Muscle-specific miRNAs Research Articles

Changes in circulating microRNAs levels with exercise modality

Here, we studied muscle-specific and muscle-related miRNAs in plasma of exercising humans. Our aim was to determine whether they are affected by eccentric and/or concentric exercise modes and could be biomarkers of muscle injuries or possible signaling molecules. On two separate days, nine healthy subjects randomly performed two 30-min walking exercises, one downhill (high eccentric component) and one uphill (high concentric component). Perceived exertion and heart rate were higher during the uphill exercise, while subjective pain and ankle plantar flexor strength losses within the first 48-h were higher following the downhill exercise. Both exercises increased serum creatine kinase and myoglobin with no significant differences between conditions. Plasma levels of circulating miRNAs assessed before, immediately after, and at 2-, 6-, 24-, 48-, and 72-h recovery showed that 1) hsa-mir-1, 133a, 133b, and 208b were not affected by concentric exercise but significantly increased during early recovery of eccentric exercise (2 to 6 h); 2) hsa-mir-181b and 214 significantly and transiently increased immediately after the uphill, but not downhill, exercise. The muscle-specific hsa-mir-206 was not reliably quantified and cardiac-specific hsa-mir-208a remained undetectable. In conclusion, changes in circulating miRNAs were dependent on the exercise mode. Circulating muscle-specific miRNAs primarily responded to a downhill exercise (high eccentric component) and could potentially be alternative biomarkers of muscle damage. Two muscle-related miRNAs primarily responded to an uphill exercise (high exercise intensity), suggesting they could be markers or mediators of physiological adaptations.

MiRNA analysis for the assessment of exercise and amino acid effects on human skeletal muscle.

The study of micro RNA (miRNA) expression and function, a largely unexplored area of human muscle biology, may provide novel data regarding the development of targeted approaches that optimize skeletal muscle responses to exercise and amino acid manipulations. miRNAs are ubiquitously expressed, small noncoding RNAs that modulate posttranscriptional gene expression. Quantifying miRNA expression and predicting function as regulators of both single targets and complex networks is technically challenging and requires a combined approach of bioinformatics, molecular, and systems biology. Recent evidence suggests that the expression of muscle-specific miRNAs (myomirs), including miR-1, miR-133a/b, miR-206, and miR-499, is modulated by essential amino acid ingestion, endurance exercise, and endurance exercise training. The expression of miRNAs has also been implicated in the anabolic intracellular signaling and muscle hypertrophic response associated with resistance exercise training. Although these findings are intriguing, comprehensive human trials assessing functional outcomes associated with changes in miRNA expression in response to exercise and nutrition interventions have not been conducted. This article reviews the current understanding of miRNA biology and includes analytical techniques used to detect miRNA expression and methods to predict function. The intent is to provide the framework for future research studies that use miRNA analysis in an effort to elucidate optimal exercise and nutritional countermeasures for the prevention of muscle loss.

Circulating miRNAs reflect early myocardial injury and recovery after heart transplantation

BackgroundMicroRNAs (miRNAs) are short, single-stranded and non-coding RNAs, freely circulating in human plasma and correlating with vary pathologies. In this study, we monitored early myocardial injury and recovery after heart transplantation by detecting levels of circulating muscle-specific miR-133a, miR-133b and miR-208a.Methods7 consecutive patients underwent heart transplantation in Fuwai hospital and 14 healthy controls were included in our study. Peripheral vein blood was drawn from patients on the day just after transplantation (day 0), the 1st, 2nd, 3rd, 7th and 14th day after transplantation respectively. Serum from peripheral blood was obtained for cardiac troponin I (cTnI) measurement. Plasma was centrifuged from peripheral blood for measuring miR-133a, miR-133b and miR-208a by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The plasma concentration of miRNAs were calculated by absolute quantification method. The sensitivity and specificity of circulating miRNAs were revealed by receiver operating characteristic curve (ROC) analysis. Correlations between miRNAs and cTnI / perioperative parameters were analyzed.ResultsSimilar to cTnI, miR-133a, miR-133b and miR-208a all showed dynamic changes from high to low levels early after operation. The Sensitivity and specificity of miRNAs were: miR-133a (85.7%,100%), miR-208a (100%,100%), and miR-133b (90%,100%). Correlations between miRNAs and cTnI were statistically significant (p < 0.05), especially for miR-133b (R2 = 0.813, p < 0.001). MiR-133b from Day 0-Day 2 (r > 0.98, p < 0.01), and cTnI from Day 1- Day 3 (r > 0.86, p < 0.05) had strong correlations with bypass time, particularly parallel bypass time. Obviously, miR-133b had a better correlation than cTnI. Circulating miR-133b correlated well with parameters of heart function such as central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP), cardiac output (CO) and inotrope support, while cTnI only correlated with 3 of the 4 parameters mentioned above. MiR-133b also had strong correlations with ventilation time (r > 0.99, p < 0.001) and length of ICU stay (r > 0.92, p < 0.05), both of which reflected the recovery after operation. The correlation coefficients of miR-133b were also higher than that of cTnI.ConclusionsThe dynamic change in circulating muscle-specific miRNAs, especially miR-133b can reflect early myocardial injury after heart transplantation. And miR-133b may have advantages over cTnI in forecasting graft dysfunction and recovery of patients after operation.

Increased skeletal muscle-specific microRNA in the blood of patients with COPD

BackgroundSkeletal muscle weakness in chronic obstructive pulmonary disease (COPD) carries a poor prognosis, therefore a non-invasive marker of this process could be useful. Reduced expression of muscle-specific microRNA (myomiRs) in...

MicroRNA Regulation and Role in Stem Cell Maintenance, Cardiac Differentiation and Hypertrophy

There are currently 1527 known microRNAs (miRNAs) in human, each of which may regulate hundreds or thousands of target genes. miRNA expression levels vary between cell types; for example, miR- 302 and miR-290 families are highly enriched in embryonic stem cells, while miR-1 is a muscle specific miRNA. miRNA biosynthesis and function are highly regulated and this regulation may be cell type specific. The processing enzymes and factors that recognize features in sequence and secondary structure of the miRNA play key roles in regulating the production of mature miRNA. Mature miRNA enriched in stem cells control stem cell self-renewal as well as their differentiation. Though specific miRNAs have been shown to control differentiation towards various lineages such as neural or skin cells, some of the most well characterized miRNAs have been found in promoting the formation of cardiac cells. In addition, miRNAs also play a critical role in cardiomyocyte hypertrophy, especially in a pathological context. Such miRNAs are predicted to be therapeutic targets for treating cardiovascular diseases. In this review we will discuss how miRNAs act to maintain the stem cell state and also explore the current knowledge of the mechanisms that regulate miRNAs. Furthermore, we will discuss the emerging roles of miRNAs using cardiomyocyte differentiation and maturation as a paradigm. Emphasis will also be given on some of the less ventured areas such as the role of miRNAs in the physiological maturation of cardiomyocytes. These potentially beneficial miRNAs are likely to improve cardiac function in both in vivo and in vitro settings and thus provide additional strategy to treat heart diseases and more importantly serve as a good model for understanding cardiomyocyte maturation in vitro.

MiR‐133a ameliorates cardiac stem cells survival and differentiation in Insulin2 mutant diabetic mice

The Ins2 +/− (orthologous to human Insulin) mutation predisposes to diabetic complications and mitigates cardiac stem cell differentiation into cardiomyocytes that foster cardiomyopathy. MicroRNA (miR)‐133a, a muscle specific miRNA that regulates cardiac hypertrophy and fibrosis, is attenuated in diabetic hearts. However, the role of miR‐133a in cardiac stem cell survival and differentiation is unclear. We hypothesized that miR‐133a improves cardiac stem cell survival and differentiation and mitigates diabetic cardiomyopathy. To test the hypothesis, miR‐133a was overexpressed in WT (C57BL/6J) and diabetic Akita (Ins2+/‐) through lentivirus. The levels of miR‐133a, c‐kit and differentiation markers (Actn2, Oct4 and Tnnt2) were determined by individual miRNA assay, RT‐QPCR, Western blot and confocal microscopy in four groups: 1) WT, 2) Akita, 3) WT +miR‐133a, and 4) Akita + miR‐133a. The cardiac dysfunction was assessed by M‐Mode echocardiography. The results revealed that miR‐133a, c‐kit, Actn2, Oct4 and Tnnt2 were up regulated in Akita + miR‐133a as compared to Akita. The % FS is increased in Akita after miR‐133a treatment (Akita‐ 38.6±1.2; Akita +miR‐133a ‐ 50.8 ±5.1). These findings elicit a novel mechanism of miR‐133a mediated cardiac stem cell survival and differentiation and cardiac protection in diabetic mice.

MicroRNAs Involved in Skeletal Muscle Differentiation

MicroRNAs (miRNAs) negatively regulate gene expression by promoting degradation of target mRNAs or inhibiting their translation. Previous studies have expanded our understanding that miRNAs play an important role in myogenesis and have a big impact on muscle mass, muscle fiber type and muscle-related diseases. The muscle-specific miRNAs, miR-206, miR-1 and miR-133, are among the most studied and best characterized miRNAs in skeletal muscle differentiation. They have a profound influence on multiple muscle differentiation processes, such as alternative splicing, DNA synthesis, and cell apoptosis. Many non-muscle-specific miRNAs are also required for the differentiation of muscle through interaction with myogenic factors. Studying the regulatory mechanisms of these miRNAs in muscle differentiation will extend our knowledge of miRNAs in muscle biology and will improve our understanding of the myogenesis regulation.

Identification and profiling of conserved and novel microRNAs from Chinese Qinchuan bovine longissimus thoracis

BackgroundMicroRNAs (miRNAs) are a family of ~22 nucleotide small RNA molecules that regulate gene expression by fully or partially binding to their complementary sequences. Recently, a large number of miRNAs and their expression patterns have been identified in various species. However, to date no miRNAs have been reported to modulate muscle development in beef cattle.ResultsTotal RNAs from the Chinese Qinchuan bovine longissimus thoracis at fetal and adult stages were used to construct small RNA libraries for Solexa SBS technology sequencing. A total of 15,454,182 clean reads were obtained from the fetal bovine library and 13,558,164 clean reads from the adult bovine library. In total, 521 miRNAs including 104 novel miRNA candidates were identified. Furthermore, the nucleotide bias, base edit and family of the known miRNAs were also analyzed. Based on stem-loop qPCR, 25 high-read miRNAs were detected, and the results showed that bta-miRNA-206, miRNA-1, miRNA-133, miRNAn12, and miRNAn17 were highly expressed in muscle-related tissue or organs, suggesting that these miRNAs may play a role in the development of bovine muscle tissues.ConclusionsThis study confirmed the authenticity of 417 known miRNAs, discovered 104 novel miRNAs in bos taurus, and identified five muscle-specific miRNAs. The identification of novel miRNAs significantly expanded the repertoire of bovine miRNAs and could contribute to further studies on the muscle development of cattle.

Regulation of IRS1/Akt insulin signaling by microRNA-128a during myogenesis

Skeletal muscle possesses a strong ability to regenerate following injury, a fact that has been largely attributed to satellite cells. Satellite cells are skeletal muscle stem cells located beneath the basal lamina of the myofiber, and are the principal cellular source of growth and regeneration in skeletal muscle. MicroRNAs (miRNAs) play key roles in modulating several cellular processes by targeting multiple mRNAs that comprise a single or multiple signaling pathway. Several miRNAs have been shown to regulate satellite cell activity, such as miRNA-489, which functions to maintain satellite cells in a quiescent state. Although muscle-specific miRNAs have been identified, many of the molecular mechanisms that regulate myogenesis that are regulated by miRNAs still remain unknown. In this study, we have shown that miR-128a is highly expressed in brain and skeletal muscle, and increases during myoblast differentiation. MiR-128a was found to regulate the target genes involved in insulin signaling, which include Insr (insulin receptor), Irs1 (insulin receptor substrate 1) and Pik3r1 (phosphatidylinositol 3-kinases regulatory 1) at both the mRNA and protein level. Overexpression of miR-128a in myoblasts inhibited cell proliferation by targeting IRS1. By contrast, inhibition of miR-128a induced myotube maturation and myofiber hypertrophy in vitro and in vivo. Moreover, our results demonstrate that miR-128a expression levels are negatively controlled by tumor necrosis factor α (TNF-α). TNF-α promoted myoblast proliferation and myotube hypertrophy by facilitating IRS1/Akt signaling via a direct decrease of miR-128a expression in both myoblasts and myotubes. In summary, we demonstrate that miR-128a regulates myoblast proliferation and myotube hypertrophy, and provides a novel mechanism through which IRS1-dependent insulin signaling is regulated in skeletal muscle.

Identification of differences in microRNA transcriptomes between porcine oxidative and glycolytic skeletal muscles

BackgroundMicroRNAs (miRNAs) are a type of non-coding small RNA ~22 nucleotides in length that regulate the expression of protein coding genes at the post-transcriptional level. Glycolytic and oxidative myofibers, the two main types of skeletal muscles, play important roles in metabolic health as well as in meat quality and production in the pig industry. Previous expression profile studies of different skeletal muscle types have focused on these aspects of mRNA and proteins; nonetheless, an explanation of the miRNA transcriptome differences between these two distinct muscles types is long overdue.ResultsHerein, we present a comprehensive analysis of miRNA expression profiling between the porcine longissimus doris muscle (LDM) and psoas major muscle (PMM) using a deep sequencing approach. We generated a total of 16.62 M (LDM) and 18.46 M (PMM) counts, which produced 15.22 M and 17.52 M mappable sequences, respectively, and identified 114 conserved miRNAs and 89 novel miRNA*s. Of 668 unique miRNAs, 349 (52.25%) were co-expressed, of which 173 showed significant differences (P < 0.01) between the two muscle types. Muscle-specific miR-1-3p showed high expression levels in both libraries (LDM, 32.01%; PMM, 20.15%), and miRNAs that potentially affect metabolic pathways (such as the miR-133 and -23) showed significant differences between the two libraries, indicating that the two skeletal muscle types shared mainly muscle-specific miRNAs but expressed at distinct levels according to their metabolic needs. In addition, an analysis of the Gene Ontology (GO) terms and KEGG pathway associated with the predicted target genes of the differentially expressed miRNAs revealed that the target protein coding genes of highly expressed miRNAs are mainly involved in skeletal muscle structural development, regeneration, cell cycle progression, and the regulation of cell motility.ConclusionOur study indicates that miRNAs play essential roles in the phenotypic variations observed in different muscle fiber types.

Muscle-Specific Microrna miR-208 Regulates Calcium Handling Release in Cardiomyocytes by Targeting Phosphodiesterase 4D and Calcineurin

MicroRNAs are small non-coding RNAs which act as endogenous regulators of gene expression, have gained much attention in the past several years due to increasing evidence of their involvement in numerous pathological processes including cardiac disease. Several microRNAs have been implicated in heart failure (HF) and sudden cardiac death (SCD) via regulation of calcium handling in cardiac myocytes. Upregulation of muscle-specific miRNA miR-208 has been associated with adverse clinical outcomes including acute failure and SCD in patients with dilated cardiomyopathy, while therapeutic inhibition of miR-208 was demonstrated to improve survival and cardiac function in the rat model of HF. However, the molecular mechanisms underlying such functional effects of miR-208 dysregulation remain largely unexplored. We hypothesize that miR-208 is an important regulator of calcium handling by targeting components of phosphorylation-dephosphorylation system in cardiac myocytes. An experimental model of miR-208 overexpression in rat ventricular myocytes was generated via adenoviral infection. Its effects on Ca2+ handling and pro-arrhythmic activity was investigated using confocal microscopy for Ca2+ imaging, western blot analysis, and luciferase reporter assays. Under conditions of beta-adrenergic stimulation we observed decreased Ca2+ transient amplitude, reduced SR Ca2+ content, faster onset of pro-arrhythmic spontaneous Ca2+ waves, and increased spark frequency in miR-208 overexpressing cells. Additionally, there was increased phosphorylation of a negative regulator of SERCA, phospholamban, and ryanodine receptors as well as decreased levels of putative targets phosphodiesterase 4D and calcineurin in miR-208 overexpressed myocytes. We can conclude that miR-208 affects Ca2+ handling by regulating the expression of proteins involved in phosphorylation-dephosphorylation of ryanodine receptors and phospholamban. This mechanism may contribute to the increased risk of arrhythmia and SCD in cardiac disease states accompanied by miR-208 upregulation including HF.

Mutual antagonism between IP(3)RII and miRNA-133a regulates calcium signals and cardiac hypertrophy.

Inositol 1,4,5'-triphosphate receptor II (IP(3)RII) calcium channel expression is increased in both hypertrophic failing human myocardium and experimentally induced models of the disease. The ectopic calcium released from these receptors induces pro-hypertrophic gene expression and may promote arrhythmias. Here, we show that IP(3)RII expression was constitutively restrained by the muscle-specific miRNA, miR-133a. During the hypertrophic response to pressure overload or neurohormonal stimuli, miR-133a down-regulation permitted IP(3)RII levels to increase, instigating pro-hypertrophic calcium signaling and concomitant pathological remodeling. Using a combination of in vivo and in vitro approaches, we demonstrated that IP(3)-induced calcium release (IICR) initiated the hypertrophy-associated decrease in miR-133a. In this manner, hypertrophic stimuli that engage IICR set a feed-forward mechanism in motion whereby IICR decreased miR-133a expression, further augmenting IP(3)RII levels and therefore pro-hypertrophic calcium release. Consequently, IICR can be considered as both an initiating event and a driving force for pathological remodeling.

Bone Morphogenetic Protein/SMAD Signaling Orients Cell Fate Decision by Impairing KSRP-Dependent MicroRNA Maturation

MicroRNAs (miRNAs) are essential regulators of development, physiology, and evolution, and their biogenesis is strictly controlled at multiple levels. Regulatory proteins, such as KSRP, modulate rates and timing of enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Here, we show that KSRP silencing in mesenchymal C2C12 cells produces a change in the transcriptome largely overlapping that induced by bone morphogenetic protein 2 (BMP2) signaling activation. This induces osteoblastic differentiation while preventing myogenic differentiation. KSRP silencing- and BMP2-dependent myogenic miRNA (myomiR) maturation blockade is required for osteoblastic differentiation of C2C12 cells. Our results demonstrate that phosphorylated R-SMAD proteins, the transducers of BMP2 signal, associate with phosphorylated KSRP and block its interaction with primary myomiRs. This abrogates KSRP-dependent myomiR maturation, with SMAD4, SMAD5, and SMAD9 silencing being able to rescue KSRP function. Thus, SMAD-induced blockade of KSRP-dependent myomiR maturation is critical for orienting C2C12 cell differentiation toward osteoblastic lineage.

Dynamic expression of miR-206-3p during mouse skin development is independent of keratinocyte differentiation.

MicroRNA-206 (miR-206), the homolog of which in mice is termed miR-206-3p, is a muscle-specific miRNA known to be important in the development of skeletal muscle, and is involved in smooth muscle innervation of the airway through the post‑transcriptional suppression of brain‑derived neurotrophic factor (Bdnf). miR‑206‑3p is also expressed at significant levels in adult and embryonic skin; however, its functional roles in adult skin and during skin development remain to be fully elucidated. In the present study, the spatiotemporal expression of miR‑206‑3p and its target‑gene, Bdnf, during mouse skin development were investigated. The expression level of miR‑206‑3p increased from 13.5days postcoitus (dpc), peaked at 17.5dpc and declined following birth. The observed temporal profile of the expression of miR‑206‑3p was accompanied by an inverse change in the protein expression levels of BDNF. However, the mRNA expression levels of Bdnf did not parallel those of BDNF protein. The localization of the expression of miR‑206‑3p was similar, or located near that of ubiquitin carboxyl‑terminal hydrolase L1 during skin development. An invitro keratinocyte model demonstrated no significant differences between primary and differentiated keratinocytes in the expression levels of either miR‑206‑3p (P=0.227) or Bdnf (mRNA, P=0.118; mature BDNF, P=0.106; pro‑BDNF, P=0.905). These findings indicate a potential role for miR‑206‑3p in cutaneous innervation, which largely relies on BDNF neurotrophic support and is independent of keratinocyte differentiation. The results of the present study suggested that this novel mechanism may be targeted for developing potential therapeutic approaches.

MiR-351 transiently increases during muscle regeneration and promotes progenitor cell proliferation and survival upon differentiation

MicroRNAs (miRNAs) regulate many biological processes including muscle development. However, little is known regarding miRNA regulation of muscle regeneration. Murine tibialis anterior muscle was evaluated after cardiotoxin-induced injury and used for global miRNA expression analysis. From day 1 through day 21 following injury, 298 miRNAs were significantly changed at least at one time point, including 86 miRNAs that were altered >10-fold compared with uninjured skeletal muscle. Temporal miRNA expression patterns included inflammation-related miRNAs (miR-223 and -147) that increased immediately after injury; this pattern contrasted to that of mature muscle-specific miRNAs (miR-1, -133a, and -499) that abruptly decreased following injury followed by upregulation in later regenerative events. Another cluster of miRNAs were transiently increased in the early days of muscle regeneration including miR-351, a miRNA that was also transiently expressed during myogenic progenitor cell (MPC) differentiation in vitro. Based on computational predictions, further studies demonstrated that E2f3 was a target of miR-351 in myoblasts. Moreover, knockdown of miR-351 expression inhibited MPC proliferation and promoted apoptosis during MPC differentiation, whereas miR-351 overexpression protected MPC from apoptosis during differentiation. Collectively, these observations suggest that miR-351 is involved in both the maintenance of MPC proliferation and the transition into differentiated myotubes. Thus, a novel, time-dependent sequence of molecular events during muscle regeneration has been identified; miR-351 inhibits E2f3 expression, a key regulator of cell cycle progression and proliferation, and promotes MPC proliferation and protects early differentiating MPC from apoptosis, important events in the hostile tissue environment after acute muscle injury.

Abstract 364: Valproic Acid Induced Reprogramming of Adult Somatic Cells for Cardiac Differentiation

Aims: Owing to the ethical concerns for use of embryonic stem cells (ESC), adult somatic cells are attractive stem cell sources for reprogramming to pluripotency. We report here non-viral approach for reprogramming of skeletal myoblasts (SM) using a small molecule. Methods and Results: SM purified from young male Oct3/4-GFP+ transgenic mouse were treated for 5 days with valproic acid (VPA), a histone deacetylase (HDAC) inhibitor. Three weeks later, GFP+ colonies of SM derived iPSC (Sk-iPS) resembling with mouse embryonic stem cells were observed and propagated in vitro. SiPS were positive for alkaline phosphatase, had normal karyotype, expressed SSEA1, and induced teratomas in nude mice containing tissue comprising all three germ layers. RT PCR analysis showed that Sk-iPS cells expressed Oct4, Sox2, KLF4, c-Myc, Nanog and ESC specific pluripotency genes. HDAC1 activity was significantly reduced in Sk-iPs generated with valproic acid treatment as compared to ES cells. Sk-iPS derived embryoid bodies (EBs) yielded spontaneously contracting cardiomyocytes with morphological, molecular, and ultrastructural features of developing cardiomyocytes. These cells were also positive for early and late cardiac markers such as myosin heavy chain, Gata4, Mef-2c and Nkx2.5, Connexin-43 (P&lt;0.01vs native SM). Micro RNA (miR) profiling showed abolition of let-7 family in Sk-iPS whereas ESC specific family of miR-290-295 was upregulated which indicated that Sk-iPS possessed miR profile similar to ESC. However muscle specific miRNAs (miR -133, -206) were identified in Sk-iPS cells as compared to ES cells indicating that the Sk-iPS retained the epigenetic memory of myogenic origin. Conclusions: We conclude that SM with endogenous expression of Sox2, KLF4, and cMyc are suitable candidates to generate iPS cells without viral vectors using a single small molecule.

Dicer‐deletion in the detrusor reduces contractility and expression of L‐type Ca2+ channels

MicroRNAs (miRNAs) are small noncoding RNAs which modulate gene expression. Production of most mature miRNAs requires the endonuclease Dicer. Here we investigated the role of miRNAs in the urinary bladder using conditional smooth muscle‐specific Dicer knockout (KO) mice. miR‐145, miR‐22, miR‐125b‐ 5p, miR27a, and miR‐1 were the most highly expressed miRNAs in the detrusor. The knockdown level for these miRNAs ranged between 68 and 99%. Using strip preparations we found that the muscarinic component of the neurogenic contraction was decreased in Dicer‐deficient bladder. A substantially reduced stress in response to high K+ was seen. The expression of L‐type Ca2+ channels (LTCC) was reduced, and only modest reductions of other differentiation markers (desmin, calponin) were noted. Assessment of micturition patterns revealed changes in both frequency and spatial voiding distribution in KO mice. We conclude that the biomechanical consequences of Dicer deletion in the detrusor are dominated by reduced LTCC expression. Deletion of Dicer‐dependent and smooth muscle‐specific miRNAs moreover results in a reduced cholinergic component of neurogenic activation. The effects of Dicer‐deletion on contractility and neuro‐effector transmission act in concert to cause a disturbed micturition pattern in vivo. Supported by Swedish Research Council, Faculty of Medicine at Lund University.

MicroRNA-133 inhibits cell proliferation, migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor

It has been shown that regulation of EGFR expression in prostate cancer cells is mostly at the transcriptional level. microRNA-133 (miR-133) has long been recognized as a muscle-specific miRNA which may regulate myoblast differentiation and participate in many myogenic diseases. Recently, it has been reported that miR-133 is also involved in other tumors, such as bladder cancer, esophageal cancer and may regulate cell motility in these cancer cells. In the present study, we examined the expression and effects of miR-133 in two hormone-insensitive prostate cancer cell lines. The expression of miR-133a and miR-133b were analyzed by quantitative RT-PCR. After transfection of miR-133a and miR-133b, cell viability assay, luciferase assay, western blot analysis, cell migration and invasion assay were conducted in Du145 and PC3 cells. In this study, we showed that miR‑133a and miR-133b are expressed at the detection limit in two hormone-insensitive prostate cancer cell lines, PC3 and DU145. Ectopic expression of miR-133 inhibited cell proliferation, migration and invasion in these cells. We also provide the first evidence that miR-133 may target EGFR. Our study provided the first glimpse of the functional role of miR-133 in two hormone-independent prostate cancer cell lines. These results may add to our knowledge on the molecular basis of prostate cancer progression.

Insulin-Like Growth Factor-1 Receptor Is Regulated by microRNA-133 during Skeletal Myogenesis

BackgroundThe insulin-like growth factor (IGF) signaling pathway has long been established as playing critical roles in skeletal muscle development. However, the underlying regulatory mechanism is poorly understood. Recently, a large family of small RNAs, named microRNAs (miRNAs), has been identified as key regulators for many developmental processes. Because miRNAs participate in the regulation of various signaling pathways, we hypothesized that miRNAs may be involved in the regulation of IGF signaling in skeletal myogenesis.Methodology/Principal FindingsIn the present study, we determined that the cell-surface receptor IGF-1R is directly regulated by a muscle-specific miRNA, microRNA-133 (miR-133). A conserved and functional binding site for miR-133 was identified in the 3′untranslated region (3′UTR) of IGF-1R. During differentiation of C2C12 myoblasts, IGF-1R protein, but not messenger RNA (mRNA) expression, was gradually reduced, concurrent with the upregulation of miR-133. Overexpression of miR-133 in C2C12 cells significantly suppressed IGF-1R expression at the posttranscriptional level. We also demonstrated that both overexpression of miR-133 and knockdown of IGF-1R downregulated the phosphorylation of Akt, the central mediator of the PI3K/Akt signaling pathway. Furthermore, upregulation of miR-133 during C2C12 differentiation was significantly accelerated by the addition of IGF-1. Mechanistically, we found that the expression of myogenin, a myogenic transcription factor reported to transactivate miR-133, was increased by IGF-1 stimulation.Conclusion/SignificanceOur results elucidate a negative feedback circuit in which IGF-1-stimulated miR-133 in turn represses IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis. These findings also suggest that miR-133 may be a potential therapeutic target in muscle diseases.

MicroRNA-1 and -133 Increase Arrhythmogenesis in Heart Failure by Dissociating Phosphatase Activity from RyR2 Complex

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis.