miR-181c Regulates the Mitochondrial Genome,Bioenergetics, and Propensity for Heart Failure In Vivo

MicroRNAs (miRNAs) are small non-coding RNAs, which inhibit the stability and/or translation of a mRNA. miRNAs have been found to play a powerful role in various cardiovascular diseases. Recently, we have demonstrated that a microRNA (miR-181c) can be encoded in the nucleus, processed to the mature form in the cytosol, translocated into the mitochondria, and ultimately can regulate mitochondrial gene expression. However the in vivo impact of miR-181c is unknown. Here we report an in-vivo method for administration of miR-181c in rats, which leads to reduced exercise capacity and signs of heart failure, by targeting the 3′-end of mt-COX1 (cytochrome c oxidase subunit 1). We cloned miR-181c and packaged it in lipid-based nanoparticles for systemic delivery. The plasmid DNA complexed nanovector shows no apparent toxicity. We find that the mRNA levels of mitochondrial complex IV genes in the heart, but not any other mitochondrial genes, are significantly altered with miR-181c overexpression, suggesting selective mitochondrial complex IV remodeling due to miR-181c targeting mt-COX1. Isolated heart mitochondrial studies showed significantly altered O₂-consumption, ROS production, matrix calcium, and mitochondrial membrane potential in miR-181c-treated animals. For the first time, this study shows that miRNA delivered to the heart in-vivo can lead to cardiac dysfunction by regulating mitochondrial genes.     

The Axl-Regulating Tumor Suppressor miR-34a Is Increased in ccRCC but Does Not Correlate with Axl mRNA or Axl Protein Levels

Background

High expression of the receptor tyrosine kinase Axl is associated with poor prognosis in patients with Renal Cell Carcinoma (RCC), the most common malignancy of the kidney. The miR-34a has been shown to directly regulate Axl in cancer cells. The miR-34a is a mediator of p53-dependent tumor suppression, and low expression of miR-34a has been associated with worse prognosis in several cancers. Our aim was to elucidate whether miR-34a or the other members of the miR-34 family (miR-34b/c) regulate Axl in RCC.

Methodology and Results

Using western blot, flow cytometry, and RT-qPCR, we showed that Axl mRNA and protein are downregulated in 786-O cells by miR-34a and miR-34c but not by miR-34b. A luciferase reporter assay demonstrated direct interaction between the Axl 3’ UTR and miR-34a and miR-34c. The levels of miR-34a/b/c were measured in tumor tissue in a cohort of 198 RCC patients, and the levels of miR-34a were found to be upregulated in clear cell RCC (ccRCC) tumors, but not associated with patient outcome. Neither of the miR-34 family members correlated with Axl mRNA, soluble Axl protein in serum, nor with immunohistochemistry of Axl in tumor tissue. In addition, we measured mRNA levels of a known miR-34a target, HNF4A, and found the HNF4A levels to be decreased in ccRCC tumors, but unexpectedly correlated positively rather than negatively with miR-34a.

Conclusions

Although miR-34a and miR-34c can regulate Axl expression in vitro, our data indicates that the miR-34 family members are not the primary regulators of Axl expression in RCC.     

心肌细胞过表达miR-27b导致小鼠发生心肌纤维化和线粒体损伤

以往的miRNA芯片研究结果显示, miR-27b在人类心脏疾病标本和压力负荷引起的小鼠心肌肥厚模型中表达水平明显升高, 提示其在心脏疾病发生过程中发挥了重要功能。为研究miR-27b在心脏组织中的功能, 文章建立了在心肌细胞特异性 a-肌球蛋白重链(a-MHC)启动子(5.5 kb)控制下过表达miR-27b的转基因小鼠。通过Real-time PCR检测, 发现miR-27b前体和成熟体表达水平在转基因小鼠心脏组织中明显升高。miR-27b转基因小鼠不仅出现心肌肥厚, 还表现出明显的心肌纤维化。进一步研究表明心肌纤维化的关键调节分子金属基质蛋白酶13(MMP13)是miR-27b的靶分子, 在miR-27b转基因小鼠中MMP13显著下调, 胶原分子I和 III则显著上调。此外, 还发现miR-27b转基因小鼠会出现心脏超微结构的损伤。以上研究结果表明, miR-27b可能通过抑制MMP13促进心肌纤维化。     

MicroRNA Profiling of Pericardial Fluid Samples from Patients with Heart Failure

Aims

Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages.

Methods and Results

Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population.

Conclusion

Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.     

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.     

MiR-216a: a link between endothelial dysfunction and autophagy

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.     

MicroRNA-216b inhibits heat stress-induced cell apoptosis by targeting Fas in bovine mammary epithelial cells

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.     

Long non-coding RNA Pvt1 modulates the pathological cardiac hypertrophy via miR-196b-mediated OSMR regulation

Cardiac hypertrophy is the uppermost risk factor for the development of heart failure, leading to irreversible cardiac structural remodeling and sudden death. As a major mediator of cardiac remodeling, oncostatin M (OSM) and its receptor, OSMR, attract plenty of interest. Recent studies have demonstrated key effects of noncoding RNAs on myocardial remodeling. However, whether noncoding RNAs that regulate the expression of OSMR would regulate the process of remodeling remain unclear. Herein, we observed that long noncoding RNA (lncRNA) Pvt1 expression showed to be significantly elicited by aortic banding (AB) operation in vivo and by angiotensin (Ang II) treatment in vitro. Pvt1 knockdown significantly attenuated the myocardial hypertrophy caused by pressure overload within rats and the cardiac myocyte hypertrophy caused by Ang II in vitro. Moreover, Pvt1 knockdown also decreased cellular myomesin and B-raf, which was involved in OSM function in cardiac remodeling. Based on online tools prediction, miR-196b may simultaneously target Pvt1 and OSMR 3′ untranslated region (UTR). In rat H9c2 cells and primary cardiac myocyte, Pvt1 and miR-196b exerted negative regulatory effects on each other and miR-196b negatively regulated OSMR expression. Pvt1 directly targeted miR-196b to relieve miR-196b-induced OSMR suppression via acting as a competing endogenous RNA (ceRNA). Moreover, the effect of miR-196b suppression upon the B-raf was opposite to Pvt1 knockdown, and miR-196b suppression might significantly attenuate the effect of Pvt1 knockdown. In summary, Pvt1/miR-196b axis modulating cardiomyocyte hypertrophy and remodeling via OSMR. Our findings provide a rationale for further studies on the potential therapeutic benefits of Pvt1 function and mechanism in cardiac and cardiomyocyte hypertrophy by a lncRNA-miRNA-mRNA network.     

Regulation of Cardiac Expression of the Diabetic Marker MicroRNA miR-29

Diabetes mellitus (DM) is an independent risk factor for heart disease and its underlying mechanisms are unclear. Increased expression of diabetic marker miR-29 family miRNAs (miR-29a, b and c) that suppress the pro-survival protein Myeloid Cell Leukemia 1(MCL-1) is reported in pancreatic β-cells in Type 1 DM. Whether an up-regulation of miR-29 family miRNAs and suppression of MCL-1 (dysregulation of miR-29-MCL-1 axis) occurs in diabetic heart is not known. This study tested the hypothesis that insulin regulates cardiac miR-29-MCL-1 axis and its dysregulation correlates with DM progression. In vitro studies with mouse cardiomyocyte HL-1 cells showed that insulin suppressed the expression of miR-29a, b and c and increased MCL-1 mRNA. Conversely, Rapamycin (Rap), a drug implicated in the new onset DM, increased the expression of miR-29a, b and c and suppressed MCL-1 and this effect was reversed by transfection with miR-29 inhibitors. Rap inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling in HL-1 cells. Moreover, inhibition of either mTORC1 substrate S6K1 by PF-4708671, or eIF4E-induced translation by 4E1RCat suppressed MCL-1. We used Zucker diabetic fatty (ZDF) rat, a rodent model for DM, to test whether dysregulation of cardiac miR-29-MCL-1 axis correlates with DM progression. 11-week old ZDF rats exhibited significantly increased body weight, plasma glucose, insulin, cholesterol, triglycerides, body fat, heart weight, and decreased lean muscle mass compared to age-matched lean rats. Rap treatment (1.2 mg/kg/day, from 9-weeks to 15-weeks) significantly reduced plasma insulin, body weight and heart weight, and severely dysregulated cardiac miR-29-MCL1 axis in ZDF rats. Importantly, dysregulation of cardiac miR-29-MCL-1 axis in ZDF rat heart correlated with cardiac structural damage (disorganization or loss of myofibril bundles). We conclude that insulin and mTORC1 regulate cardiac miR-29-MCL-1 axis and its dysregulation caused by reduced insulin and mTORC1 inhibition increases the vulnerability of a diabetic heart to structural damage.     

Damage associated molecular pattern molecule-induced microRNAs (DAMPmiRs) in human peripheral blood mononuclear cells

Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10(-4) and p<3.7×10(-3)), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1(+/+)) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1(-/-) MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic "PAMPmiR". Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K(+) channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.     

心肌细胞过表达miR-27b导致小鼠发生心肌纤维化和线粒体损伤

以往的miRNA芯片研究结果显示, miR-27b在人类心脏疾病标本和压力负荷引起的小鼠心肌肥厚模型中表达水平明显升高, 提示其在心脏疾病发生过程中发挥了重要功能。为研究miR-27b在心脏组织中的功能, 文章建立了在心肌细胞特异性 a-肌球蛋白重链(a-MHC)启动子(5.5 kb)控制下过表达miR-27b的转基因小鼠。通过Real-time PCR检测, 发现miR-27b前体和成熟体表达水平在转基因小鼠心脏组织中明显升高。miR-27b转基因小鼠不仅出现心肌肥厚, 还表现出明显的心肌纤维化。进一步研究表明心肌纤维化的关键调节分子金属基质蛋白酶13(MMP13)是miR-27b的靶分子, 在miR-27b转基因小鼠中MMP13显著下调, 胶原分子I和 III则显著上调。此外, 还发现miR-27b转基因小鼠会出现心脏超微结构的损伤。以上研究结果表明, miR-27b可能通过抑制MMP13促进心肌纤维化。     

miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy

Multi-drug resistance is a major challenge to hepatocellular carcinoma (HCC) treatment, and the over-expression or deletion of microRNA (miRNA) expression is closely related to the drug-resistant properties of various cell lines. However, the underlying molecular mechanisms remain unclear. CCK-8, EdU, flow cytometry, and transmission electron microscopy were performed to determine cell viability, proliferation, apoptosis, autophagic flow, and nanoparticle characterization, respectively. In this study, the results showed that the expression of miR-26b was downregulated following doxorubicin treatment in human HCC tissues. An miR-26b mimic enhanced HCC cell doxorubicin sensitivity, except in the absence of p53 in Hep3B cells. Delivery of the proteasome inhibitor, MG132, reversed the inhibitory effect of miR-26b on the level of p53 following doxorubicin treatment. Tenovin-1 (an MDM2 inhibitor) protected p53 from ubiquitination-mediated degradation only in HepG2 cells with wild type p53. Tenovin-1 pretreatment enhanced HCC cell resistance to doxorubicin when transfected with an miR-26b mimic. Moreover, the miR-26b mimic inhibited doxorubicin-induced autophagy and the autophagy inducer, rapamycin, eliminated the differences in the drug sensitivity effect of miR-26b. In vivo, treatment with sp94dr/miR-26b mimic nanoparticles plus doxorubicin inhibited tumor growth. Our current data indicate that miR-26b enhances HCC cell sensitivity to doxorubicin through diminishing USP9X-mediated p53 de-ubiquitination caused by DNA damaging drugs and autophagy regulation. This miRNA-mediated pathway that modulates HCC will help develop novel therapeutic strategies.     

microRNA在常见致死性心脏病中的改变

微小RNA(micro RNA,mi RNA)是一类真核生物内源性非编码单链的小RNA分子,长度大约为19-23个核苷酸,拥有高度的保守性,不编码蛋白质,也是近年来研究最热门的一个新领域,通过与靶m RNA特异性结合来调节基因表达,且表达都具有组织特异性。最近,许多研究表明mi RNA在心血管系统疾病和肿瘤疾病方面的相关研究都取得了突破性的进展,mi RNA在肿瘤疾病中是通过调节癌基因及抑癌基因而调控肿瘤的生物学过程,在心血管系统疾病中与心肌肥厚及心肌再生等过程有密切的关系,包括冠状动脉疾病、心肌肥大、心肌梗死、心律失常、高血压和心力衰竭等疾病,且在心脏病学中扮演着及其重要的角色。Mi RNA的表达量增加或者减少对心血管疾病都有影响,该文对新近有关的mi RNA在心血管系统疾病中的研究进展、诊断、治疗以及预后予以综述。     

miR-34a Modulates Angiotensin II-Induced Myocardial Hypertrophy by Direct Inhibition of ATG9A Expression and Autophagic Activity

Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3′-UTR, but not to the mutated 3′-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity.     

miR-106b在阿尔茨海默病中的作用机制

目的 探讨miR-106b在阿尔茨海默病(Alzheimer's disease,AD)发病中的作用.方法 取3月龄和6月龄APPswe/PSΔE9小鼠脑组织,进行microRNA芯片的检测;利用real-time PCR检测3、6、9月龄APPswe/PSΔE9小鼠脑组织中miR-106b的表达,对芯片检测结果进行验证;通过构建miR-106b稳定转染细胞系和miR-106b knockdown研究miR-106b与TGFBR2表达之间的关系; 构建TGFBR2 3'UTR-荧光素酶报告载体,验证miR-106b是否可以直接调控TGFBR2蛋白的表达;采用Western blot的方法检测APPswe/ΔPSΔE9小鼠和对照小鼠脑组织中TGFBR2蛋白的表达情况.结果 miR-106b在3月龄和6月龄AD模型小鼠脑组织中表达升高,在9月龄模型小鼠脑组织中表达降低;通过体外实验,我们发现miR-106b与TGFBR2蛋白的表达呈负相关;荧光素酶报告实验表明TGFBR2 3'UTR序列中包含miR-106b的结合位点;TGFBR2蛋白在3、6、9、12月龄AD模型小鼠脑组织中表达均降低.结论 miR-106b可能通过调控TGFBR2蛋白的表达影响TGF-β信号通路,从而参与AD的发病.