<Emphasis Type="Italic">miR-3075</Emphasis> Inhibited the Migration of Schwann Cells by Targeting Cntn2

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.     

Astragaloside IV alleviates Schwann cell injury in diabetic peripheral neuropathy by regulating microRNA-155-mediated autophagy

BackgroundMicroRNA-155(miR-155) is closely associated with diabetic peripheral neuropathy (DPN). Astragaloside IV (AST) is a significant extract of Astragalus membranaceus, which has been found to be effective in the treatment of DPN. However, whether astragaloside IV alleviate DPN via regulating miR-155-mediated autophagy remains unclear.PurposeThis study was designed to evaluate the effects of AST on DPN myelin Schwann cells injury and explore the mechanism of AST in treating DPN for the first time.MethodsGK rats fed with high-fat diet and RSC96 cells cultured in high glucose were used to establish DPN Schwann cells injury in vivo and in vitro model. The effects of AST on DPN were explored through blood glucose detection, nerve function detection, pathological detection and the expression of Neuritin detected by immunohistochemical. To study the effect of AST on the DPN Schwann cells autophagy and the upstream PI3K/Akt/mTOR pathway, the expressions of beclin-1 and LC3 were detected by western blot (WB) in sciatic nerves and by immunofluorescence (IFC) in RSC96 cells. The real-time polymerase chain reaction (RT-PCR) was applied to detect the expressions of miR-155, ATG5, ATG12 both in vivo and in vitro. The binding effect of miR-155 and target gene PI3KCA was verified by luciferase reporter gene assay. The expressions of PI3K, p-Akt/Akt, p-mTOR/mTOR were detected by WB and the expressions of PI3KCA were detected by RT-PCR in vitro. The apoptosis was detected by flow cytometry. Meanwhile, the influence of miR-155 overexpression and knocked down on the above indicators was also detected in RSC96 cells. At last, further mechanism experiments were conducted to verify the mechanism of AST regulating the autophagy and apoptosis of RSC96 cells.ResultsAST reduced blood glucose levels, alleviated peripheral nerve myelin sheath injury, and improved neurological function in DPN rats. In addition, AST enhanced the autophagy activity and alleviated the apoptosis in RSC96 cell. Mechanism study shown that AST promote autophagy via regulating miR-155-mediated PI3K/Akt/mTOR signaling pathways. AST reduced RSC96 cells apoptosis by promoting autophagy.ConclusionAST alleviate the myelin sheath injury of DPN caused by the apoptosis of Schwann cells via enhancing autophagy, which was attributed to inhibiting the activation of the PI3K/Akt/mTOR signaling pathway by upregulating miR-155 expression.     

miR-204/miR-211 downregulation contributes to Candidemia-induced kidney injuries via derepression of Hmx1 expression

Aims

This study was aimed to exploit the role of heme oxygenase Hmx1 and the potential miRNA mechanisms in the kidney injuries induced by urinary tract infection by Candida species/Candidemia.

Main methods

We employed a mouse model of systemic Candidiasis by injection of the Candida albicans strain SC5314 into C57BL/6 mice. Kidney injuries were assessed by measuring serum cystatin C (CysC), serum β₂-microglobulin (β₂-MG) and blood urea nitrogen (BUN). Validation of miRNA target gene was conducted by luciferase reporter gene assay, Western blot analysis and real-time RT-PCR.

Key findings

We showed here that Candidemia caused significant downregulation of microRNAs miR-204 and miR-211. In sharp contrast, Hmx1 expression was remarkably upregulated, particularly at the protein level. Computational analysis predicted Hmx1 as a target gene for both miR-204 and miR-211 that share the same seed site sequence. We then experimentally validated the targeting relationship between miR-204/miR-211 and Hmx1, which explains the reciprocal changes of expression of miR-204/miR-211 and Hmx1 in Candidemia. Administration of miR-204/miR-211 mimics substantially downregulated Hmx1 and mitigated the severity of the kidney injuries induced by Candidemia, as reflected by improved renal glomerular filtration rate (GFR) determined by serum cystatin C (CysC), serum β₂-microglobulin (β₂-MG) and blood urea nitrogen (BUN). Knockdown of miR-204/miR-211 worsened while forced expression of miR-204/miR-211 ameliorated kidney injuries in mice with systemic Candidiasis.

Significance

Our findings indicate that miR-204/miR-211 downregulation accounts at least partially for the Hmx1 upregulation and the miR-204/miR-211–Hmx1 signaling axis may contribute to immune-suppression in the host thereby the Candidemia-induced kidney dysfunction.     

miR-140-3p suppresses the proliferation and migration of macrophages

Macrophages benefit myelin debris removal, blood vessel formation, and Schwann cell activation following peripheral nerve injury. Identifying factors that modulate macrophage phenotype may advantage the repair and regeneration of injured peripheral nerves. microRNAs (miRNAs) are important regulators of many physiological and pathological processes, including peripheral nerve regeneration. Herein, we investigated the regulatory roles of miR-140-3p, a miRNA that was differentially expressed in injured rat sciatic nerves, in macrophage RAW264.7 cells. Observations from EdU proliferation assay demonstrated that elevated miR-140-3p decreased the proliferation rates of RAW264.7 cells while suppressed miR-140-3p increased the proliferation rates of RAW264.7 cells. Transwell-based migration assay showed that up-regulated and down-regulated miR-140-3p led to elevated and reduced migration abilities, respectively. However, the abundances of numerous phenotypic markers of M1 and M2 macrophages were not significantly altered by miR-140-3p mimic or inhibitor transfection. Bioinformatic analysis and miR-140-3p-induced gene suppression examination suggested that Smad3 might be the target gene of miR-140-3p. These findings illuminate the inhibitory effects of miR-140-3p on the proliferation and migration of macrophages and contribute to the cognition of the essential roles of miRNAs during peripheral nerve regeneration.     

1,25(OH)2D3 attenuates pulmonary arterial hypertension via microRNA-204 mediated Tgfbr2/Smad signaling

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by high pulmonary artery pressure. It is reported that microRNA-204 (miR-204) plays an important role in the development of PAH. Calcitriol [1,25-dihydroxyvitamin D3, 1,25(OH)₂D₃] mediates multiple pathophysiological processes. The aim of the current study was to explore the role of 1,25(OH)₂D₃ in PAH. PAH was induced in rats and rat pulmonary arterial endothelial cells (PAECs) were isolated as in vitro PAH model. The mean pulmonary artery pressure, morphologic changes, and expressions of transforming growth factor-beta1 (Tgfbr2), Smad2/7, alpha smooth muscle actin (α-SMA), and p21 were then measured. Furthermore, the effect of 1,25(OH)₂D₃ on rat PAECs with or without hypoxia treatment was also assessed by measuring the proliferation, migration, and cell cycle distribution of PAECs. The potential targets of miR-204 were also predicted and validated with a dual-luciferase reporter system. Then the role of miR-204 and Tgfbr2 in the anti-PAH effect of 1,25(OH)₂D₃ was further explored by modulating the expression of the two genes. The overall pulmonary hypertension and hypoxia-induced proliferation and migration of PAECs were attenuated by administration of 1,25(OH)₂D₃, which was associated with the suppressed expressions of Tgfbr2, α-SMA, and Smad7 and induced expressions of miR-204, p21 and Smad2 both in vitro and in vivo. Moreover, the luciferase reporter assay identified Tgfbr2 as a novel direct target of miR-204. Both overexpression of miR-204 and inhibition of Tgfbr2 would strengthen the effect of 1,25(OH)₂D₃ administration. Findings outlined in the current study demonstrated that 1,25(OH)₂D₃ was a promising therapeutic modality for treatment of PAH, function of which was exerted through miR-204 mediated Tgfbr2 signaling.     

IGF-1 prevents oxidative stress induced-apoptosis in induced pluripotent stem cells which is mediated by microRNA-1

Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H₂O₂) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H₂O₂ showed increases in miR-1 expression, mitochondria dysfunction, cytochrome-c release and apoptosis, Addition of IGF-1 into the iPS cell cultures reduced the H₂O₂ cytotoxicity. Prediction algorithms showed that 3′-untranslated regions of IGF-1 gene as a target of miR-1. Moreover, miR-1 mimic, but not miR-1 mimic negative control, diminished the protective effect of IGF-1 on H₂O₂-induced mitochondrial dysfunction, cytochrome-c release and apoptosis in iPS cells. In conclusion, IGF-1 inhibits H₂O₂-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. IGF-1′s effect is, at least partially, regulated by miR-1 in iPS cells.     

MicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2

MicroRNAs (MiRs) are short noncoding RNAs that can regulate gene expression. It has been reported that miR-21 suppresses apoptosis in activated T cells, but the molecular mechanism remains undefined. Tumor suppressor Tipe2 (or tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2)) is a newly identified anti-inflammatory protein of the TNFAIP8 family that is essential for maintaining immune homeostasis. We report here that miR-21 is a direct target of nuclear factor-κB and could regulate Tipe2 expression in a Tipe2 coding region-dependent manner. In activated T cells and macrophages, Tipe2 expression was markedly downregulated, whereas miR-21 expression was upregulated. Importantly, Tipe2-deficient T cells were significantly less sensitive to apoptosis. Conversely, overexpression of Tipe2 in EL-4 T cells increased their susceptibility to activation-induced apoptosis. Therefore, Tipe2 provides a molecular bridge between miR-21 and cell apoptosis; miR-21 suppresses apoptosis in activated T cells at least in part through directly targeting tumor suppressor gene Tipe2.     

socs7, a target gene of microRNA-145, regulates interferon-β induction through STAT3 nuclear translocation in bladder cancer cells

We recently reported that microRNA (miR)-145 is downregulated and induces apoptosis in human bladder cancer cells. Also, it is suggested that the ectopic expression of miR-145 induces apoptosis with the induction of TRAIL expression in several cancer cells. Here, we demonstrated a novel mechanism of apoptosis induction by miR-145 in bladder cancer cells. Exogenous miR-145 in T24 and NKB1 cells markedly increased the expression levels of interferon (IFN)-β, 2′–5′-oligoadenylate synthetase 1, which lies upstream of 2′–5′ oligoadenylates/RNase L system, and TRAIL, and induced apparent caspase-dependent apoptosis that was suppressed by cotreatment with a pan-caspase inhibitor; moreover, these expression levels were reduced by cotreatment with an miR-145 inhibitor. The apoptosis did not depend on Toll-like receptor 3 (TLR3) expression, because TLR3-silencing failed to inhibit IFN-β induction by miR-145. Then, we focused on the suppressor of cytokine signaling 7 (socs7), whose expression level was upregulated in bladder cancer cells compared with its level in normal human urothelial cells, as a putative target gene involved in IFN-β induction by miR-145. Expectedly, exogenous miR-145 decreased the expression level of SOCS7, and socs7-silencing enhanced IFN-β induction by transfection with a TLR3 ligand, polyinosinic acid-polycytidylic acid (PIC). The results of a luciferase reporter assay revealed that miR-145 targeted socs7. In addition, socs7-silencing significantly decreased the level of p-Akt and suppressed the growth of T24 cells. Furthermore, exogenous miR-145 or socs7-silencing promoted nuclear translocation of STAT3. In conclusion, the machinery of IFN-β induction through the regulation of SOCS7 by miR-145 was closely associated with the induction of apoptosis. Moreover, exogenous miR-145 promoted IFN-β induction by targeting socs7, which resulted in the nuclear translocation of STAT3. Additionally, our data indicate that SOCS7 functioned as an oncogene, the finding that revealed a novel mechanism of carcinogenesis in bladder cancer cells.     

MiR-204 regulates type 1 IP3R to control vascular smooth muscle cell contractility and blood pressure

MiR-204 is expressed in vascular smooth muscle cells (VSMC). However, its role in VSMC contraction is not known. We determined if miR-204 controls VSMC contractility and blood pressure through regulation of sarcoplasmic reticulum (SR) calcium (Ca²⁺) release. Systolic blood pressure (SBP) and vasoreactivity to VSMC contractile agonists (phenylephrine (PE), thromboxane analogue (U46619), endothelin-1 (ET-1), angiotensin-II (Ang II) and norepinephrine (NE) were compared in aortas and mesenteric resistance arteries (MRA) from miR-204^−/− mice and wildtype mice (WT). There was no difference in basal systolic blood pressure (SBP) between the two genotypes; however, hypertensive response to Ang II was significantly greater in miR-204^−/− mice compared to WT mice. Aortas and MRA of miR-204^−/− mice had heightened contractility to all VSMC agonists. In silico algorithms predicted the type 1 Inositol 1, 4, 5-trisphosphate receptor (IP₃R1) as a target of miR-204. Aortas and MRA of miR-204^−/− mice had higher expression of IP₃R1 compared to WT mice. Difference in agonist-induced vasoconstriction between miR-204^−/− and WT mice was abolished with pharmacologic inhibition of IP₃R1. Furthermore, Ang II-induced aortic IP₃R1 was greater in miR-204^−/− mice compared to WT mice. In addition, difference in aortic vasoconstriction to VSMC agonists between miR-204^−/− and WT mice persisted after Ang II infusion. Inhibition of miR-204 in VSMC in vitro increased IP₃R1, and boosted SR Ca²⁺ release in response to PE, while overexpression of miR-204 downregulated IP₃R1. Finally, a sequence-specific nucleotide blocker that targets the miR-204-IP₃R1 interaction rescued miR-204-induced downregulation of IP₃R1. We conclude that miR-204 controls VSMC contractility and blood pressure through IP₃R1-dependent regulation of SR calcium release.     

Study on the Mechanism of BMSCs in Regulating NF-κB Signal Pathway by Targeting miR-449a to Improve the Inflammatory Response to Peripheral Nerve Injury

Objective:To evaluate the mechanism of Bone Marrow Mesenchymal Stem Cells (BMSCs) in regulating NF-κB signal pathway by targeting miR-449a.Methods:Stem cells were transfected by over-expressing and inhibiting miR-449a to detect the levels and viability of miR-449a in stem cells after transfection. Stem cells and neurons were co-cultured in vitro to evaluate the in vitro mechanism of stem cells over-expressing miR-449a on neurons.Results:After the addition of neurons, the neuronal activity of miR-449a over-expression group increased significantly, the expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, and the inflammatory cytokines (TNF-α and IL-1β) decreased significantly (P<0.05). In vivo experiments in rats also showed that rats were unresponsive, did not chirp or elude after being stimulated. After stem cell therapy, the weight and response of rats gradually returned to normal levels. miR-449a expression significantly increased in the stem cell + miR-449a over-expression group, expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, inflammatory cytokines (TNF-α and IL-1β) significantly decreased, and cell activity significantly increased (P<0.05).Conclusions:BMSCs can modulate NF-κB signaling pathway by targeting miR-449a, so as to reduce the inflammatory response to peripheral nerve injury and repair nerve injury.     

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation

Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D₃, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D₃ administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition.     

miR-223 contributes to the AGE-promoted apoptosis via down-regulating insulin-like growth factor 1 receptor in osteoblasts

Advanced glycation end products (AGEs) have been confirmed to induce bone quality deterioration in diabetes mellitus (DM), and to associate with abnormal expression of miRNAs in DM patients or in vitro. Recently, miRNAs have been recognized to mediate the onset or progression of DM. In the present study, we investigated the regulation on miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells, with real-time quantitative PCR assay. And then we examined the inhibition of insulin-like growth factor 1 receptor (IGF-1R) expression by miR-223, via targeting of the 3′ UTR of IGF-1R with real-time quantitative PCR, western blotting and luciferase reporter assay. Then we explored the regulation of miR-223 and IGF-1R levels, via the lentivirus-mediated miR-223 inhibition and IGF-1R overexpression in the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It was demonstrated that AGE-BSA treatment with more than 100 μg/ml significantly up-regulated miR-223 level, whereas down-regulated IGF-1R level in MC3T3-E1 cells. And the up-regulated miR-223 down-regulated IGF-1R expression in both mRNA and protein levels, via targeting the 3′ UTR of IGF-1R. Moreover, though the AGE-BSA treatment promoted apoptosis in MC3T3-E1 cells, the IGF-1R overexpression or the miR-223 inhibition significantly attenuated the AGE-BSA-promoted apoptosis in MC3T3-E1 cells. In summary, our study recognized the promotion of miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells. The promoted miR-223 targeted IGF-1R and mediated the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It implies that miR-223 might be an effective therapeutic target to antagonize the AGE-induced damage to osteoblasts in DM.     

TIMP-1 suppressed by miR-138 participates in endoplasmic reticulum stress-induced osteoblast apoptosis in osteoporosis

The aim of this study was to investigate the role of miR-138 in osteoporosis and its underlying mechanism. Hydrogen peroxide (H₂O₂) was used to induce osteoporotic injury of osteoblasts. The cell viability and apoptosis of MC3T3-E1 cells was assessed using MTT assay and flow cytometry, respectively. The cell transfection was carried out to modulate the expression levels of miR-138 and TIMP-1 in MC3T3-E1 cells. Luciferase reporter gene assay was performed to determine the interaction between miR-138 and TIMP-1 3′UTR. In the present study, H₂O₂ inhibited osteoblasts growth and induced intracellular endoplasmic reticulum (ER) stress accompanied by high expression of miR-138. We also confirmed that miR-138 promoted osteoblasts apoptosis in vitro and in vivo. MiR-138 was further indicated to inhibit osteoblast survival via negative regulating TIMP-1 expression. Moreover, the downregulated TIMP-1 also mediated the ER stress-induced apoptosis of osteoblasts. We confirmed that miR-138 and ER stress were induced in osteoporosis and then promoted the apoptosis of osteoblasts, at least in part, through TIMP-1.     

miR-146a down-regulation alleviates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cytotoxicity of PC12 cells by regulating MCL1/JAK/STAT pathway

Oxidative stress and miRNAs have been confirmed to play an important role in neurological diseases. The study aimed to explore the underlying effect and mechanisms of miR-146a in H₂O₂-induced injury of PC12 cells. Here, PC12 cells were stimulated with 200 μM of H₂O₂ to construct oxidative injury model. Cell injury was evaluated on the basis of the changes in cell viability, migration, invasion, apoptosis, and DNA damage. Results revealed that miR-146a expression was up-regulated in H₂O₂-induced PC12 cells. Functional analysis showed that down-regulation of miR-146a alleviated H₂O₂-induced cytotoxicity in PC12 cells. Dual-luciferase reporter and western blot assay verified that MCL1 was a direct target gene of miR-146a. Moreover, anti-miR-146a-mediated suppression on cell cytotoxicity was abated following MCL1 knockdown in H₂O₂-induced PC12 cells. Furthermore, MCL1 activated JAK/STAT signaling pathway and MCL1 overexpression attenuated H₂O₂-induced cytotoxicity in PC12 cells by JAK/STAT signaling pathway. In conclusion, this study suggested that suppression of miR-146a abated H₂O₂-induced cytotoxicity in PC12 cells via regulating MCL1/JAK/STAT pathway.     

5-ht2a Receptors in rat Sciatic Nerves and Schwann Cell Cultures

Pharmacological approaches and optical recordings have shown that Schwann cells of a myelinating phenotype are activated by 5-HT upon its interaction with the 5-HT_2A receptor (5-HT_2AR). In order to further characterize the expression and distribution of this receptor in Schwann cells, we examined rat sciatic nerve and cultured rat Schwann cells using probes specific to 5-HT_2AR protein mRNA. We also examined the endogenous sources of 5-HT in rat sciatic nerve by employing both histochemical stains and an antibody that specifically recognizes 5-HT. Rat Schwann cells of a myelinating phenotype contained both 5-HT_2AR protein and mRNA. In the healthy adult rat sciatic nerve, 5-HT_2ARs were evenly distributed along the outermost portion of the Schwann cell plasma membrane and within the cytoplasm. The most prominent source of 5-HT was within granules of the endoneurial mast cells, closely juxtaposed to Schwann cells within myelinating sciatic nerves. These results support the hypothesis that the 5-HT receptors expressed by rat Schwann cells in vivo are activated by the release of 5-HT from neighboring mast cells.     

Neural stem cell-derived exosomes suppress neuronal cell apoptosis by activating autophagy via miR-374-5p/STK-4 axis in spinal cord injury

Objectives:To evaluate the roles of MicroRNAs (miRNAs) enclosed in the neuron-derived exosomes in the recovery of the spinal cord injury (SCI) and the mechanism.Methods:The exosomes were isolated from neural stem cells (NSCs) and characterized by transmission electron microscopy (TEM) and NanoSight system (NTA). For in vivo experiments, Basso Mouse Scale, beam walking, and inclined plane tests were used to determine the behavioral symptoms of the SCI mice. For in vitro experiments, H₂O₂ treated HT22 cells were used to simulate SCI cells and cocultured with exosomes to analyze the cell apoptosis using TUNEL assays and flow cytometry. Apoptosis- and autophagy-related protein expression was detected by western blot and the green fluorescent protein (GFP)-LC3 assay was used to detect the level of autophagy. In addition, luciferase assay was performed to assess the relationship between miR-374-5p and SKT-4.Results:Exosomes from NSCs alleviated spinal cord injury by triggering autophagy flux and suppressing apoptosis. Besides, miR-374-5p was highly expressed in these exosomes and was responsible for the decent in injured neural cell apoptosis by activating autophagy flux. The SKT-4 was the target gene regulated by miR-374-5p in this exosomal protective function to SCI cells.Conclusion:The elevated level of miR-374-5p in neuronal exosomes could enhance spinal cord injury recovery by activating autophagy.     

MiR-28 inhibits cardiomyocyte survival through suppressing PDK1/Akt/mTOR signaling

MicroRNAs play critical roles in regulating cell survival under multiple pathological conditions of heart diseases. Oxidative stress-induced apoptosis contributes greatly to heart ischemia-reperfusion injury. Herein, we describe a novel regulatory role of miR-28 on the survival of cardiomyocytes. We show that miR-28 was upregulated in cardiomyocytes treated with hydrogen peroxide (H₂O₂). MiR-28 gain of function sensitized cell apoptosis, whereas miR-28 loss of function partially rescued cell apoptosis induced by H₂O₂. Importantly, we observed a significant reduction in Akt/mammalian target of rapamycin (mTOR) signaling activity after miR-28 treatment. Luciferase activity assay and western blot analysis both revealed that, phosphoinositide-dependent kinase-1 (PDK1), which is critical for Akt activation, was directly and negatively modulated by miR-28. Our results therefore indicate that miR-28 regulates oxidative stress-induced cell apoptosis in heart muscle cells, which possibly involves a PDK1/Akt/mTOR-dependent mechanism. MIR-28 could serve as a critical therapeutic target to diminish oxidative stress-induced cell death in the heart.     

MicroRNA-101 protects cardiac fibroblasts from hypoxia-induced apoptosis via inhibition of the TGF-β signaling pathway

Cardiac fibroblasts (CFs) are the most numerous cells in the heart and are recognized primarily for their ability to maintain both the structural integrity and the physiological functions of the heart. The transforming growth factor beta (TGF-β) signaling pathway is reportedly involved in the modulation of CF functions, including apoptosis. Recent studies have indicated that microRNA-101 (miR-101) attenuates the TGF-β signaling pathway, either by inhibiting the expression of TGFβ1 or by targeting transforming growth factor-β receptor type I (TGFβRI). The present study aimed to determine whether miR-101 protects CFs from hypoxia-induced apoptosis and to investigate the mechanisms underlying its protective effects. The CCK-8 test, electron microscopy and TUNEL assay results demonstrated that miR-101a/b significantly inhibited hypoxia-induced CF apoptosis. The results of Western blotting, quantitative RT-PCR and immunofluorescence assays indicated that miR-101a dramatically inhibited the hypoxia-induced up-regulation of both TGFβRI and p-Smad 3 but not TGFβ1 in CFs. Additionally, miR-101a significantly reversed the hypoxia-induced up-regulation of Bax and Caspase-3, the down-regulation of Bcl-2 and the activation of Caspase-3 in CFs. Moreover, miR-101a markedly inhibited the intracellular Ca²⁺ ([Ca²⁺]_i) overload caused by hypoxia. Taken together, our results suggest that miR-101a protects CFs against hypoxia-induced apoptosis by inhibiting the TGF-β signaling pathway, which may be a potential therapeutic target for heart injury.