β effects on endothelial cells. Microarray transcriptome analysis of MeCP2-overexpressing endothelial cells identified several deregulated genes important for endothelial cell function including sirtuin1 (Sirt1). In vivo experiments using endothelial cell-specific MeCP2 null or Sirt1 transgenic mice confirmed the involvement of MeCP2/Sirt1 in the regulation of angiogenic functions of endothelial cells. Additional experiments identified that MeCP2 inhibited endothelial angiogenic characteristics partly by epigenetic silencing of Sirt1., Conclusions: TGF-β impairs endothelial angiogenic responses partly by downregulating miR-30a-3p and subsequent derepression of MeCP2-mediated epigenetic silencing of Sirt1.

Heart failure is a leading cause of death in industrialized nations especially in an aging population. The recent improvements in cardiac revascularization therapy reduced death rates because of myocardial infarction but steadily increased the number of individuals developing cardiac remodeling and heart failure in the future. Conceptual novel approaches entering the clinics to treat cardiac remodeling and heart failure remain scarce. MicroRNAs emerged as powerful and dynamic modifiers of cardiovascular diseases. In this review, the current approaches using microRNAs as novel diagnostic and therapeutic strategies for cardiac remodeling and heart failure are highlighted. Other gene regulatory mechanisms presented include long (>200 bp) noncoding RNAs that function as an additional regulatory machinery of the genome controlling both transcriptional and post-transcriptional events also in the cardiovascular system.

Diabetes mellitus due to its high prevalence and associated complications is a major socioeconomic health problem. Diabetes is characterized by multiple macro- and microvascular complications (e.g. diabetic nephropathy, cardiomyopathy, neuropathy, retinopathy). Research efforts aim to elucidate pathophysiological mechanisms contributing to the disease process. MicroRNAs are endogenous small single stranded molecules regulating targets through mRNA cleavage or translational inhibition. MicroRNAs regulate many biological cellular functions and are often deregulated during diseases. The aim of the present article is to summarize the current knowledge of the impact of microRNAs on the development of diabetes and its associated complications including endothelial and vascular smooth muscle cell dysfunction, diabetic cardiomyopathy, diabetic nephropathy, regulation of pancreatic beta cell function as well as skeletal muscle and hepatic involvement.

MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression by base-pairing to the 3'-UTR of the target mRNA. Recently, miRNAs have been shown to regulate cancer metastasis, however, central molecular mechanisms of this ability still need to be investigated. Epithelial to mesenchymal transition (EMT), which is characterized especially by repression of E-cadherin expression and increased cell motility, is an essential component of cancer metastasis and progression. In the present study, we found that Snai1, a known transcriptional repressor of E-cadherin and modulator of EMT, is post-transcriptionally targeted by miRNA-30a in non-small cell lung cancer (NSCLC). Consistent with this, microRNA-30a expression was found inversely proportional to the invasive potential of various NSCLC cell lines, correlating positively with E-cadherin (epithelial marker) and negatively with N-cadherin (mesenchymal marker) expression. Forced re-introduction of miR-30a significantly altered cell morphology, in vitro invasion and migration of invasive cell lines, this being paralleled by a downregulation of Snai1 and upregulation of E-cadherin expression. Using a chicken embryonic metastasis assay, we found that miR-30a suppresses in vivo distant metastasis to the lungs and liver. Finally, we screened the expression of miR-30a in 64 consecutively resected NSCLC patients and found that, in 81% of the patients, expression of miR-30a was downregulated significantly (p < 0.0001) in tumors compared to corresponding normal tissues. These results suggest that miR-30a targets Snai1, inhibits invasion and metastasis, and is downregulated in NSCLC., (Copyright © 2011 UICC.)

Aims: Impaired myocardial sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) activity is a hallmark of failing hearts, and SERCA2a gene therapy improves cardiac function in animals and patients with heart failure (HF). Deregulation of microRNAs has been demonstrated in HF pathophysiology. We studied the effects of therapeutic AAV9.SERCA2a gene therapy on cardiac miRNome expression and focused on regulation, expression, and function of miR-1 in reverse remodelled failing hearts., Methods and Results: We studied a chronic post-myocardial infarction HF model treated with AAV9.SERCA2a gene therapy. Heart failure resulted in a strong deregulation of the cardiac miRNome. miR-1 expression was decreased in failing hearts, but normalized in reverse remodelled hearts after AAV9.SERCA2a gene delivery. Increased Akt activation in cultured cardiomyocytes led to phosphorylation of FoxO3A and subsequent exclusion from the nucleus, resulting in miR-1 gene silencing. In vitro SERCA2a expression also rescued miR-1 in failing cardiomyocytes, whereas SERCA2a inhibition reduced miR-1 levels. In vivo, Akt and FoxO3A were highly phosphorylated in failing hearts, but reversed to normal by AAV9.SERCA2a, leading to cardiac miR-1 restoration. Likewise, enhanced sodium-calcium exchanger 1 (NCX1) expression during HF was normalized by SERCA2a gene therapy. Validation experiments identified NCX1 as a novel functional miR-1 target., Conclusion: SERCA2a gene therapy of failing hearts restores miR-1 expression by an Akt/FoxO3A-dependent pathway, which is associated with normalized NCX1 expression and improved cardiac function.

Objective: MicroRNAs are a class of small ribonucleotides regulating gene/protein targets by transcript degradation or translational inhibition. Transforming growth factor-β (TGF-β) is involved in cardiac fibrosis partly by stimulation of endothelial-to-mesenchymal transition (EndMT). Here, we investigated whether microRNA (miR)-21, a microRNA enriched in fibroblasts and involved in general fibrosis, has a role in cardiac EndMT., Methods and Results: TGF-β treatment of endothelial cells significantly increased miR-21 expression and induced EndMT characterized by suppression of endothelial and increase of fibroblast markers. Overexpression of miR-21 alone also stimulated EndMT. Importantly, miR-21 blockade by transfection of specific microRNA inhibitors partly prevented TGF-β-induced EndMT. Mechanistically, miR-21 silenced phosphatase and tensin homolog in endothelial cells, resulting in activation of the Akt-pathway. Akt inhibition partly restored TGF-β-mediated loss of endothelial markers during EndMT. In vivo, pressure overload of the left ventricle led to increased expression of miR-21 in sorted cardiac endothelial cells, which displayed molecular and phenotypic signs of EndMT. This was attenuated by treatment of mice subjected to left ventricular pressure overload with an antagomir against miR-21., Conclusions: TGF-β-mediated EndMT is regulated at least in part by miR-21 via the phosphatase and tensin homolog/Akt pathway. In vivo, antifibrotic effects of miR-21 antagonism are partly mediated by blocking EndMT under stress conditions.

Pathological growth of cardiomyocytes (hypertrophy) is a major determinant for the development of heart failure, one of the leading medical causes of mortality worldwide. Here we show that the microRNA (miRNA)-212/132 family regulates cardiac hypertrophy and autophagy in cardiomyocytes. Hypertrophic stimuli upregulate cardiomyocyte expression of miR-212 and miR-132, which are both necessary and sufficient to drive the hypertrophic growth of cardiomyocytes. MiR-212/132 null mice are protected from pressure-overload-induced heart failure, whereas cardiomyocyte-specific overexpression of the miR-212/132 family leads to pathological cardiac hypertrophy, heart failure and death in mice. Both miR-212 and miR-132 directly target the anti-hypertrophic and pro-autophagic FoxO3 transcription factor and overexpression of these miRNAs leads to hyperactivation of pro-hypertrophic calcineurin/NFAT signalling and an impaired autophagic response upon starvation. Pharmacological inhibition of miR-132 by antagomir injection rescues cardiac hypertrophy and heart failure in mice, offering a possible therapeutic approach for cardiac failure.

The small regulatory RNA microRNA-21 (miR-21) plays a crucial role in a plethora of biological functions and diseases including development, cancer, cardiovascular diseases and inflammation. The gene coding for pri-miR-21 (primary transcript containing miR-21) is located within the intronic region of the TMEM49 gene. Despite pri-miR-21 and TMEM49 are overlapping genes in the same direction of transcription, pri-miR-21 is independently transcribed by its own promoter regions and terminated with its own poly(A) tail. After transcription, primiR- 21 is finally processed into mature miR-21. Expression of miR-21 has been found to be deregulated in almost all types of cancers and therefore was classified as an oncomiR. During recent years, additional roles of miR-21 in cardiovascular and pulmonary diseases, including cardiac and pulmonary fibrosis as well as myocardial infarction have been described. MiR-21 additionally regulates various immunological and developmental processes. Due to the critical functions of its target proteins in various signaling pathways, miR-21 has become an attractive target for genetic and pharmacological modulation in various disease conditions.

Curcumin has promising potential in cancer prevention and therapy by interacting with proteins and modifying their expression and activity, which includes transcription factors, inflammatory cytokines and factors of cell survival, proliferation and angiogenesis. miR-21 is overexpressed in many tumours, promoting progression and metastasis. In the present study, we examined the potential of curcumin to regulate miR-21, tumour growth, invasion and in vivo metastasis in colorectal cancer. In Rko and HCT116 cells, we identified two new transcriptional start sites of the miR-21 gene and delineated its promoter region. PMA stimulation induced miR-21 expression via motifs bound with AP-1 (activator protein 1) transcription factors. Curcumin treatment reduced miR-21 promoter activity and expression in a dose-dependent manner by inhibiting AP-1 binding to the promoter, and induced the expression of the tumour suppressor Pdcd4 (programmed cell death protein 4), which is a target of miR-21. Curcumin-treated Rko and HCT116 cells were arrested in the G2/M phase with increasing concentrations. Furthermore, curcumin inhibited tumour growth, invasion and in vivo metastasis in the chicken-embryo-metastasis assay [CAM (chorionallantoic membrane) assay]. Additionally, curcumin significantly inhibited miR-21 expression in primary tumours generated in vivo in the CAM assay by Rko and HCT116 cells (P<0.00006 and P<0.035 respectively). Taken together, this is the first paper to show that curcumin inhibits the transcriptional regulation of miR-21 via AP-1, suppresses cell proliferation, tumour growth, invasion and in vivo metastasis, and stabilizes the expression of the tumour suppressor Pdcd4 in colorectal cancer.

The development of metastases is the main reason for cancer-related death in non-small cell lung cancer (NSCLC). The initiation of metastasis involves an increase in cell motility mediated by the loss of cell-cell adhesion caused by E-cadherin repression, in a process commonly known as epithelial-to-mesenchymal transition. A role for microRNA-200 family members in regulating epithelial-to-mesenchymal transition has recently been indicated but data about their expression in lung tumors is still unavailable. The present study investigated the expression of miR-200c in a panel of NSCLC cell lines (n = 9), and a strong inverse correlation with invasion was detected. Reintroduction of miR-200c into highly invasive/aggressive NSCLC cells induced a loss of the mesenchymal phenotype by restoring E-cadherin and reducing N-cadherin expression, and inhibited in vitro cell invasion as well as in vivo metastasis formation. Moreover, miR-200c overexpression restored the sensitivity of NCI-H1299 cells to cisplatin and cetuximab. Hypermethylation of the promoter region was found to be responsible for the loss of miR-200c in invasive cells, as evaluated by 5-aza-2'-deoxycytidine treatment, methylation-specific PCR, and bisulfite sequencing. In primary tumor specimens obtained from 69 patients with consecutively resected NSCLC, lower miR-200c expression levels were found to be associated with a poor grade of differentiation (P = 0.04), a higher propensity to lymph node metastases (P < 0.01), and with a lower E-cadherin expression (P = 0.01). These data indicate that the loss of miR-200c expression induces an aggressive, invasive, and chemoresistant phenotype, and that assessment of its expression could contribute to a better clinicopathologic definition of patients with NSCLC., (© 2010 AACR.)

Actinomycin-D (Act-D) and other inhibitors of RNA synthesis induce extensive and rapid apoptosis in the lepidopteran insect cells. Interestingly, a similar effect is not observed in the case of protein synthesis shutdown, implying that certain RNA species may be critically required for cell survival. In order to assess whether depletion of certain anti-apoptotic microRNAs may result in insect cell apoptosis induced by these transcriptional inhibitors, we inhibited two antiapoptotic microRNAs, viz. bantam and miR-14 (microRNA-14), with known functions in insect systems, by transfecting lepidopteran Sf9 cell line (derived from Spodoptera frugiperda) with sequence-specific inhibitory anti-miRs. Our results indicate that miR-14 is indeed required for constitutive cell survival as its inhibition caused considerable apoptosis. Importantly, exogenous supplementation with the mimics of miR-14 precursor molecules could partially inhibit the Act-D-induced Sf9 cell death. Further, our results indicate that miR-14 may function downstream of mitochondrial cytochrome c release in preventing Act-D-induced apoptosis, implying possible inhibitory interactions with caspases as reported previously in other organisms. While the microRNA species are known to regulate cell death in Drosophila, which belongs the insect order Diptera, the present study demonstrates a definitive antiapoptotic role of miR-14 in lepidopteran apoptosis as well. Our study also indicates that additional microRNA species may be regulating lepidopteran cell survival and death, thus warranting further in-depth investigations into these important mechanisms of cell death. Since lepidopteran cells are an excellent model for general stress resistance, this study presents important information about their stress response mechanisms.

Role of cytochrome-c in insect cell apoptosis is highly controversial, with many earlier reports suggesting lack of involvement of mitochondrial factors in Drosophila while more recent studies have indicated otherwise, thus warranting more in-depth studies of insect cell apoptosis. In the present study, we investigated mitochondrial involvement during actinomycin-D induced apoptosis in Sf9 Lepidopteran cells. Cytochrome-c was released from mitochondria very early during apoptosis, and was preceded quickly by ROS generation and cardiolipin peroxidation. Albeit cytochrome-c release and apoptosis induction were inhibited by bongkrkicacid (BKA) it appears that the release is independent of permeability transition pore (PTP) as it preceded mitochondrial Ca(2+) buildup and mitochondrial membrane potential (MMP) loss. Further, the release was found to be unaffected by PTP inhibitor cyclosporin-A. Bax inhibitory peptide BiP-P5 could effectively block both cytochrome-c release and apoptosis induction indicating dependence on Bax-channel formation. Inhibition of apoptosis by FSBA, a nucleotide analog that inhibits apoptosome formation through Apaf1 binding, suggested activity of apoptosome similar to mammalian cells. Mitochondria isolated from treated cells activated caspases in the cytosolic fraction of untreated cells while mitochondrial lysates of treated or untreated cells had similar effect. Sequestering cytochrome-c in mitochondrial lysates inhibited DEVDase activity, and addition of purified cytochrome-c and dATP to Sf9 cytosolic fraction induced DEVDase activity, suggesting that cytochrome-c may be exclusively required for Lepidopteran apoptosis. This is the first detailed study demonstrating mitochondrial regulation of Lepidopteran insect cell apoptosis, and reiterates its homology with mammalian cell apoptosis while showing distinctive differences from earlier reports in Drosophila.