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MicroRNAs (miRNAs) are increasingly reported to have important roles in diverse biological and pathological processes. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the miR-1 function in the heart are only beginning to emerge. In this study, we investigated miR-1 expression and its potential role in the mouse model of viral myocarditis (VMC). The expression levels of miR-1 and its target gene Connexin 43 (Cx43) were measured by real-time PCR and western blotting, respectively. The miR-1 expression levels were significantly increased in cardiac myocytes from VMC mice in comparison with control samples (relative expression: 10 ± 2.5 vs. 31 ± 7.6, P < 0.05). Among the target genes of miR-1, the expression Cx43 protein was significantly reduced in such mice while there was no significant difference in the its mRNA levels. Our results revealed an inverse correlation between miR-1 levels and Cx43 protein expression in VMC samples. Using a bioinformatics-based approach, we found two identical potential binding sites were found in mouse miR-1 and Cx43 3'- untranslated region, this confirms a possible regulatory role of miR-1. In cultured, miRNA transfected myocardial cells, we show overexpression of miR-1 accompanied by a decrease in Cx43 protein's expression. There was only a slight (not statistically significant) drop in Cx43 mRNA levels. Our results indicate that miR-1 is involved in VMC via post-transcriptional repression of Cx43, and might constitute potentially valuable data for the development of a new approach in the treatment of this disease.

Acute membrane damage due to traumatic brain injury (TBI) is a critical precipitating event. However, the subsequent effects of the mechanical trauma, including mitochondrial and lysosomal membrane permeability (MOMP and LMP) remain elusive. The main objective of the current study was to assess the role of a putative membrane-resealing agent poloxamer 188 (P188) in MOMP and LMP in response to a well-defined mechanical insult. Using an in vitro cell shearing device (VCSD), mechanical injury resulted in immediate disruption of membrane integrity in cultured primary neurons, and neurons were treated with P188 or a cathepsin B inhibitor (CBI) after VCSD 10 min. The protective effect of P188 on cultured primary neurons was first detected visually with a light microscope, and measured by MTT assay and LDH assay. The validity of monitoring changes in mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining, and Western blot for cytochrome c and truncated Bid (tBid) in purified mitochondria was also performed. In addition, lysosomal integrity was detected by blotting for cathepsin B and tBid in purified lysosomes. Our results showed post-injury P188 treatment moderated the dissipation of ΔΨm in mitochondria, and inhibited VCSD-induced cytochrome c release from mitochondria as well as cathepsin B from lysosomes. Cathepsin B inhibition (CBI) could also increase cell viability, maintain mitochondrial membrane potential, and repress VCSD-induced release of cytochrome c from mitochondria to cytosol. Both P188 and CBI treatment decreased the cytosolic accumulation of tBid in supernatant of purified lysosomes, and the amount of mitochondrial localized tBid. These data indicate injured neurons have undergone mitochondrial and lysosomal membrane permeability damage, and the mechanism can be exploited with pharmacological interventions. P188's neuroprotection appears to involve a relationship between cathepsin B and tBid-mediated mitochondrial initiation of cell death.

Previous data demonstrate that JMJD2A is a cancer-associated gene and may be involved in human breast cancer by demethylation of H3K9me3. The aim of this study was to investigate depressive effects on JMJD2A by transfection with JMJD2A-sepcific siRNA in human breast cancer cell line MDA-MB-231 and effects on cell proliferation, invasion and migration. JMJD2A-specific siRNA was chemically synthesised and transfected into human breast cancer cell line MDA-MB-231. Expression levels of JMJD2A were detected by quantitative real-time PCR and Western blot analysis. Cells proliferation was evaluated by using flow cytometric anlysis and MTT assay. The abilities of invasion and migration were evaluated by cell migration and invasion assay with Boyden chambers. The results showed that the transfection was successful and expression levels of JMJD2A mRNA and protein in siRNA group were both down-regulated. By MTT assay, the mean actual absorbance in siRNA group was significantly lower than that in blank control group (P < 0.05) and negative control group (P < 0.05). In addition, the percentage of cells in G0/G1 phase in siRNA group was significantly more than that in blank control group (P < 0.05) and negative control group (P < 0.05). Furthermore, by cell invasion and migration assay, the decreased number of migrated cells in siRNA group was observed (P < 0.05). These data imply that silencing JMJD2A gene could result in cell cycle change and proliferation inhibition, and lead to suppress tumor cell invasion and migration. It provides a new perspective in understanding the pleiotropic functions of JMJD2A and its contribution to human breast cancer.

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Abstract. MicroRNAs (miRNAs) comprise a broad class of small non-coding RNAs that control the expression of complementary target messenger RNAs. The dysregulation of miRNAs by several mechanisms has been described in various disease states, including cardiac disease. Although an etiological link between viral myocarditis (VMC) and idiopathic dilated cardiomyopathy (DCM) has long been recognized, the true extent of this association is uncertain. Previous studies of the two diseases have focused on protein degradation systems. In the present study, miR-21 expression and its potential role in VMC and DCM was investigated. The expression levels of miR-21, its target gene sprouty homolog 1 (SPRY1) and mitogen-activated protein kinase (MAPK) were measured by quantitative polymerase chain reaction. The protein levels of SPRY1 and MAPK were also determined by western blotting. miR-21 levels were significantly increased in cardiac myocytes from VMC and DCM in comparison with control samples. The levels of SPRY1 were decreased and MAPK activity was increased. Using a bioinformatics-based approach, an identical potential binding site was identified in mouse miR-21 and the SPRY 3' untranslated region (3' UTR), suggesting a regulatory role for miR-21. In cultured, miRNA-transfected myocardial cells, the overexpression of miR-21 was associated with a decrease in SPRY1 protein expression and an increased expression of the MAPK protein. These findings revealed that changes in the expression of miRNAs may contribute to the pathogenesis of VMC to DCM and establish the therapeutic efficacy of miRNA targeted intervention in a cardiovascular disease setting.

Aconitine, a diterpenoid alkaloids derived from Aconitum plants, is widely employed to treat various diseases. The aim of the present study was to investigate the apoptotic effect of aconitine in H9c2 cardiac cells. H9c2 cell apoptosis induced by aconitine was detected by a Cell Counting kit-8 assay, DAPI staining, Annexin V-FITC/propidium iodide double staining and western blotting. The effects of aconitine on reactive oxygen species levels and mitochondrial membrane potential were confirmed by fluorescence microscopy and flow cytometry. In addition, ATP contents were determined using a ATP-dependent bioluminescence assay kit. The levels of peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) expression and apoptosis-associated proteins including Caspase-3, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and Cytochrome c were also assessed. Taken together, the results indicated that aconitine may inhibit cell viability, decrease PGC-1α expression, induce mitochondrial dysfunctions, upregulate Cytochrome c, Bax and Caspase-3, and downregulate Bcl-2, suggesting that aconitine may induce apoptosis through mitochondria-mediated signaling pathways in H9c2 cells.