Academic literature on the topic 'MiR-9a'

Background/Aims: Previous studies have suggested that autophagy is activated in distinct cerebrovascular diseases, including stroke. However, the underlying regulatory mechanism of autophagy under stroke remained elusive. Accumulating evidence indicates that dysfunctions of microRNAs (miRNAs) are involved in the pathological process of stroke. Therefore, this study was taken to identify the effect of microRNA-9a-5p (miR-9a-5p) on autophagy in rats following stroke. Methods: The rat model of focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) surgery; The neurological outcomes were defined by neurological evaluation and infarct volume; The western blotting and immunofluorescence assays were used to detected the protein levels of microtubule-associated protein 1 light chain 3 (LC3) and autophagy related 5 (ATG5); The mRNA level of miR-9a-5p, LC3 and ATG5 were quantified by real-time RT-PCR; The luciferase activities of ATG5 and miR-9a-5p was detected by luciferase assay. Results: We showed here that the level of miR-9a-5p was decreased in the ischemic region of rats after MCAO. Overexpression of miR-9a-5p by miR-9a-5p agomir reduced infarct volume and alleviated neurological deficit. Moreover, we found that autophagy was activated by miR-9a-5p inhibition and inactivated by miR-9a-5p overexpression both in the MCAO rat and in SY-5Y cell lines, and unchanged by miR-masks as indicated by LC3 expression. Furthermore, the protein level of ATG5 was decreased by miR-9a-5p overexpression, but increased by miR-9a-5p inhibition and unchanged by miR-masks transfection. In addition, the luciferase assay data showed that miR-9a-5p suppressed the luciferase activity of 3’UTR of ATG5, whereas the repressive effect was relieved by mutation of binding sites. Conclusion: Our study demonstrated that miR-9a-5p may play a critical role in regulating the process of autophagy through targeting ATG5 expression, and overexpression of miR-9a-5p may be a potential approach in alleviating ischemia injury induced by MCAO.

Abstract The Drosophila melanogaster peripheral nervous system (PNS) comprises the sensory organs that allow the fly to detect environmental factors such as temperature and pressure. PNS development is a highly specified process where each sensilla originates from a single sensory organ precursor (SOP) cell. One of the major genetic orchestrators of PNS development is Senseless, which encodes a zinc finger transcription factor (Sens). Sens is both necessary and sufficient for SOP differentiation. Senseless expression and SOP number are regulated by the microRNA miR-9a. However, the reciprocal dynamics of Senseless and miR-9a are still obscure. By coupling single-molecule FISH with immunofluorescence, we are able to visualize transcription of the mir-9a locus and expression of Sens simultaneously. During embryogenesis, we show that the expression of mir-9a in SOP cells is rapidly lost as Senseless expression increases. However, this mutually exclusive expression pattern is not observed in the third instar imaginal wing disc, where some Senseless-expressing cells show active sites of mir-9a transcription. These data challenge and extend previous models of Senseless regulation and show complex co-expression dynamics between mir-9a and Senseless. The differences in this dynamic relationship between embryonic and larval PNS development suggest a possible switch in miR-9a function. Our work brings single-cell resolution to the understanding of dynamic regulation of PNS development by Senseless and miR-9a.

This study assessed the mechanism of tanshinone A in regulating biological characteristics of Hematopoietic Stem Cell (HSC) in liver cirrhosis through targeting of miR-9a-5p. HSC cells were divided into negative control group and stimulated miR-9a-5p inhibitor group. Transfection was performed according to specification of the kit. Expression of miR-9a-5p was assessed with Real-time polymerase chain reaction (PCR). Cell proliferation was tested with flow cytometry (FCM), and α-smooth muscle actin (SMA) and Type I collagen expressions were detected with Western Blot assay. Caspase-3 activity was tested with spectrophotometry, while variation of inflammatory factor was detected with enzyme-linked immunosorbent assay (ELISA). There was higher miR-9a-5p level in HSC induced by Chemokine (C-C motif) ligands 4 (CCL-4). Biological characteristics of HSC induced by CCL-4 was restrained by down-regulation of miR-9a-5p, and presentation quantity of α-SMA and Type I collagen was reduced. So, occurrence of inflammation and migration of HSC could be restrained. The presentation quantity of Type I collagen was reduced with tanshinone A, and expression of miR-9a-5p was reduced. HSC characteristics in liver cirrhosis were affected by tanshinone A probably through regulating miR-9a-5p. It could provide a brand-new selection for treatment on liver cirrhosis.

ATP-sensitive K+ (KATP) channels regulate plasma membrane excitability. The Kir6.1/SUR2B isoform of KATP channels is expressed in vascular smooth muscles and plays an important role in vascular tone regulation. This KATP channel is targeted by several reactive species. One of them is methylglyoxal (MGO), which is overly produced with persistent hyperglycemia and contributes to diabetic vascular complications. We have previously found that MGO causes posttranscriptional inhibition of the KATP channel, aggravating vascular tone regulation. Here we show evidence for the underlying molecular mechanisms. We screened microRNA databases and found several candidates. Of them, miR-9a-3p, increased its expression level by ∼240% when the cultured smooth muscle cell line was exposed to micromolar concentrations of MGO. Treatments with exogenous miR-9a-3p downregulated the SUR2B but not Kir6.1 mRNA. Antisense nucleotides of miR-9a-3p alleviated the effects of MGO. Quantitative PCR showed that the targeting sites of the miR-9a-3p were likely to be in the coding region of SUR2B. The effects of miR-9a-3p were mostly eliminated when the potential targeting site in SUR2B was site-specifically mutated. Our functional assays showed that KATP currents were impaired by miR-9a-3p induced with MGO treatment. These results suggest that MGO exposure raises the expression of miR-9a-3p, which subsequently downregulates the SUR2B mRNA, compromising KATP channel function in vascular smooth muscle.

Purpose. Present research is aimed at exploring the effect of miR-9a-5p on mitochondrial autophagy and alleviating cellular oxidative stress injury in ischemic stroke. Methods. SH-SY5Y cells were cultured with oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemia/reperfusion. The cells were treated in an anaerobic incubator (95% N2, 5% CO2) for 2 h and then reoxygenated in the normoxic condition for 24 h with 2 ml of normal medium. Cells were transfected with miR-9a-5p mimic/inhibitor or negative control. The RT-qPCR assay was utilized to measure the mRNA expression. Western blot was utilized to evaluate the protein expression. The CCK-8 assay was conducted to detect cell viability. Flow cytometry was applied to examine apoptosis and the cell cycle. The ELISA assay was applied to measure the contents of SOD and MDA in mitochondria. Autophagosomes were observed via electron microscopy. Results. By comparison with the control group, the miR-9a-5p expression in the OGD/R group obviously declined. Mitochondrial crista breaks, vacuole-like changes, and increased autophagosome formation were observed in the OGD/R group. OGD/R injury enhanced oxidative stress damage and mitophagy. When transfected with the miR-9a-5p mimic, mitophagosome production of SH-SY5Y cells decreased and oxidative stress injury was inhibited. However, the miR-9a-5p inhibitor obviously increased mitophagosome production and enhanced oxidative stress injury. Conclusion. miR-9a-5p protects against ischemic stroke by inhibiting OGD/R-induced mitochondrial autophagy and alleviating cellular oxidative stress injury.

Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p < 0.01 for all), respectively, distinguishing rats with mTBI from naïve rats with 100% sensitivity and specificity. DdPCR further identified a subpopulation of mTBI patients with plasma miR-9-3p (n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at <2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population.

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

The muscular system is a highly complex and important system in the body. Proper muscle physiology is critical for locomotion, digestion, circulation, reproduction as well as for metabolic and immune homeostasis. Defects in muscle development, structure or function result in muscle disorders and diseases. Chapter 1 reviews the important events of muscle development and growth as well as the various processes that are involved in the regulation of the same. The muscle disorders that occur due to the mis -regulation of these processes are discussed. Specifically, the significance of microRNAs in muscle development and function in the context of cardiac hypertrophy has been described. Chapter 1 also explains the importance of mitochondrial morphology and function for normal tissue functioning along with the dynamic processes that mediate changes in mitochondrial shape and size, namely fusion and fission. Thus, the first chapter discusses what is known and unknown about the roles played by microRNAs in and the regulators of mitochondrial dynamics during muscle development, and highlights questions being addressed in the present thesis. The advantages of using the Drosophila indirect flight muscle (IFMs) as a model system to address the unanswered questions are also enumerated in this chapter. The main features of IFM development and the similarities with vertebrate muscle development have been highlighted. Chapter 2 details the various Drosophila melanogaster lines, genetic tools and the experimental techniques used in this study. In the context of muscle function, the spatial and temporal regulation of the expression and assembly of structural proteins into structural units (sarcomeres) is crucial. The derailing of this process has been shown to result in number of muscle defects. One among them is cardiac hypertrophy which is characterized by mis-regulation of structural protein levels. Recently, miR-9 was shown to be involved in cardiac hypertrophy, however the role played by miR-9 in the regulation of muscle proteins is not known. Chapter 3 explains the novel findings regarding the role of miR-9a (Drosophila homolog) in the regulation IFM development. Results from IFM-specific over-expression of miR-9a during early muscle development indicate that miR-9a may have a role in repressing the regulators of dorsal longitudinal muscles (DLMs – a subset of the IFMs) patterning. The results discussed in this chapter also reveal that the over-expression of miR-9a exclusively in the IFMs during myofibrillogenesis rendered the flies flightless and the muscles showed hypercontraction -an auto-destructive process resulting from mis-regulated acto-myosin interactions. Bioinformatics analysis predicted 27 putative targets of miR-9a in muscles and Troponin-T (TnT), a structural protein component of the thin filament complex required for regulation of muscle contraction, was identified as putative target of miR-9a . Based on the observations that TnT levels are reduced when miR-9a is over-expressed and that overexpression of TnT, which lacked the miR-9a binding site, resulted in rescue of miR-9a over-expression phenotype, Chapter 3 concludes by stating that Troponin T is a major target of miR-9a in the IFMs. This finding along with the fact that human cardiac Troponin T (TNNT2) possesses a miR-9 binding site indicates that miR-9 could be involved in regulating the Troponin T levels during cardiac hypertrophy. Maintenance of mitochondrial quality and quantity is vital particularly in an energetically active tissue such as muscle. Mutations in the genes encoding regulators of mitochondrial dynamics have been shown to result in degenerative diseases. However, the process of mitochondrial fusion and fission are not well studied in vivo , especially during tissue development. In Chapter 4, the changes in mitochondrial morphology across IFM development have been described for the first time. Since all the major events of myogenesis during IFM development have been well demarcated and can be spatio-temporally tracked, it serves as a good model to investigate the mitochondria dynamics and roles of molecular players. Mitochondrial morphology was observed to be thin and continuous in the early stages of development, circular during mid-pupal phase and large and tubular during late pupal stage, indicating the occurrence of both mitochondrial fusion and fission during myogenesis. Further, Chapter 4 details the effect of knock down of the regulators of mitochondrial fusion and fission, namely Mitochondrial associated regulatory factor (Marf) and Dynamin related protein 1 (Drp1) during development of the IFMs. Genetic studies that revealed the importance of these regulators in mammalian development and human diseases are also mentioned. The results presented in Chapter 4 show that the knock down of Marf during development of the IFMs resulted in abnormal mitochondrial morphology and dysfunctional mitochondria that undergo mitophagy. While, Marf expression was found to be vital during early in IFM development, it did not appear to be as necessary during later in development. Knock down of Marf during the myofibrillogenesis phase of IFM development did not result in any defect in mitochondrial morphology function and myofibril ultrastructure. Importantly, it is shown in Chapter 4 that when Marf was depleted from early in development, adult flies exhibited abnormal sarcomeric structures, were incapable of flight and had greatly reduced life span. On the other hand, knock down of Drp1, the regulator of fission did not affect the mitochondrial morphology, muscle function and myofibril ultrastructure. Therefore, for the first time, this study reports that the spatiotemporal regulation of mitochondrial fusion and not fission appears to be critical for IFM development, maintenance, and function. In conclusion, the present study offers the following novel insights into the regulation of IFM development and the how specific developmental events influence IFM function; I) The major target of miR-9a in IFMs is Troponin T, whose levels must be regulated during myofibrillogenesis in order to achieve stoichiometric balance essential for muscle contraction. II) The expression of Marf, a mediator of mitochondrial fusion is crucial during a window of time in IFM development in order to achieve normal mitochondria morphology and function as well as structurally sound, functional muscle fibres in adult.