MicroRNA-199a-5p aggravates angiotensin II–induced vascular smooth muscle cell senescence by targeting Sirtuin-1 in abdominal aortic aneurysm

Vascular smooth muscle cells (VSMCs) senescence contributes to abdominal aortic aneurysm (AAA) formation although the underlying mechanisms remain unclear. This study aimed to investigate the role of miR-199a-5p in regulating VSMC senescence in AAA. VSMC senescence was determined by a senescence-associated β-galactosidase (SA-β-gal) assay. RT-PCR and Western blotting were performed to measure miRNA and protein level, respectively. The generation of reactive oxygen species (ROS) was evaluated by H2DCFDA staining. Dual-luciferase reporter assay was used to validate the target gene of miR-199a-5p. VSMCs exhibited increased senescence in AAA tissue relative to healthy aortic tissue from control donors. Compared with VSMCs isolated from control donors (control-VSMCs), those derived from patients with AAA (AAA-VSMCs) exhibited increased cellular senescence and ROS production. Angiotensin II (Ang II) induced VSMC senescence by promoting ROS generation. The level of miR-199a-5p expression was upregulated in the plasma from AAA patients and Ang II–treated VSMCs. Mechanistically, Ang II treatment significantly elevated miR-199a-5p level, thereby stimulating ROS generation by repressing Sirt1 and consequent VSMC senescence. Nevertheless, Ang II–induced VSMC senescence was partially attenuated by a miR-199a-5p inhibitor or Sirt1 activator. Our study revealed that miR-199a-5p aggravates Ang II–induced VSMC senescence by targeting Sirt1 and that miR-199a-5p is a potential therapeutic target for AAA.     

MiR-130a-5p contributed to the progression of endothelial cell injury by regulating FAS

MicroRNAs (miRNAs) play critical roles in the development of vascular diseases. However, the effects of miR- 130a-5p and its functional targets on atherosclerosis (AS) are still largely unknown. In this regard, our aim is to explore the potentially important role of miR-130a-5p and its target gene during the progression of endothelial cell injury. We first found oxidized low-density lipoprotein (ox-LDL) induced FAS and cell apoptosis in HUVECs. Subsequently, miR-130a-5p expression was verified to be downregulated after ox-LDL treatment and negatively correlated with FAS, and FAS was identified as substantially upregulated in the ox-LDL-treated HUVEC cells. After that, the knockdown of FAS and overexpression of miR-130a-5p together were observed to aggregate ox-LDL-induced reduction of cell viability and apoptosis, cell cycle progression, cell proliferation, cell migration and invasion. In conclusion, we detected that miR-130a-5p contributed to the progression of endothelial cell injury by regulating of FAS, which may provide a new and promising therapeutic target for AS.Key words: Atherosclerosis, ox-LDL, miR-130a-5p, FAS     

Interfering histone deacetylase 4 inhibits the proliferation of vascular smooth muscle cells via regulating MEG3/miR-125a-5p/IRF1

In this study, we investigated the role ofhistone deacetylase 4 (HDAC4) and MEG3/miR-125a-5p/interferonregulatoryfactor 1 (IRF1) on vascular smooth muscle cell (VSMCs)proliferation. Platelet derived growth factor (PDGF)-BB was used toinduce the proliferation and migration of VSMCs. The expressionsof MEG3, miR-125a-5p, HDAC4 and IRF1in VSMCs were detectedby qRT-PCR and western blot, respectively. ChIP assay was usedto determine the relationship between MEG3 and HDAC4. Doubleluciferase reporter assay was used to test the regulation betweenmiR-125-5p and IRF1. Results showed that PDGF-BB decreasedthe expression of MEG3 and IRF1, while increased the expressionof miR-125a-5p and HDAC4. In addition, HDAC4 knockdowninhibited the proliferation and migration of VSMCs via upregulatingMEG3 and downregulating miR-125a-5p. MiR-125a-5p inhibitorcould repress the proliferation and migration of VSMCs andalleviate intimal hyperplasia (IH) by directly upregulating IRF1expression. These results suggested that HDAC4 interferenceinhibited PDGF-BB-induced VSMCs proliferation via regulatingMEG3/miR-125a-5p/IRF1 axis, and then alleviated IH.     

miR-448-3p alleviates diabetic vascular dysfunction by inhibiting endothelial–mesenchymal transition through DPP-4 dysregulation

Diabetes mellitus (DM) often causes vascular endothelial damage and alters vascular microRNA (miR) expression. miR-448-3p has been reported to be involved in the development of DM, but whether miR-448-3p regulates diabetic vascular endothelial dysfunction remains unclear. To investigate the molecular mechanism of diabetic vascular endothelial dysfunction and the role of miR-448-3p therein, Sprague-Dawley rats were injected with streptozotocin (STZ) to establish diabetic animal model and the rat aortic endothelial cells were treated with high glucose to establish diabetic cell model. For the treatment group, after the induction of diabetes, the miR-448-3p levels in vivo and in vitro were upregulated by adeno-associated virus serotype 2 (AAV2)-miR-448-3p injection and miR-448-3p mimic transfection, respectively. Our results showed that AAV2-miR-448-3p injection alleviated the body weight loss and blood glucose level elevation induced by STZ injection. The miR-448-3p level was significantly decreased and the dipeptidyl peptidase-4 (DPP-4) messenger RNA level was increased in diabetic animal and cell models, which was reversed by miR-448-3p treatment. Moreover, the diabetic rats exhibited endothelial damage and endothelial–mesenchymal transition (EndMT), while AAV2-miR-448-3p injection relieved those situations. In vitro experiments demonstrated that miR-448-3p overexpression in endothelial cells alleviated endothelial damage by inhibiting EndMT through blocking the transforming growth factor-β/Smad pathway. We further proved that miR-448-3p negatively regulated DPP-4 by binding to its 3′-untranslated region, and DPP-4 overexpression reversed the effect of miR-448-3p overexpression on EndMT. Overall, we conclude that miR-448-3p overexpression inhibits EndMT via targeting DPP-4 and further ameliorates diabetic vascular endothelial dysfunction, indicating that miR-448-3p may serve as a promising therapeutic target for diabetic endothelial dysfunction.     

Estimation of time-dependent microRNA expression patterns in brain tissue,leukocytes, and blood plasma of rats under photochemically induced focal cerebral ischemia

miRNA expression over different time periods (24 and 48 h) using the quantitative RT-PCR and deep sequencing has been evaluated in a model of photochemically induced thrombosis. A combination of two approaches allowed us to determine the miRNA expression patterns caused by ischemia. Nine miRNAs, including let-7f-5p, miR-221-3p, miR-21-5p, miR-30c-5p, miR-30a-3p, miR-223-3p, miR-23a-3p, miR-22-5p, and miR-99a-5p, were differentially expressed in brain tissue and leukocytes of rats 48 h after onset of ischemia. In addition, six miRNAs were differentially expressed in the brain tissue and blood plasma of rats 24 h after exposure, among which miR-145-3p and miR-375-3p were downregulated and miR-19a-3p, miR-92a-3p, miR-188-5p, and miR-532-5p were upregulated. In our opinion, miR-188-5p and miR-532-5p may be considered to be new potential markers of ischemic injury. The level of miRNA expression tended to increase 48 h after the onset of ischemia in brain tissue and leukocytes, which reflects not only the local response in brain tissue due to inflammation, vascular endothelial dysfunction, and disorders of the permeability of the blood–brain barrier, but also the systemic response of the organism to multifactor molecular processes induced by ischemic injury.     

DNA聚合酶δ结合蛋白38是microRNA-291a-5p的一个靶基因

DNA聚合酶δ结合蛋白38 (DNA Polymerase delta-interacting protein 38,PDIP38) 是2003年新鉴定的一个基因,目前认为其可能在DNA修复、有丝分裂以及血管平滑肌细胞迁移中起重要作用。根据本实验室前期在胚胎干细胞中对该基因的研究,认为microRNA可能在PDIP38的调控过程中发挥了重要作用。为证实这种推论,运用生物信息学方法预测发现在胚胎干细胞中高表达的microRNA——microRNA-291a-5p (miR-291a-5p) 与PDIP38的开放阅读框 (ORF) 有一个配对非常理想的靶位点,通过构建该靶位点的报告基因载体以及ORF表达载体,分别进行荧光素酶报告基因分析以及细胞转染和Western blotting方法。结果证明miR-291a-5p能够直接调节PDIP38的蛋白表达。进一步运用real-time PCR和Western blotting分析证明了在胚胎干细胞中miR-291a-5p能够调节内源PDIP38的蛋白表达而对其mRNA表达无影响,这些都证明PDIP38确实是miR-291a-5p的一个靶基因。     

microRNA-26a-5p affects myocardial injury induced by coronary microembolization by modulating HMGA1

Coronary microembolization (CME) occurs when atherosclerotic plaque debris is detached during the treatment of acute coronary syndrome with Percutaneous Coronary Intervention (PCI). The complications of distal microvascular embolism, including local myocardial inflammation, are the main causes of myocardial damage and are a strong predictor of poor long-term prognosis and major cardiac adverse events. microRNAs (miRNAs) are involved in the pathophysiological processes of cardiovascular inflammatory diseases. Dysregulation of microRNA (miR)-26a-5p, in particular, is associated with a variety of cardiovascular diseases. However, the role of miR-26a-5p in CME-induced myocardial injury is unclear. In this study, we developed an animal model of CME by injecting microembolic balls into the left ventricle of rats and found that miR-26a-5p expression decreased in myocardial tissue in response. Using a miR-26a-5p mimic, echocardiography, hematoxylin-eosin staining, and Western blot analysis we found that the diminished cardiac function and myocardial inflammation induced by CME is alleviated by miR-26a-5p overexpression. Furthermore, our results show that inhibitors of miR-26a-5p have the opposite effect. In addition, in vitro experiments using real-time PCR, Western blot analysis, and a dual luciferase reporter gene show that HMGA1 is a target gene of miR-26a-5p. Thus, overexpression of miR-26a-5p could be a novel therapy to improve CME-induced myocardial damage.     

The lncRNA small nucleolar RNA host gene 5 regulates trophoblast cell proliferation,invasion, and migration via modulating miR-26a-5p/N-cadherin axis

Pre-eclampsia (PE) is a pregnancy-specific disease characterized by the occurrence of hypertension and proteinuria after two weeks of gestation. Long noncoding RNAs (lncRNAs) are emerging as key regulators in PE development. This study aims to investigate the role of lncRNA, small nucleolar RNA host gene 5 (SNHG5), in the pathogenesis of PE. The expression of SNHG5 was significantly downregulated in placental tissues from patients with severe PE compared normal controls. Overexpression of SNHG5 promoted trophoblast (HTR-8/SVneo) cell proliferation, invasion, and migration, and flow cytometry results showed that SNHG5 overexpression inhibited apoptosis and caused a decrease of cell population at the G ₀/G ₁ phase and an increase of cell population at the S phase, while knockdown of SNHG5 had the opposite effects. The interaction between SNHG5 and miR-26a-5p was predicted by bioinformatics analysis and confirmed by luciferase reporter assay and RNA immunoprecipitation, and miR-26a-5p was negatively regulated by SNHG5; miR-26a-5p expression was upregulated in PE placental tissues and was inversely correlated with SNHG5 expression. Furthermore, miR-26a-5p was predicted to target the 3′ untranslated region of N-cadherin, which was confirmed by luciferase reporter assay, and miR-26a-5p overexpression suppressed N-cadherin expression in HTR-8/SVneo cells. N-cadherin mRNA expression was downregulated in PE placental tissues and was positively correlated with SNHG5 expression. Both overexpression of miR-26a-5p and knockdown of N-cadherin suppressed HTR-8/SVneo cell invasion and migration, and also attenuated the effects of SNHG5 on the cellular functions of HTR-8/SVneo cells. In conclusion, our study suggested that SNHG5 promotes trophoblast cell proliferation, invasion, and migration at least partly via regulating the miR-26a-5p/N-cadherin axis.     

LncRNA SNHG12 promotes proliferation and epithelial mesenchymal transition in hepatocellular carcinoma through targeting HEG1 via miR-516a-5p

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.     

The lncRNA ZEB1-AS1 sponges miR-181a-5p to promote colorectal cancer cell proliferation by regulating Wnt/β-catenin signaling

Long noncoding RNAs (lncRNAs) are important regulators of the biological functions and underlying molecular mechanisms of colorectal cancer (CRC). However, the role of the lncRNA ZEB1-AS1 in CRC is not thoroughly understood. In this study, we found that ZEB1-AS1 was markedly upregulated in CRC. ZEB1-AS1 knockdown significantly suppressed CRC cell proliferation and induced apoptosis, whereas enhanced expression of ZEB1-AS1 had the opposite effect. Bioinformatics analysis identified miR-181a-5p as a candidate target of ZEB1-AS1. Moreover, we found an inverse correlation between ZEB1-AS1 and miR-181a-5p expression in CRC tissue. Inhibition of miR-181a-5p significantly upregulated ZEB1-AS1, whereas overexpression of miR-181a-5p had the opposite effect, suggesting that ZEB1-AS1 is negatively regulated by miR-181a-5p. Using luciferase reporter and RIP assays, we found that miR-181a-5p directly targets ZEB1-AS1. Importantly, ZEB1-AS1 may act as an endogenous ‘sponge’ to regulate miRNA targets by competing for miR-181a-5p binding. In summary, our findings provide the evidence supporting the role of ZEB1-AS1 as an oncogene in CRC. Our study also demonstrates that miR-181a-5p targets not only protein-coding genes but also the lncRNA ZEB1-AS1.     

MicroRNA-19a-3p promotes rheumatoid arthritis fibroblast-like synoviocytes via targeting SOCS3

Rheumatoid arthritis (RA) is a common chronic autoimmune disease and effective treatment for RA is still lacking. In this study, the regulatory role of miR-19a-3p in RA was investigated. Quantitative polymerase chain reaction analysis of human blood samples showed that the level of miR-19a-3p was significantly lower in the RA patients compared with that in healthy patients (P < 0.05). In RA fibroblast-like synoviocytes (RAFLS), miR-19a-3p and suppressor of cytokine signaling 3 (SOCS3) were also downregulated and upregulated, respectively, compared with those of normal FLS. Transfection of miR-19a-3p mimic in RAFLS inhibited cell proliferation and promoted cell apoptosis. TargetScan identified SOCS3 as a target of miR-19a-3p, which was confirmed by dual-luciferase assay. Western blot indicated that SOCS3 protein level was significantly decreased after miR-19a-3p overexpression. Moreover, SOCS3 silencing through siRNA transfection also enhanced cell proliferation, meanwhile inhibiting RAFLS apoptosis. In addition, SOCS3 overexpression abrogated the effects of miR-19a-3p overexpression on cell proliferation and apoptosis, corroborating that SOCS3 acts as a downstream effector in the miR-19a-3p-mediated function of RAFLS. These findings suggest that miR-19a-3p plays an important role in RA, and the miR-19a-3p/SOCS3 axis may become a potential therapeutic target for RA.     

MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.     

An antagonism between the AKT and beta-adrenergic signaling pathways mediated through their reciprocal effects on miR-199a-5p

We have recently reported that downregulation of miR-199a-5p is necessary and sufficient for inducing upregulation of its targets, including hypoxia-inducible factor-1alpha (Hif-1α) and Sirt1, during hypoxia preconditioning (HPC). Conversely, others and we have reported that miR-199a-5p is upregulated during cardiac hypertrophy. Thus, the objective of this study was to delineate the signaling pathways that regulate the expression of miR-199a-5p and its targets, and their role in myocyte survival during hypoxia. Since HPC is mediated through activation of the AKT pathway, we questioned if AKT is sufficient for inducing downregulation of miR-199a-5p. Our present study shows that overexpression of a constitutively active AKT (caAKT) induced 70% reduction in miR-199a-5p and was associated with a robust increase in HiF-1α (10 ± 2 fold) and Sirt1 (4 ± 0.8 fold) that was reversed by overexpression of miR-199a-5p. Similarly, insulin receptor-stimulated activation of the AKT pathway induced downregulation of miR-199a-5p and upregulation of its targets. In contrast, β-adrenergic receptor (βAR) activation in vitro and in vivo, induced 1.8–3.5-fold increase in miR-199a-5p. Accordingly, we predicted that βAR would antagonize AKT-induced, miR-199a-5p-dependent, upregulation of Hif-1α and Sirt1. Indeed, pre-treating the myocytes with isoproterenol before applying HPC, caAKT, or insulin resulted in 87 ± 3%, 75 ± 15%, and 100% reductions in Hif-1α expression, respectively, and sensitized the cells to hypoxic injury. Thus, activation of beta-adrenergic signaling counteracts the survival effects of the AKT pathway via upregulating miR-199a-5p.     

miR-216b-5p regulates proliferation and apoptosis of ox-LDL-stimulated VSMCs and HUVECs via IGF2

Atherosclerosis (AS) is one of the principal causes of cardiovascular disorder. Reportedly, vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) play key roles in AS development, and microRNAs (miRNAs) regulate their functions. The function of miR-216b-5p in AS remains unknown. Human VSMCs and human HUVECs were treated with ox-LDL to establish the in vitro model of AS. MiR-216b-5p and IGF2 expressions in VSMCs and HUVECs were probed by qRT-PCR and western blot. The viability, cell cycle progression, and apoptosis of VSMCs and HUVECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine, and flow cytometry assays, respectively. The binding sites between IGF2 3′UTR and miR-216b-5p were validated by dual-luciferase reporter assay. miR-216b-5p expression was declined in ox-LDL-induced VSMCs and HUVECs. In VSMCs, miR-216b-5p overexpression inhibited excessive proliferation and induced apoptosis. MiR-216b-5p could markedly restrain the viabiblity of VSMCs induced by ox-LDL and enhanced the viability of HUVECs. Additionally, IGF2 was confirmed as the direct target of miR-216b-5p and transfection of IGF2 overexpression plasmids rescued the effects of miR-216b-5p on VSMCs and HUVECs. miR-216b-5p alleviates the dysfunction of VSMCs and HUVECs caused by ox-LDL via repressing IGF2, and exerts protective functions to block the development of AS.     

Licochalcone A reverses NNK-induced ectopic miRNA expression to elicit in vitro and in vivo chemopreventive effects

BackgroundChemoprevention is the best cost-effective way regarding cancers. MicroRNAs (miRNAs) have been reported to be differentially expressed during the development of lung cancer. However, if lung cancer prevention can be achieved through modulating miRNAs expression so far remains unknown.PurposeTo discover ectopically expressed miRNAs in NNK-induced lung cancer and clarify whether Licochalcone A (lico A) can prevent NNK-induced lung cancer by modulating miRNA expression.Study design and methodsA/J mice were used to construct a lung cancer model by intraperitoneal injection with physiological saline NNK (100 mg/kg). Chemopreventive effects of lico A against lung cancer at 2 mg/kg and 20 mg/kg doses were evaluated in vivo. MicroRNA array and RT-qPCR were used to assess the expression levels of miRNAs. MLE-12 cells were treated with 0.1 mg/ml NNK, stimulating the ectopic expression pattern of miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p. miR-144-3p mimics and inhibitors were used to manipulate miR-144-3p levels. The effects of lico A (10 μM) on cell cycle distribution, apoptosis, and the expression of CK19, RASA1, miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p in NNK-treated MLE-12 cells were studied.ResultsThe expression levels of miR-144-3p, miR-20a-5p, and miR-29c-3p increased, while those of let-7d-3p and miR-328-3p decreased in both NNK-induced A/J mice and MLE-12 cells. Lico A could reverse the NNK-induced ectopic miRNA (miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p) expression both in vivo and in vitro and elicit in vivo lung cancer chemopreventive effect against NNK. In MLE-12 cells, the overexpression of miR-144-3p elicited the same effect as NNK regarding the expression of lung cancer biomarker CK19; the silencing of miR-144-3p reversed the effect of NNK on cell cycle distribution and apoptosis. Lico A could reverse the effect of NNK on the expression of miR-144-3p, CK19, and RASA1 (predicted target of miR-144-3p).ConclusionThe present study suggests that miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p were involved in the in vivo pathogenesis of NNK-induced lung cancer, and lico A could reverse the effect of NNK both in vivo and in vitro to elicit lung cancer chemopreventive effects through, at least partially, these five ectopically expressed miRNAs, especially miR-144-3p.