USP7, negatively regulated by miR-409-5p, aggravates hypoxia-induced cardiomyocyte injury.

Hypoxia-induced apoptosis is linked to the pathogenesis of myocardial infarction (MI) and heart failure. Ubiquitin-specific peptidase 7 (USP7) is related to catabolic/pro-apoptotic signaling. However, its role in cardiomyocyte injury is unclear. In this study, we aimed to investigate the role and the underlying regulatory mechanism of USP7 in MI. H9c2 cardiomyocytes were cultured in hypoxia to establish an in vitro model of myocardial hypoxic/ischemic injury. Sprague-Dawley (SD) rats were used to establish animal models with MI. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays were performed to evaluate the expression levels of miR-409-5p, USP7, and p53, respectively. After USP7 and miR-409-5p were selectively regulated in H9c2 cells, the inflammatory response, apoptosis, and cell viability were detected by ELISA, flow cytometry, and MTT assay, respectively. The interaction between USP7 and miR-409-5p was determined by bioinformatics analysis, qRT-PCR, Western blot, and dual-luciferase reporter assay. LVEF, LVIDd, and LVIDs of rats after MI were also measured. USP7 expression was markedly elevated while miR-409-5p expression was significantly down-regulated in H9c2 cells under hypoxic culture. Augmentation of USP7 expression led to a dramatic promotion of hypoxia-induced apoptosis of cardiomyocytes, accompanied by an increase in the secretion of the cytokines IL-1ß, TNF-α, and IL-6. Myocardial injury markers LDH, cTnI, and CK-MB expressions were also increased. Besides, overexpression of USP7 aggravated left ventricular remodeling and decreased left ventricular function of the rats. Conversely, the up-regulation of miR-409-5p expression protected H9c2 cells from apoptosis and inhibited the release of cytokines and myocardial injury. Left ventricular remodeling and left ventricular function were also improved by miR-409-5p overexpression. Furthermore, USP7 was identified as a target of miR-409-5p and the overexpression of miR-409-5p reversed the effects of USP7 on H9c2 cells. USP7 exacerbates myocardial ischemic injury by promoting inflammation and apoptosis of cardiomyocytes, and the up-regulation of its expression is partly caused by the down-regulation of miR-409-5p expression.

Expression of miR-409-5p in gestational diabetes mellitus and its relationship with insulin resistance.

Expression of miR-409-5p in gestational diabetes mellitus (GDM) and its relationship with insulin resistance were explore. One hundred and forty-nine pregnant women who underwent antenatal examination in Taizhou First People's Hospital were divided into a GDM group and a control group according to whether they had GDM or not. Serum miR-409-5p expression of the two groups was detected, and the levels of glycosylated hemoglobin (HbAlc) and other GDM-related biochemical indicators were measured. Fasting plasma glucose (FPG) was determined by glucose oxidase method, fasting insulin (FINS) was detected by radioimmunoassay, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. The relationship between miR-409-5p and other biochemical indicators and insulin resistance was analyzed, and logistic multivariate regression was employed to analyze the risk factors of GDM. miR-409-5p was highly expressed in the serum of GDM patients. HbAlc, FPG, FINS, and HOMA-IR in pregnant women in the GDM group were markedly higher than those in the control group. The serum miR-409-5p in GDM pregnant women showed a positive correlation with HbAlc, FPG, FINS, and HOMA-IR (P<0.05). The insulin resistance group presented remarkably higher serum miR-409-5p level than the non-insulin resistance group. Moreover, it was found that elevated miR-409-5p, FINS, and HOMA-IR were all independent risk factors for the onset of GDM. miR-409-5p is highly expressed in the serum of patients with GDM, and it is positively correlated with insulin resistance index of GDM patients, which may be a potential target for clinical diagnosis and treatment of GDM.

MicroRNA-409-5p promotes retinal neovascularization in diabetic retinopathy.

BACKGROUND: Retinal neovascularization, which is characterized by the increased proliferation, migration, and tube formation of retinal microvascular endothelial cells (RMECs), contributes to the progression of diabetic retinopathy (DR). MiR-409-5p has been reported to be upregulated in peripheral blood of DR patients and in vascular endothelial growth factor (VEGF)-induced RMECs. However, the role of miR-409-5p in retinal neovascularization of DR remains unelucidated. METHOD: The expression of miR-409-5p was measured in retinal tissues of streptozocin-induced and db/db diabetic mice, in high glucose-induced mouse RMECs (mRMECs), and in vitreous fluid of proliferative DR patients. Antagomir of miR-409-5p was intravitreally injected into diabetic mice. Proliferation, migration, and tube formation were detected using cell counting kit-8 assay, transwell assay, and microscope observation, respectively. Luciferase reporter assay was used to detect the direct interaction between miR-409-5p and peroxisome proliferator-activated receptor-α (PPARα). RESULT: MiR-409-5p was upregulated in retinal tissues of diabetic mice, in high glucose-induced mRMECs, and in vitreous fluid of proliferative DR patients. The knockdown of miR-409-5p attenuated retinal neovascularization in vivo. The overexpression of miR-409-5p promotes the proliferation, migration, and tube formation, and increased VEGF expression and secretion, while the knockdown of miR-409-5p suppressed the VEGF-induced retinal neovascularization in vitro. PPARα is a downstream target of miR-409-5p, and PPARα overexpression negated the promotion of miR-409-5p overexpression on the proliferation, migration, and tube formation of mRMECs. CONCLUSION: Our findings demonstrated that miR-409-5p acted as a neovasculogenic factor in DR, and anti-miR-409-5p therapy may provide a novel strategy in treating DR.

MiR-409-5p as a Regulator of Neurite Growth Is Down Regulated in APP/PS1 Murine Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a heterogeneous neurodegenerative disease. Recent studies suggest that miRNA expression changes are associated with the development of AD. Our previous study showed that the expression level of miR-409-5p was stably downregulated in the early stage of APP/PS1 double transgenic mice model of AD. We now report that miR-409-5p impairs neurite outgrowth, decreases neuronal viability, and accelerates the progression of Aß1 - 42-induced pathologies. In this study, we found that Aß1 - 42 peptide significantly decreased the expression of miR-409-5p, which was consistent with the expression profile of miR-409-5p in the APP/PS1 mice cortexes. Plek was confirmed to be a potential regulatory target of miR-409-5p by luciferase assay and Western blotting. Overexpression of miR-409-5p has an obvious neurotoxicity in neuronal cell viability and differentiation, whereas Plek overexpression could partially rescue neurite outgrowth from this toxicity. Some cytoskeleton regulatory proteins have been found to be related to AD pathogenesis. Our data show some clues that cytoskeletal reorganization may play roles in AD pathology. The early downregulation of miR-409-5p in AD progression might be a self-protective reaction to alleviate the synaptic damage induced by Aß, which may be used as a potential early biomarker of AD.

MicroRNA-409-5p is upregulated in breast cancer and its downregulation inhibits cancer development through downstream target of RSU1.

We investigated the expression and function of miR-409-5p in human breast cancer. Quantitative real-time polymerase chain reaction was conducted to evaluate endogenous miR-409-5p expression in breast cancer tumors and breast cancer cell lines. Lentiviral transduction was performed to stably downregulate miR-409-5p in breast cancer cell lines MDA-MB-231 and MCF-7 and cells. The effects of miR-409-5p downregulation on breast cancer proliferation, migration, and xenograft development were then evaluated. Downstream target gene of miR-409-5p, Ras suppressor protein 1, was examined by dual-luciferase activity assay, quantitative real-time polymerase chain reaction, and western blot in lentiviral-transduced breast cancer cells. Ras suppressor protein 1 was also inhibited in miR-409-5p-downregulated breast cancer cells to examine its functional effect on breast cancer proliferation and migration. MiR-409-5p was aberrantly upregulated in both breast cancer tumors and cell lines. Lentiviral transduction successfully downregulated endogenous miR-409-5p expression as well as suppressed proliferation, migration, and xenograft development in MDA-MB-231 and MCF-7 cells. Ras suppressor protein 1 was confirmed to be directly targeted by miR-409-5p in breast cancer cells. Small interfering RNA-mediated Ras suppressor protein 1 inhibition reversely promoted cancer proliferation and migration in miR-409-5p-downregualted breast cancer cells. MiR-409-5p is downregulated in breast cancer and its inhibition has anti-cancer effect on breast cancer development both in vitro and in vivo. The regulatory effect of miR-409-5p inhibition is likely through the inverse upregulation of Ras suppressor protein 1 in breast cancer.