MicroRNA-7a-5p ameliorates diabetic peripheral neuropathy by regulating VDAC1/JNK/c-JUN pathway.

AIMS: The pathogenesis of diabetic peripheral neuropathy (DPN) is complex, and its treatment is extremely challenging. MicroRNA-7a-5p (miR-7a-5p) has been widely reported to alleviate apoptosis and oxidative stress in various diseases. This study aimed to investigate the mechanism of miR-7a-5p in DPN. METHODS: DPN cell model was constructed with high-glucose-induced RSC96 cells. Cell apoptosis and viability were detected by flow cytometry analysis and cell counting kit-8 (CCK-8) assay respectively. The apoptosis and Jun N-terminal kinase (JNK)/c-JUN signalling pathway-related proteins expression were detected by Western blotting. The intracellular calcium content and oxidative stress levels were detected by flow cytometry and reagent kits. Mitochondrial membrane potential was evaluated by tetrechloro-tetraethylbenzimidazol carbocyanine iodide (JC-1) staining. The targeting relationship between miR-7a-5p and voltage-dependent anion-selective channel protein 1 (VDAC1) was determined by RNA pull-down assay and dual-luciferase reporter gene assay. The streptozotocin (STZ) rat model was constructed to simulate DPN in vivo. The paw withdrawal mechanical threshold (PTW) was measured by Frey capillary line, and the motor nerve conduction velocity (MNCV) was measured by electromyography. RESULTS: MiR-7a-5p expression was decreased, while VDAC1 expression was increased in HG-induced RSC96 cells and STZ rats. In HG-induced RSC96 cells, miR-7a-5p overexpression promoted cell proliferation, inhibited apoptosis, down-regulated calcium release, improved mitochondrial membrane potential and repressed oxidative stress response. MiR-7a-5p negatively regulated VDAC1 expression. VDAC1 knockdown improved cell proliferation activity, suppressed cell apoptosis and mitochondrial dysfunction by inhibiting JNK/c-JUN pathway activation. MiR-7a-5p overexpression raised PTW, restored MNCV and reduced oxidative stress levels and nerve cell apoptosis in STZ rats. CONCLUSION: MiR-7a-5p overexpression ameliorated mitochondrial dysfunction and inhibited apoptosis in DPN by regulating VDAC1/JNK/c-JUN pathway.

Taurine promotes estrogen synthesis by regulating microRNA-7a2 in mice ovarian granulosa cells.

Taurine, acting as a free amino acid, is widely distributed and plays multiple functions, including its regulating effect on estrogen synthesis in ovary. However, the mechanisms of taurine regulating estrogen synthesis in granulosa cells are not well understood. In this study, we identify whether microRNA-7a2 (miR-7a2) is involved in the signaling of taurine regulating estrogen synthesis in mouse granulosa cells for the first time. The results demonstrated that taurine transporter (TauT) co-localized with miR-7a in mouse ovarian granulose cells. Further, taurine treatment markedly enhanced the expression of miR-7a and Cyp19a1 in mouse ovaries and increased serum 17ß-estradiol (E2) concentration. Meanwhile, miR-7a2 knockout reversed the effect of taurine on E2. In addition, Golgi apparatus protein 1 (Glg1), a downstream target gene of miR-7a2, was significantly down-regulated by taurine, while Glg1 knockdown markedly increased the Cyp19a1 expression and E2 synthesis. Moreover, taurine affected miR-7a expression via activating p38 signaling. These results suggest that taurine promotes E2 synthesis through p38/miR-7a/Glg1/Cyp19a1 signaling pathway, which is crucial to understand the function and mechanism of taurine on estrogen synthesis.

Increased Expression of miR-7a-5p and miR-592 during Expansion of Rat Dental Pulp Stem Cells and Their Implication in Osteogenic Differentiation.

Dental pulp stem cells (DPSCs) possess strong osteogenic differentiation potential and are promising cell sources in regenerative medicine. However, such differentiation capacity progressively declines during their in vitro expansion. MicroRNAs (miRNAs) play important roles in modulating stem cell differentiation. This study aimed (1) to determine if miR-7a-5p and miR-592 are involved in maintaining and regulating osteogenic differentiation of DPSCs, and (2) to explore their potential regulatory pathways. We found that the expression of miR-7a-5p and miR-592 was significantly upregulated during the expansion of rat DPSCs (rDPSCs). Overexpression of these miRNAs inhibited the osteogenic/odontogenic differentiation of rDPSCs, as evidenced by calcium deposition and osteogenic/odontogenic gene expression. RT-qPCR determined that miR-592 could downregulate heat shock protein B8, whose expression is reduced during the expansion of rDPSCs. Furthermore, RNA-seq and bioinformatics analysis identified significant signaling pathways of miR-7a-5p and miR-592 in regulating osteogenic differentiation, including TNF, MAPK, and PI3K-Akt pathways. We conclude that upregulating miR-7a-5p and miR-592 suppresses the osteogenic differentiation of rDPSCs during their in vitro expansion, likely via TNF, MAPK, and PI3K-Akt pathways. The results may shed light on application of miR-7a-5p and miR-592 for maintaining osteo-differentiation potential in stem cells for bone regeneration and bone-related disease treatment.

Down-regulation of Xist and Mir-7a-5p improves LPS-induced myocardial injury.

Background: X-inactive specific transcript (Xist) is a lncRNA, which plays a significant role in X-chromosome inactivation, regulates cell proliferation in tumor cells, and inhibits apoptosis in acute myocardial infarction. On the other hand, miR-7a-5p is involved in cardiomyocytes injury in myocardial ischemia/reperfusion. However, their roles in LPS-induced damage remain unclear. Objectives: This study aimed at using siRNA transfection and lentivirus infection to regulate the expression of xist and miR-7a-5p, and to evaluate their effects on LPS-induced myocardial damage. Method: Mice cardiomyocytes (MCM) cells were divided into six groups, namely the control group, the LPS group, the LPS + lncRNA- group, the LPS + lncRNA+ group, the LPS + miRNA- group, and the LPS + miRNA+ group. Quantitative real-time PCR (qRT-PCR) was performed to assay for the RNA expressions of xist, miR-7a-5p, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and recombinant mitochondrial transcription factor A (Tfam) in all the groups. The ATP level was determined using the adenosine triphosphate (ATP) assay kit according to the manufacturer's instructions. Flow cytometry was performed to estimate the level of apoptosis and proliferation in cells in each group. Results: The level of xist in the myocardial cells was markedly higher in the LPS group compared with the control group; however, it was reduced in the LPS+ lncRNA- group. There was no significant difference in the expression of xist among the LPS+miRNA-, LPS+miRNA+, and LPS groups. Moreover, the expression of mir-7a-5p was significantly reduced in myocardial cells in the LPS group, and moderately reduced in the LPS+ miRNA- group, but remarkably elevated in the LPS+ miRNA+ group (P<0.05). The expression of mir-7a-5p was comparably similar in the LPS+ lncRNA- group, LPS+ lncRNA+ group, and LPS groups. Further, the levels of PGC-1a, and Tfam were determined. In the LPS group, the expression of PGC-1α was significantly reduced but elevated in the LPS+lncRNA- and LPS+ miRNA- groups (P<0.05). There was no significant difference in the level of PGC-1α among the LPS, LPS+ lncRNA+, and LPS+ miRNA+ groups. The expression of Tfam was markedly reduced in the LPS group (P < 0.05), but elevated after the suppression of xist and mir-7a-5p. The expression of Tfam was not significantly different among the LPS group, LPS+ lncRNA+ and LPS+ miRNA+ groups. Notably, overexpression of mir-7a-5p had a mild effect on the expression of Tfam in the LPS+ miRNA+ group compared with the control group. Besides, ATP expression in the LPS group was markedly reduced, but elevated after the inhibition of xist and mir-7a-5p. Suppressing the expression of xist or mir-7a-5p resulted in reduced cell apoptosis and increased cell proliferation. Conclusions: In this study, we established that down-regulation of xist and mir-7a-5p reduces apoptosis in response to LPS.

Edwardsiella tarda-induced miR-7a functions as a suppressor in PI3K/AKT/GSK3ß signaling pathway by targeting insulin receptor substrate-2 (IRS2a and IRS2b) in Paralichthys olivaceus.

To study the effect of Edwardsiella tarda infection on miRNAs expression profile in Japanese flounder, fish were injected intraperitoneally with E. tarda. The miRNAs involved in regulating immune responses were analyzed by high-throughput sequencing. A total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed (DE miRNAs) after E. tarda infection, indicating that they were immune-related miRNAs. To further examine the relationship between the miRNAs and their predicted target mRNAs, a total of 22 predicted target mRNAs, mainly related to endocytic signaling pathway, NF-κB signaling pathway, and p53 signaling pathway, were detected with miRNA mimics in HEK-293T cells by dual-luciferase reporter experiments. Finally, we confirmed that insulin receptor substrate-2 (IRS2a and IRS2b) were regulated by miR-7a. And the target sites of the 3' untranslated region (UTR) of IRS2a and IRS2b were verified by dual-luciferase reporter experiments. Furthermore, we found that the E. tarda and LPS significantly increased host miR-7a expression. In vivo and in vitro studies revealed that IRS2-mediated PI3K/AKT/GSK3ß signaling pathway was suppressed. Taken together, these results implied that miR-7a might be a key regulator of PI3K/AKT/GSK3ß signaling pathway via suppressing the IRS2a and IRS2b genes.

A causal relationship between the neurotherapeutic effects of miR182/7a and decreased expression of PRDM5.

BACKGROUND: Spinal cord injury (SCI) is terrible damage resulting in the deficiencies and necrosis of neurology and causes infinite inconvenience to sufferers. The therapy of SCI still meets a larger number of problems. Therefore, the underlying mechanism and novel therapy of acute SCI (ASCI) are urgent to explore. MATERIALS AND METHODS: The SCI model was established in rats. The expression of miR-182/miR-7a and PRDM5 at mRNA level was detected by quantitative real-time PCR and the protein expression of PRDM5 and c-caspase 3 was assessed by western blotting assays. The apoptosis of spinal cord neurons (SCN) was assessed on flow cytometry. The transfection of cells was performed by Lipofectamine 2000 kit. The relationship between PRDM5 and miR-182/miR-7a was examined by Luciferase assay. RESULTS: The expression of PRDM5 was up-regulated at either mRNA (2.212 folds) or protein level after SCI in rats, and knockdown of PRDM5 in SCN declined the c-caspase3 expression. In addition, the expression of miR-182 and miR-7a was decreased by 44.6% and 39.3% after SCI in rats. Moreover, the expression of miR-182 and miR-7a were negatively correlated with the level of PRDM5 expression, and the expression of PRDM5 was inhibited due to the increase of miR-182 and/or miR-7a expression. Moreover, both miR-182 and miR-7a could regulate PRDM5 to control SCN apoptosis. According to the BBB score increased 2 folds, the intrathecal injection of miR-182 and miR-7a improved the neurological function of rats. CONCLUSION: Inhibition of PRDM5 which was apparently negative correlation with miR-182 and miR-7a could suppress the neurons apoptosis to attenuate acute spinal cord injury in rats.

Taurine promotes insulin synthesis by enhancing Isl-1 expression through miR-7a/RAF1/ERK1/2 pathway.

It has been well established that the circulating taurine affects the insulin synthesis in pancreatic islet ß-cells, whereas miR-7a and LIM-homeodomain transcription factor Isl-1 are important intracellular factors regulating insulin transcription and synthesis. However, it still remains unknown whether taurine regulates insulin synthesis by affecting miR-7a and/or Isl-1 expressions in mouse pancreatic islet ß-cells. The present study was thus proposed to identify the effects of taurine on the expressions of miR-7a and/or Isl-1 and their relations to insulin synthesis in mouse pancreatic islet ß-cells by using miR-7a2 knockout (KO) and taurine transporter (TauT) KO mouse models and the related in vitro experiments. The results demonstrated that taurine supplement significantly decreased the pancreas miR-7a expression, but sharply upregulated the pancreas Isl-1 and insulin expressions, and serum insulin levels. However, the enhanced effects of taurine on Isl-1 expression and insulin synthesis were mitigated in the TauT KO and miR-7a2 KO mice. In addition, our results confirmed that taurine markedly increased pancreas RAF1 and ERK1/2 expressions. Collectively, the present study firstly demonstrates that taurine regulates insulin synthesis through TauT/miR-7a/RAF1/ERK1/2/Isl-1 signaling pathway, which are crucial for our understanding the mechanisms of taurine affecting insulin synthesis, and also potential for establishing the therapeutic strategies for diabetes and the diseases related to metabolism.

Floralozone regulates MiR-7a-5p expression through AMPKα2 activation to improve cognitive dysfunction in vascular dementia.

BACKGROUND: The pathogenesis of vascular dementia (VD) is complex, and currently, no effective treatments have been recommended. Floralozone is a colorless liquid first discovered in Lagotis Gaertn. Recently, its medicinal value has been increasingly recognized. Our previous study has demonstrated that Floralozone can improve cognitive dysfunction in rats with VD by regulating the transient receptor potential melastatin 2 (TRPM2) and N-methyl-D-aspartate receptor (NMDAR) signaling pathways. However, the mechanism by which Floralozone regulates TRPM2 and NMDAR to improve VD remains unclear. AMP-activated protein kinase (AMPK) is an energy regulator in vivo; however, its role of AMPK activation in stroke remains controversial. MiR-7a-5p has been identified to be closely related to neuronal function. PURPOSE: To explore whether Floralozone can regulate the miR-7a-5p level in vivo through AMPKα2 activation, affect the TRPM2 and NR2B expression levels, and improve VD symptoms. METHODS: The VD model was established by a modified bilateral occlusion of the common carotid arteries (2-VO) of Sprague-Dawley (SD) rats and AMPKα2 KO transgenic (AMPKα2-/-) mice. Primary hippocampal neurons were modeled using oxygen and glucose deprivation (OGD). Morris water maze (MWM) test, hematoxylin-eosin staining (HE staining), and TUNEL staining were used to investigate the effects of Floralozone on behavior and hippocampal morphology in rats. Minichromosome maintenance complex component 2(MCM2) positive cells were used to investigate the effect of Floralozone on neurogenesis. Immunofluorescence staining, qRT-PCR, and western blot analysis were used to investigate the effect of Floralozone on the expression levels of AMPKα2, miR-7a-5p, TRPM2, and NR2B. RESULTS: The SD rat experiment revealed that Floralozone improved spatial learning and memory, improved the morphology and structure of hippocampal neurons, reduced apoptosis of hippocampal neurons and promoted neurogenesis in VD rats. Floralozone could increase the miR-7a-5p expression level, activate AMPKα2 and NR2B expressions, and inhibit TRPM2 expression in hippocampal neurons of VD rats. The AMPKα2 KO transgenic (AMPKα2-/-) mice experiment demonstrated that Floralozone could regulate miR-7a-5p, TRPM2, and NR2B expression levels through AMPKα2 activation. The cell experiment revealed that the TRPM2 and NR2B expression levels were regulated by miR-7a-5p, whereas the AMPKα2 expression level was not. CONCLUSION: Floralozone could regulate miR-7a-5p expression level by activating the protein expression of AMPKα2, control the protein expression of TRPM2 and NR2B, improve the morphology and structure of hippocampus neurons, reduce the apoptosis of hippocampus neurons, promote neurogenesis and improve the cognitive dysfunction.

MiR-7a-5p Attenuates Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis by Targeting VDAC1.

MicroRNA-7a-5p (miR-7a-5p) is closely related to apoptosis and plays an important role in ischemia/reperfusion (I/R) injury. Whether miR-7a-5p is involved in hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis is unknown. Therefore, this study aims to evaluate the role of miR-7a-5p in cardiomyocyte H9C2 cells in response to H/R stimulation. The results of RT-qPCR demonstrated that the expression level of miR-7a-5p was significantly down-regulated in H/R-treated H9C2 cells. MTT assay revealed that the cell viability was notably decreased in H/R group. Flow cytometric analysis found that the ratio of apoptotic cells was increased markedly following H/R. Enforced miR-7a-5p expression increased cell viability and decreased the apoptotic rate. Western blot analysis revealed that the expressions of pro-apoptotic proteins cleaved caspase-3 and Bax were down-regulated, while the expression of anti-apoptotic protein Bcl-2 was up-regulated in H/R-treated H9C2 cells transfected with miR-7a-5p mimic. On the contrary, miR-7a-5p downexpressing promoted apoptosis in H/R-treated H9C2 cells. Furthermore, the bioinformatics prediction manifested voltage-dependent anion channel 1 (VDAC1) was a potential target for miR-7a-5p, and dual-luciferase reporter assay confirmed that miR-7a-5p targeted VDAC1 3' untranslated regions, which leads to the repressed expressions of VDAC1 mRNA and protein. Knockdown of VDAC1 potentiated the protective effects of miR-7a-5p against H/R-induced cell injury. In conclusion, our results demonstrated that miR-7a-5p is involved in H/R-induced cardiomyocyte apoptosis through targeting VDAC1. MiR-7a-5p/VDAC1 axis might be utilized as hopeful biomarkers to reveal the potential mechanism of myocardial I/R injury.

Msi2-mediated MiR7a-1 processing repression promotes myogenesis.

BACKGROUND: Most of the microRNAs (MiRs) involved in myogenesis are transcriptional regulated. The role of MiR biogenesis in myogenesis has not been characterized yet. RNA-binding protein Musashi 2 (Msi2) is considered to be one of the major drivers for oncogenesis and stem cell proliferation. The functions of Msi2 in myogenesis have not been explored yet. We sought to investigate Msi2-regulated biogenesis of MiRs in myogenesis and muscle stem cell (MuSC) ageing. METHODS: We detected the expression of Msi2 in MuSCs and differentiated myotubes by quantitative reverse transcription PCR (RT-qPCR) and western blot. Msi2-binding partner human antigen R (HuR) was identified by immunoprecipitation followed by mass spectrometry analysis. The cooperative binding of Msi2 and HuR on MiR7a-1 was analysed by RNA immunoprecipitation and electrophoresis mobility shift assays. The inhibition of the processing of pri-MiR7a-1 mediated by Msi2 and HuR was shown by Msi2 and HuR knockdown. Immunofluorescent staining, RT-qPCR and immunoblotting were used to characterize the function of MiR7a-1 in myogenesis. Msi2 and HuR up-regulate cryptochrome circadian regulator 2 (Cry2) via MiR7a-1 was confirmed by the luciferase assay and western blot. The post-transcriptional regulatory cascade was further confirmed by RNAi and overexpressing of Msi2 and HuR in MuSCs, and the in vivo function was characterized by histopathological and molecular biological methods in Msi2 knockout mice. RESULTS: We identified a post-transcription regulatory cascade governed by a pair of RNA-binding proteins Msi2 and HuR. Msi2 is enriched in differentiated muscle cells and promotes MuSC differentiation despite its pro-proliferation functions in other cell types. Msi2 works synergistically with another RNA-binding protein HuR to repress the biogenesis of MiR7a-1 in an Msi2 dose-dependent manner to regulate the translation of the key component of the circadian core oscillator complex Cry2. Down-regulation of Cry2 (0.6-fold, vs. control, P < 0.05) mediated by MiR7a-1 represses MuSC differentiation. The disruption of this cascade leads to differentiation defects of MuSCs. In aged muscles, Msi2 (0.3-fold, vs. control, P < 0.01) expression declined, and the Cry2 protein level also decreases (0.5-fold, vs. control, P < 0.05), suggesting that the disruption of the Msi2-mediated post-transcriptional regulatory cascade could attribute to the declined ability of muscle regeneration in aged skeletal muscle. CONCLUSIONS: Our findings have identified a new post-transcriptional cascade regulating myogenesis. The cascade is disrupted in skeletal muscle ageing, which leads to declined muscle regeneration ability.

MicroRNA-7a inhibits Isl1 expression to regulate insulin secretion by targeting Raf1 and Mapkap1 in NIT-1 cells.

Both microRNA-7a (miR-7a) and LIM-homeodomain transcription factor ISL1 are important factors regulating insulin transcription and secretion, but the functional relationship and the interacting mechanisms between miR-7a and ISL1 in pancreatic islet ß-cells remain unknown. The aims of this study were thus to identify the potential interactions and signaling communication between miR-7a and ISL1 in regulating insulin transcription and secretion in the cultured NIT-1 cells. The results show that miR-7a inhibitor upregulates Isl-1 and insulin gene expressions, and the insulin secretion. Whereas miR-7a mimics inhibit ISL1 and insulin gene expressions, and decreases the insulin secretion. Furthermore, we identified the target gene of miR-7a using dual-luciferase reporter assay, and the results demonstrate that Raf1 and Mapkap1 is a direct target gene of miR-7a, modeling RAF1/MEK/ERK1/2 and mTORC2/AKT signaling pathway to regulate Isl1 expression, and thus influencing insulin expression and secretion. Our results indicate that therapeutic inhibition of miR-7a function could be of relevance for preserving the function of pancreatic ß-cells during the course of diabetes development, implicating miR-7, ISL1, and/or the connecting molecules may act as novel targets for pharmacological or gene therapy in diabetes and related metabolic disease, although much detailed studies are required in the further study.

miR-7a Targets Insulin Receptor Substrate-2 Gene and Suppresses Viability and Invasion of Cells in Diabetic Retinopathy Mice via PI3K-Akt-VEGF Pathway.

INTRODUCTION: Diabetic retinopathy (DR) is one of the major leading causes for vision loss globally. Current study illustrates the role of miR-7a in DR. MATERIAL AND METHODS: Retinal pericytes (RPs) and Endothelial cells (ECs) were isolated from mouse model of DR. qRT-PCR was done for expression of miR-7a and target gene mRNA, Western blot for protein expression. Identification of miR-7a target gene was done by TargetScan and Luciferase assay. Cell viability and invasion was done by MTT and Transwell chamber assay. RESULTS: The expression of miR-7a was down-regulated whereas level of IRS-2 was unregulated in isolated RPs and ECs. Luciferase assay suggested correlation between miR-7a and IRS-2, over-expression of miR-7a using a mimic resulted in suppression in viability and invasion capacity of RPs and ECs and inhibited the protein levels of PI3K/Akt cascade and IRS-2, and however the inhibitor reversed them respectively. Transfection of siRNA targeting IRS-2 caused alteration in miR-7a mediated changes in ECs suggesting that miR-7a may decrease angiogenesis in DR by inhibiting the levels of IRS-2. CONCLUSION: miR-7a suppresses PI3K/Akt cascade via targeting IRS-2, thus decreasing the viability and invasion capacity of RPs and ECs, suggesting an interesting treatment target for DR.

LncRNA-Meg3 promotes Nlrp3-mediated microglial inflammation by targeting miR-7a-5p.

Recent studies have identified neuroinflammation as a significant contributor to the pathological process of traumatic brain injury (TBI) and as a potentially effective target for treatment. LncRNA maternally expressed gene 3 (Meg3) has further been observed to play a critical role in diverse biological processes, including microglial activation and the inflammatory response. However, its target gene and associated signaling pathway require further elucidation. This study found that lipopolysaccharide + ATP upregulated Meg3, promoted microglia activation, Nlrp3/caspase1 activation and inflammation, and markedly reduced miR-7a-5p. Overexpression of miR-7a-5p attenuated Meg3-induced microglial activation, but not Meg3 expression. Bioinformatic analysis and dual-luciferase assays indicated that Meg3 was a direct target of miR-7a-5p that negatively regulates miR-7a-5p expression. Further, we showed that Meg3 acted as a competing endogenous RNA for miR-7a-5p and induced microglial inflammation by regulating nod-like receptor protein 3 (Nlrp3) expression. Our study thus demonstrates Meg3 regulates microglia inflammation by targeting the miR-7a-5p /Nlrp3 pathway.

[Effects of dezocine on cardiac myocytes injury induced by hypoxia and reoxygenation in rats and its mechanism].

Objective: To investigate the mechanisms of dezocine on regulating H9C2 oxidative stress and apoptosis of rat cardiac myocytes induced by hypoxia-reoxygenation(H/R) by regulating the expressions of microRNA-7a- 5p(miR-7a-5p)/ubiquitin E3 ligase tripartite motif 10(TRIM10). Methods: H9C2 cells were divided into control group (cultured normally), H/R group (treated with hypoxia for 3 h and then reoxygenation for 4 h), different doses of dezocine intervention group (H9c2 cells were pretreated with dezocine at the concentrations of 10-7, 10-6 and 10-5 mmol/L for 24 h, and then treated with H/R), H/R+miR-7a-5p group (H9C2 cells were transfected with miR-7a-5p mimics and then treated with H/R), H/R+miR-NC group (H9C2 cells were transfected with miR-NC and then treated with H/R), H/R+Dezocine+anti-miR-7a-5p group (H9c2 cells transfected with anti-miR-7a-5p were pretreated with 10-5 mmol/L dezocine for 24 h, and then treated with H/R), H/R+dezocine+ anti-miR-NC Group (H9c2 cells transfected with anti-miR-NC were pretreated with 10-5 mmol/L dezocine for 24 h, and then treated with H/R). Each group of cells was set with 3 replicate wells, and the experiment was repeated 3 times. The content of malondialdehyde(MDA) and activity of superoxide dismutase(SOD) and glutathione peroxidas(GSH-Px) were detected by the enzyme-linked immunosorbent assay. The cells apoptosis was detected by flow cytometry. The protein expressions of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax) and TRIM10 were detected by Western blot, and the expressions of miR-7a-5p and TRIM10 mRNA were detected by real-time quantitative PCR(RT-qPCR). The double luciferase reporter gene experiment was used to verify the regulatory relationship between miR-7a-5p and TRIM10. Results: Compared with the control group, the MDA content, apoptosis rate, the expression of Bax protein, and the expression of TRIM10 mRNA and protein in the H/R group were all increased (P＜0.05), while the activities of SOD and GSH-Px, and the expressions of Bcl-2 protein and miR-7a-5p were all decreased (P＜0.05). Compared with the H/R group, the MDA content, apoptosis rate, the expression of Bax protein, and the expression of TRIM10 mRNA and protein in the different doses of dezocine intervention group were decreased (P＜0.05), while the activities of SOD and GSH-Px, and the expressions of Bcl-2 protein and miR-7a-5p were all increased (P＜0.05), and there were significant differences in each index between the different doses of dezocine intervention groups (P＜ 0.05). Compared with the H/R+miR-NC group, the MDA content, apoptosis rate, the protein expressions of Bax and TRIM10 in the H/R+miR-7a-5p group were decreased (P＜0.05), while the activities of SOD and GSH-Px, and the expression of Bcl-2 protein were all increased (P＜0.05). Compared with the H/R+dezocine+anti- miR-NC group, the MDA content, apoptosis rate, the protein expressions of Bax and TRIM10 in the H/R+dezocine+anti-miR-7a-5p group were all increased (P＜0.05), while the activities of SOD and GSH-Px, and the expression of Bcl-2 protein were all decreased (P＜0.05). Conclusion: Dezocine can reduce oxidative stress and apoptosis of rat cardiomyocytes H9C2 induced by H/R, which may play a role in regulating the miR-7a-5p / TRIM10 axis.

Pituitary tissue-specific miR-7a-5p regulates FSH expression in rat anterior adenohypophyseal cells.

The follicle-stimulating hormone (FSH), which is synthesized and secreted by the anterior pituitary gland, plays an important role in regulating reproductive processes. In this study, using the TargetScan program, we predicted that microRNAs (miRNAs) regulate FSH secretion. Dual-luciferase reporter assays were performed and identified miR-7a-5p. MiR-7a-5p has been reported to regulate diverse cellular functions. However, it is unclear whether miR-7a-5p binds to mRNAs and regulates reproductive functions. Therefore, we constructed a suspension of rat anterior pituitary cells and cultured them under adaptive conditions, transfected miR-7a-5p mimics or inhibitor into the cell suspension and detected expression of the FSHb gene. The results demonstrated that miR-7a-5p downregulated FSHb expression levels, while treatment with miR-7a-5p inhibitors upregulated FSHb expression levels relative to those of negative control groups, as shown by quantitative PCR analysis. The results were confirmed with a subsequent experiment showing that FSH secretion was reduced after treatment with mimics and increased in the inhibitor groups, as shown by enzyme-linked immunosorbent assay. Our results indicated that miR-7a-5p downregulates FSHb expression levels, resulting in decreased FSH synthesis and secretion, which demonstrates the important role of miRNAs in the regulation of FSH and animal reproduction.

miRNA-7a-2-3p Inhibits Neuronal Apoptosis in Oxygen-Glucose Deprivation (OGD) Model.

Neuronal apoptosis is a major pathological hallmark of the neonatal hypoxic-ischemic brain damage (HIBD); however, the role of miR-7a-2-3p in the regulation of HIBD remains unknown. The purpose of this study was to explore the possible roles of miR-7a-2-3p in brain injury using a hypoxia-ischemia model in rats and oxygen-glucose deprivation (OGD) model in vitro. Firstly, we established the hypoxia-ischemia (HI) model and verified the model using Zea Longa scores and MRI in rats. Next, the changes of miR-7a-2-3p were screened in the ischemic cortex of neonatal rats by qRT-PCR at 12, 48, and 96 h after HIBD. We have found that the expression of miR-7a-2-3p in the HI rats decreased significantly, compared with the sham group (P < 0.01). Then, we established the OGD model in PC12 cells, SH-SY5Y cells and primary cortical neurons in vitro and qRT-PCR was used to confirm the changes of miR-7a-2-3p in these cells after the OGD. In order to determine the function of miR-7a-2-3p, PC12 cells, SH-SY5Y cells and rat primary cortical neurons were randomly divided into normal, OGD, mimic negative control (mimic-NC) and miR-7a-2-3p groups. Then, Tuj1+ (neuronal marker) staining, TUNEL assay (to detect apoptotic cells) and MTT assay (to investigate cell viability) were performed. We have found that the number of PC12 cells, SH-SY5Y cells and cortical neurons in the miR-7a-2-3p groups increased significantly (P < 0.01) in comparison to the OGD groups. The survival of cortical neurons in the miR-7a-2-3p group was improved markedly (P < 0.01), while the apoptosis of neurons in the miR-7a-2-3p group was significantly decreased (P < 0.01), compared with the normal group. Lastly, we investigated the target genes of miR-7a-2-3p by using the prediction databases (miRDB, TargetScan, miRWalk, and miRmap) and verified the target genes with qRT-PCR in the HI rats. Bioinformatics prediction showed that Vimentin (VIM), pleiomorphic adenoma gene 1(PLAG1), dual specificity phosphatase 10 (DUSP10), NAD(P)H dehydrogenase, quinone 1 (NQO1) and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) might be the targets of miR-7a-2-3p and the qRT-PCR confirmed that VIM increased in the HI rats (P < 0.01). In conclusion, miR-7a-2-3p plays a crucial role in the hypoxic-ischemic injury, and is associated with regulation of VIM.

MiR-7a is an important mediator in Fas-associated protein with death domain (FADD)-regulated expression of focal adhesion kinase (FAK).

Fas-associated protein with death domain (FADD), a classical adaptor protein mediating apoptotic stimuli-induced cell death, has been reported to engage in several non-apoptotic processes such as T cell and cardiac development and tumorigenesis. Recently, there are several reports about the FADD's involvement in cell migration, however the underlying mechanism remains elusive. Here, we present a new finding that FADD could regulate the expression of FAK, a non-receptor protein tyrosine kinase overexpressed in many cancers, and played an important role in cell migration in murine MEF and melanoma cells with different metastatic potential, B16F10 and B16F1. Moreover, miR-7a, a tumor suppressor which prohibits cell migration and invasion, was up-regulated in FADD-deficient cells. And FAK was verified to be the direct target gene of miR-7a in B16F10 cells. Furthermore, we demonstrate that miR-7a was a necessary mediator in FADD-regulated FAK expression. In contrast to its classical apoptotic role, FADD interference could reduce the rate of cell migration, which could be rescued by inhibiting miR-7a expression. Taken together, our data provide a novel explanation regarding how FADD regulates cell migration in murine melanoma cells.

miRNA signature of unfolded protein response in H9c2 rat cardiomyoblasts.

BACKGROUND: Glucose and oxygen deprivation during ischemia is known to affect the homeostasis of the endoplasmic reticulum (ER) in ways predicted to activate the unfolded protein response (UPR). Activation of UPR signalling due to ER stress is associated with the development of myocardial infarction (MI). MicroRNAs (miRNAs) are key regulators of cardiovascular development and deregulation of miRNA expression is involved in the onset of many cardiovascular diseases. However, little is known about the mechanisms regulating the miRNA expression in the cardiovascular system during disease development and progression. Here we performed genome-wide miRNA expression profiling in rat cardiomyoblasts to identify the miRNAs deregulated during UPR, a crucial component of ischemia. RESULTS: We found that expression of 86 microRNAs changed significantly during conditions of UPR in H9c2 cardiomyoblasts. We found that miRNAs with known function in cardiomyoblasts biology (miR-206, miR-24, miR-125b, miR-133b) were significantly deregulated during the conditions of UPR in H9c2 cells. The expression of miR-7a was upregulated by UPR and simulated in vitro ischemia in cardiomyoblasts. Further, ectopic expression of miR-7a provides resistance against UPR-mediated apoptosis in cardiomyoblasts. The ample overlap of miRNA expression signature between our analysis and different models of cardiac dysfunction further confirms the role of UPR in cardiovascular diseases. CONCLUSIONS: This study demonstrates the role of UPR in deregulating the expression of miRNAs in MI. Our results provide novel insights about the molecular mechanisms of deregulated miRNA expression during the heart disease pathogenesis.