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MiR-30c and fatty acid synthase (fas) both play important roles in physiological processes such as lipid synthesis and fat metabolism. Predictive analysis revealed that fas is a target gene of miR-30c with multiple seed sites. Seed sites are useful to predict miRNA targeting relationships; however, detailed analyses of seed sites in fish genomes remain poorly studied. In this study, the regulatory relationship between miR-30c and fas, number and effect of seed regions, and mechanism by which miR-30c regulates lipid metabolism were evaluated in blunt snout bream (Megalobrama amblycephala). Four miR-30c target sites for fas were identified using various prediction tools. miR-30c mimics were transfected into 293 T cells, and dual-luciferase reporter assays were used to evaluate the roles of different fas target sites. When a single target site was mutated, relative luciferase activity was higher than that in the control group, with different activity levels depending on the mutation site. When multiple target sites were mutated, relative luciferase activity increased significantly as the number of mutation sites increased and was the highest when the four sites were mutated simultaneously. The miR-30c agomir was injected into the abdominal cavity of M. amblycephala at various concentrations for analyses of physiological and biochemical parameters in the liver and blood and the expression of genes related to lipid metabolism in the liver. Total cholesterol, free fatty acid, triglyceride, and low density lipoprotein levels were significantly lower after miR-30c agomir injection comparing to the control (P < 0.05). Additionally, the expression levels of genes related to lipid metabolism were significantly lower after miR-30c agomir injection than in the control (P < 0.05). In summary, this study identified four specific miR-30c target sites in the 3′ UTR of fas mRNA; the effects of these sites are cumulative, and the redundancy ensures the accurate regulation of fas during evolution. In addition, miR-30c has a negative regulatory effect on fas and regulates lipid metabolism via various genes related to this process. Therefore, the regulation of miR-30c can effectively ameliorate the side effects of a high-fat diet on liver function in M. amblycephala. [Display omitted] • miR-30c specifically bound to fas through these four seed sites and binding at multiple sites showed cumulative effects. • Redundancy of miR-30c at multiple sites of fas likely ensures precise regulation of fas during evolutionary processes. • miR-30c can improve lipid metabolism disorders in Megalobrama amblycephala by regulating the expression of fas. [ABSTRACT FROM AUTHOR]

Abstract Spinal cord ischemia/reperfusion (I/R) injury is a severe complication in many surgeries. Although microRNAs (miRNAs) are involved in I/R-caused spinal cord injury (SCI), the mechanism that underlies miR-30c interacted with SCI remains elusive. In this study, I/R surgery or oxygen-glucose deprivation (OGD) were performed to establish SCI model in vivo or in vitro, respectively. Basso, Beattie and Bresnahan (BBB) score, spinal cord infarct, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining, flow cytometry and enzyme linked immunosorbent assays (ELISA) were used to investigate SCI. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the abundances of miR-30c and sirtuin 1 (SIRT1) either in spinal cord or PC12 cells. Luciferase assay and RNA immunoprecipitation (RIP) were performed to probe the interaction between miR-30c and SIRT1. Western blot and immunofluorescence assays were used to analyze SIRT1 protein expression. Our results showed that I/R increased miR-30c expression and induced SCI, revealed by decreasing BBB score, enhancing apoptosis, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression. However, miR-30c knockdown attenuated I/R-induced SCI in vivo. Moreover, depletion of miR-30c protected PC12 cells against OGD-caused apoptosis and inflammatory response. In addition, SIRT1 was limited by miR-30c, silencing of which reversed anti-miR-30c-mediated inhibitory effect on apoptosis and secretion of inflammatory cytokines in PC12 cells after OGD treatment. Collectively, abrogation of miR-30c inhibited spinal cord ischemia reperfusion injury through targeting SIRT1, providing a promising biomarker of prognosis and therapeutic for SCI. [ABSTRACT FROM AUTHOR]

Background Multiple sclerosis is an immune-mediated inflammatory disease of central nervous system. MicroRNAs play important roles in autoimmune diseases such as MS. Objectives The aim was to evaluate the expression pattern of miR-34a, miR-199a, miR-30c and miR-19a in peripheral blood derived CD4+ T lymphocytes of both relapsing and remitting phases of MS. Methods Blood samples from 40 RRMS patients (20 in relapsing and 20 in remitting phase) and 20 healthy volunteers were taken. CD4+ T cells were isolated. The expression level of miR-34a, miR-199a, miR-30c and miR-19a, and the percentage of Th17 and Treg cells were measured. Expression of master transcription factors of Th17 and Treg cells and several targets of these miRNAs were also evaluated. Results Data indicated an increased expression of miR-34a, miR-30c and miR-19a in relapsing phase and decreased expression of miR-199a in remitting phase. ROC curve data add other prestigious information of miR-34a, miR-199a, miR-30c and miR-19a by defining relapsing and remitting phase and also healthy cases with high specificity and sensitivity at a proposed optimum cut-off point. Conclusion Collectively, we showed a correlation between the four miRNAs with different phases of MS and their possible involvement in differentiation pathways of Th17 cells, as the most important players in MS. [ABSTRACT FROM AUTHOR]

Background Aberrant expression of miRNAs was involved in tumor initiation, progression and metastasis in multiple cancers. Many kinds of microRNAs in esophageal squamous cell carcinoma (ESCC) have been researched, whereas miR-30c has not been included. Methods Firstly, we explored the expression of miR-30c in ESCC tissue and serum samples and its relations to the survival. To further investigate its effects on ESCC cells, we completed a series of experiments. We detected the effects of ectopic miR-30c expression on the proliferation, migration and invasion of ESCC cells in vitro . We identified the target role of SNAI1 in ESCC using Dual-luciferase reporter assay and western blot assay. Results The results showed miR-30c was significant down-regulated in ESCC tissues and cell lines. Clinically, we found lower miR-30c expression was significantly correlated with worse ESCC progression and survival. Also we clarified that miR-30c suppressed cell proliferation, invasion and epithelial to mesenchymal transition (EMT) of ESCC cell lines. What's more, we figured out that miR-30c inhibits ESCC biological behaviors and EMT progress by directly binding to the 3′-UTR of SNAI1 . Conclusion This study provides new insight into the mechanism responsible for the development of human ESCC. Therefore, miR-30c could be a promising biomarker and a therapeutic target for ESCC in the future. [ABSTRACT FROM AUTHOR]

Objective Hydrogen sulfide has been recognized as an important neuroprotective agent in the nervous system. The present study aimed to study the effect and the underlying mechanisms of hydrogen sulfide on spinal cord ischemia-reperfusion injury (SCII). Methods The gene mRNA and protein expression were examined by RT-PCR and western blot, respectively. Spinal cord infarct zone was analyzed by Triphenyltetrazolium chloride staining. Cell apoptosis was detected by MTT assay. The relationship between miR-30c and CasC7 was analyzed by RNA immunoprecipitation (RIP) and RNA pull-down. Results We observed that SCII rat model have a bigger spinal cord infarct zone than the control rat, but NaSH preprocessing could reduce spinal cord infarct zone in SCII rat. OGD/R induced cell apoptosis, and NaSH preprocessing reduced the OGD/R-induced SH5Y-SY cells apoptosis. CasC7 was decreased in SCII rat and OGD/R-induced SH5Y-SY cells, while miR-30c expression was increased. NaSH preprocessing upregulated CasC7 and downregulated miR-30c in OGD/R-induced SH5Y-SY cells. In OGD/R induced SH5Y-SY cells with NaSH preprocessing, knockdown of CasC7 could upregulate miR-30c expression, promote cell apoptosis and downregulate miR-30c’s target gene expression. The RIP and RNA pull-down demonstrated that CasC7 functioned as a miR-30c decoy, and miR-30c inhibitor could reverse the effect of si-CasC7. Moreover, intrathecal injection of si-CasC7 upregulated miR-30c expression and increased spinal cord infarct zone in SCII rat with NaSH preprocessing. Conclusion Hydrogen sulfide protects spinal cord by upregulating CasC7 expression in SCII rat model. [ABSTRACT FROM AUTHOR]

Tuberculous pleurisy (PLTB) is a common form of extrapulmonary tuberculosis. It often resolves without chemotherapy being hence considered a rather benign manifestation of the disease. Patients with PLTB mount an effective anti-mycobacterial response, unlike those with active pulmonary TB (pTB) that were shown to present an imbalance in plasma immune and endocrine mediators. In this work, we explored whether expression of the active isoform of the glucocorticoid receptor (hGRα) in the context of the inflammatory-anti-inflammatory responses of TB patients may be associated to microRNA levels. As expected, the inflammatory response triggered in patients coexists with increased circulating cortisol and altered hGRα levels in the peripheral blood mononuclear cells. However, while hGRα expression is significantly downregulated in PLTB, its levels in pTB patients are higher within the control values. These results point out to the existence of an additional mechanism tending to preserve hGRα levels probably to deal with the chronic inflammation observed in pTB. In this regard, we found that miR-30c is strongly downregulated in mononuclear cells of pTB patients compared to PLTB cases, showing an expression profile opposite to that seen with hGRα. Interestingly, low levels of miR-30c are specific for this active form of TB, as its expression is not altered in mononuclear cells from either healthy controls or patients with tuberculous or non-tuberculous pleurisy. Moreover, miR-30c and hGRα also showed an inverse expression pattern in M. tuberculosis -stimulated THP-1 macrophage cultures. In sum, our studies identify miR-30c as a specific correlate of pulmonary manifestations of TB, potentially involved in the altered glucocorticoid sensitivity observed in these patients. [ABSTRACT FROM AUTHOR]

Background Small non-coding microRNAs (miR) have important regulatory roles and are used as biomarkers of disease. We investigated the relationship between lipoproteins and circulating miR-30c, evaluated how they are transported in circulation and determined whether statins altered the circulating concentration of miR-30c. Methods To determine the relationship between lipoproteins and circulating miR-30c, serum samples from 79 subjects recruited from a lipid clinic were evaluated. Ultracentrifugation and nanoparticle tracking analysis was used to evaluate the transportation of miR-30c in the circulation by lipoproteins and extracellular vesicles in three healthy volunteers. Using archived samples from previous studies, the effects of 40 mg rosuvastatin (n = 22) and 40 mg pravastatin (n = 24) on miR-30c expression was also examined. RNA extraction, reverse transcription-quantitative real-time polymerase chain reaction was carried out using standard procedures. Results When stratified according to total cholesterol concentration, there was increased miR-30c expression in the highest compared to the lowest tertile (p = 0.035). There was significant positive correlation between miR-30c and total- (r = 0.367; p = 0.002) and LDL-cholesterol (r = 0.391; p = 0.001). We found that miR-30c was transported in both exosomes and on HDL3. There was a 3.8-fold increased expression of circulating miR-30c after pravastatin treatment for 1 year (p = 0.005) but no significant change with atorvastatin after 8 weeks (p = 0.145). Conclusions This study shows for the first-time in humans that circulating miR-30c is significantly, positively correlated with total- and LDL-cholesterol implicating regulatory functions in lipid homeostasis. We show miR-30c is transported in both exosomes and on HDL3 and pravastatin therapy significantly increased circulating miR-30c expression adding to the pleiotropic dimensions of statins. [ABSTRACT FROM AUTHOR]

Aims This study aimed to determine the role of miR-30c in the process of hepatic stellate cells (HSCs) activation and liver fibrosis/cirrhosis and to explore the underlying mechanism. Main methods A microarray analysis of miRNAs in HSCs was performed, and quantitative RT-PCR analyses were conducted to validate the results in HSCs, cirrhotic liver tissues and plasma. Rat HSCs were stimulated with angiotensin II (AngII) and transfected with miR-30c mimics/inhibitor to elucidate the underlying mechanism. Key findings miR-30c was down-regulated during HSCs activation and in cirrhotic liver tissues and plasma. This miRNA was found to be involved in HSCs activation by repressing the expression of one of its target genes—plasminogen activator inhibitor-1 (PAI-1), and AngII stimulation decreased the expression of miR-30c. However, the up-regulation of miR-30c by specific mimics could down-regulate the mRNA and protein expression of α-SMA, a marker of HSCs activation, in AngII-stimulated HSCs and attenuate the AngII-induced activation of HSCs. Significance miR-30c is involved in HSCs activation and might be a novel biomarker of liver fibrosis/cirrhosis. [ABSTRACT FROM AUTHOR]

Semaphorin3A (sema3a), mainly localized in the olfactory neuron layer and periglomerular layer, is essential for the normal arrangement of axons in the olfactory bulb both in embryonic and adult mice functioning through its dynamic spatiotemporal expression. The regulators that can modulate the expression of sema3a by direct interaction, however, are unknown. In order to find the regulators of sema3a in the olfactory bulb, we focused on microRNAs, well-known post-transcriptional regulators. We found that axon guidance is the main molecular and biological process ongoing in the steady-state olfactory bulb of the adult mouse by screening the abundant microRNAs and exploring their functions in the olfactory bulb via our customized microRNA arrays, Gene Ontology and Kyoto Encyclopedia of Genes annotation. Furthermore, we traced the expression of three candidate regulators (miR-30c, miR-200b, and miR-429) and sema3a by the quantitative real-time polymerase chain reaction and immunohistochemistry. The results showed that only miR-30c expression corresponded inversely with sema3a. Finally, miR-30c was verified to be a specific regulator of sema3a by dual luciferase reporter assay in vitro. Taken together, our results suggested that miR-30c is a potential regulator in axon-guidance by suppressing the expression of sema3a, which will give new insights in elucidating the mechanism of architectonic and functional maintenance of the olfactory bulb. [ABSTRACT FROM AUTHOR]

Abstract: Osteoblast differentiation is tightly regulated by post transcriptional regulators such as microRNAs (miRNAs). Several bioactive materials including nano-bioglass ceramic particles (nBGC) influence differentiation of the osteoblasts, but the molecular mechanisms of nBGC-stimulation of osteoblast differentiation via miRNAs are not yet determined. In this study, we identified that nBGC-treatment stimulated miR-30c expression in human osteoblastic cells (MG63). The bioinformatics tools identified its regulatory network, molecular function, biological processes and its target genes involved in negative regulation of osteoblast differentiation. TGIF2 and HDAC4 were found to be its putative target genes and their expression was down regulated by nBGC-treatment in MG63 cells. Thus, this study advances our understanding of nBGC action on bone cells and supports utilization of nBGC in bone tissue engineering. [Copyright &y& Elsevier]

To explore the neuroprotective effect and mechanism of miR-30c in rats with cerebral ischemia/reperfusion (CI/R) injury. in vivo, miR-30c mimic was transfected before the rats were treated with CI/R injury, and the neurological damage was evaluated. The expression of miR-30c was analyzed by RT-PCR.The area of cerebral infarction and pathological damage in CI/R rats were detected by TTC, H&E, Nissl and TUNEL staining. The expression of SOX9, BNDF and NT-3 were analyzed by immunofluorescence and western blot. The target relationship between SOX9 and miR-30c was analyzed by dual- luciferase reporter assay. in vitro, miR-30c mimic, SOX9 mimic and negative control were transfected before the OGD/R model of HT22 was established. CCK8 and flow cytometry were used to analyze the effect of miR-30c on the cell viability and apoptosis of HT22 cells in OGD/R condition. The protein expression of SOX9, BNDF and NT-3 were analyzed by immunofluorescence and western blot. The expression of miR-30c in the CI/R rats were significantly lower than that of sham group (p < 0.05). The area of cerebral infarction and the number of pathological damage and apoptosis in hippocampus were improved by miR-30c. miR-30c reduced SOX9 expression and improved BNDF and NT-3 expression in hippocampus compared with CI/R group (p < 0.05). The results of dual-luciferase reporter showed that SOX9 was the target of miR-30c. Under OGD/R condition, miR-30c mimic promoted cell viability and reduced cell apoptosis. miR-30c decreased the expression of SOX9, which was consistent with the results of immunofluorescence. Moreover, miR-30c could increase the expression of BNDF and NT-3 in HT22 cells. After transfection of SOX9 mimic, it was found that the above effects of miR-30c were all reversed. miR-30c can improve the pathological damage of rats with CI/R injury and plays a neuroprotective role both in vivo and in vitro by targeting SOX9. [ABSTRACT FROM AUTHOR]

• The miR-30c was up-regulated in miniature pig with CIAKI. • The miR-30c suppressed apoptosis on renal cells stimulated by iohexol. • In the pig model, miR-30c inhibited the CIAKI development. Contrast-induced acute kidney injury (CIAKI) is the third most common cause of hospital-acquired AKI. It has been demonstrated that microRNA-30c (miR-30c) was upregulated in the CIAKI. However, the underlying mechanism remain unclear. The CIAKI was induced in miniature pig. The expression profile of miR-30c in the kidney was evaluated by qPCR. The pathways regulated by miR-30c was identified by qPCR and western blot on renal tubular epithelial cells isolated from miniature pig. Finally, the potential therapeutic application of targeting miR-30c was assessed in the pig model of CIAKI. The miR-30c was up-regulated in miniature pig with CIAKI. The miR-30c suppressed cell apoptosis, expression of NLRP3, the secretion of IL-1β and caspase-1 p10 on renal cells stimulated by iohexol in vitro. In the pig model, miR-30c inhibited the CIAKI development. Our data demonstrated that the miR-30c induced by CIAKI could suppress cell apoptosis and kidney injury via targeting NLRP3. Therefore, targeting miR-30c might be a novel therapeutic candidate for CIAKI treatment and prevention. [ABSTRACT FROM AUTHOR]