Downregulation of EIF5A2 by miR-221-3p inhibits cell proliferation,promotes cell cycle arrest and apoptosis in medulloblastoma cells

Recently, miR-221-3p expression has been reported to be down-regulated in medulloblastoma (MB), but its functional effects remains unclear. In this study, quantitative real-time PCR (qRT-PCR) revealed significantly decreased miR-221-3p in MB cell lines. Transfection of miR-221-3p mimics reduced, or inhibitor increased cell proliferation in MB cells using MTT assay. Flow cytometry analysis indicated miR-221-3p overexpression promoted, while knockdown alleviated G0/G1 arrest and apoptosis. Luciferase reporter assay confirmed miR-221-3p directly targets the EIF5A2 gene. Moreover, restoration of EIF5A2 in the miR-221-3p-overexpressing DAOY cells significantly alleviated the suppressive effects of miR-221-3p on cell proliferation, cell cycle and apoptosis. Furthermore, miR-221-3p overexpression decreased CDK4, Cyclin D1 and Bcl-2 and increased Bad expression, which was reversed by EIF5A2 overexpression. These results uncovered the tumor suppressive role of miR-221-3p in MB cell proliferation at least in part via targeting EIF5A2, suggesting that miR-221-3p might be a potential candidate target for diagnosis and therapeutics of MB.     

miR-221 is required for endothelial tip cell behaviors during vascular development

Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented "hyper-angiogenesis" defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.     

Circ_0026579 alleviates LPS-induced WI-38 cells inflammation injury in infantile pneumonia

Circular RNA (circRNA) represents an important regulator in infantile pneumonia progression. To clarify the role of circ_0026579 in this disease, LPS was used to treat WI-38 cells to mimic inflammation injury. The levels of inflammatory factors were determined by ELISA assay. Cell proliferation and apoptosis were measured by MTT assay, EdU staining and flow cytometry. The protein levels of cyclinD1, cleaved-caspase-3 and insulin-like growth factor 2 (IGF2) were examined using Western blot analysis. Cell oxidative stress was assessed by detecting MDA level and SOD activity. The expression of circ_0026579, miR-24-3p and IGF2 were analyzed using quantitative real-time PCR, and the interaction between miR-24-3p and circ_0026579 or IGF2 was confirmed by dual-luciferase reporter assay and RIP assay. LPS induced inflammation in WI-38 cells. Circ_0026579 expression was promoted in LPS-induced WI-38 cells, and its knockdown alleviated LPS-induced WI-38 cells inflammation. MiR-24-3p was sponged by circ_0026579, and its expression was reduced by LPS. MiR-24-3p inhibitor reversed the regulation of circ_0026579 knockdown on LPS-induced WI-38 cells inflammation. IGF2 was targeted by miR-24-3p, and its expression could be enhanced by LPS. MiR-24-3p relieved the inflammation of WI-38 cells which could be abolished by IGF2 overexpression. Circ_0026579 positively regulated IGF2 expression through sponging miR-24-3p. Circ_0026579 knockdown alleviated LPS-induced WI-38 cells inflammation by miR-24-3p/IGF2 axis, suggesting that circ_0026579 might contribute to infantile pneumonia progression.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway

It has been widely reported that exosomes derived from mesenchymal stem cells (MSCs) have a protective effect on myocardial infarction (MI). However, the specific molecules which play a damaging role in MSCs shuttled miRNAs are much less explored. MiRNA-153-3p (miR-153-3p) is a vital miRNA which has been proved to modulate cell proliferation, apoptosis, angiogenesis, peritoneal fibrosis and aortic calcification. Here, we aim to study the effect and mechanism of miR-153-3p in MSC-derived exosomes on hypoxia-induced myocardial and microvascular damage. The exosomes of MSCs were isolated and identified, and the MSCs-exosomes with low expression of miR-153-3p (exo-miR-153-3p⁻) were constructed to interfere with the endothelial cells and cardiomyocytes in the oxygen-glucose deprivation (OGD) model. The viability, apoptosis, angiogenesis of endothelial cells and cardiomyocytes were determined. Additionally, ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway was detected by ELISA and/or western blot. The results illustrated that exo-miR-153-3p⁻ significantly reduced the apoptosis of endothelial cells and cardiomyocytes and promoted their viability. Meanwhile, exo-miR-153-3p⁻ can promote the angiogenesis of endothelial cells. Mechanistically, miR-153-3p regulates the VEGF/VEGFR2/PI3K/Akt/eNOS pathways by targeting ANGPT1. Intervention with VEGFR2 inhibitor (SU1498, 1 μM) remarkably reversed the protective effect of exo-miR-153-3p⁻ in vascular endothelial cells and cardiomyocytes treated by OGD. Collectively, MSCs-derived exosomes with low-expressed miR-153-3p notably promotes the activation of ANGPT1 and the VEGF/VEGFR2 /PI3K/Akt/eNOS pathways, thereby preventing the damages endothelial cells and cardiomyocytes against hypoxia.     

Mechanism of miR-455–3 in suppressing epithelial–mesenchymal transition and angiogenesis of non-small cell lung cancer cells

The tumor-suppressing role of miR-455-3p has been reported in lung cancer, but the working mechanism remains to be fully elucidated. This study aims to explore the possible mechanism of miR-455-3p in regulating epithelial–mesenchymal transition (EMT) progression and angiogenesis in non-small cell lung cancer (NSCLC) cells.The expressions of miR-455-3p, HSF1, GLS1, and EMT-related proteins (E-cadherin, N-cadherin, vimentin, and Snail-1) in both NSCLC tissues and cell lines were determined by RT-qPCR and western blot. After cell transfection, cell proliferation and angiogenesis ability on NSCLC cells were assessed by MTT and tube formation assay. The binding of miR-455-3p with HSF1 was measured by luciferase reporter gene assay, while the interaction between HSF1 and GLS1 was determined by co-immunoprecipitation assay (Co-IP).HSF1 was highly expressed in NSCLC tissues and cells. Inhibition of HSF1 expression or overexpression of miR-455-3p in NSCLC cells can suppress cell proliferation, angiogenesis ability, and EMT progression. miR-455-3p was found to negatively regulate HSF1 expression. Co-transfection of miR-455-3p overexpression and HSF1 inhibition in NSCLC cells showed that miR-455-3p can partially counteract the effect of HSF1 in NSCLC cells. HSF1 can interact with GLS1 and elevate the expression of GLS1. GLS1 can partially abolish the suppressive effect of miR-455-3p in NSCLC cells.miR-455-3p can bind HSF1 to suppress the GLS1 in NSCLC cells, therefore suppressing EMT progression and angiogenesis of NSCLC cells.     

Hypoxic bone marrow mesenchymal cell-extracellular vesicles containing miR-328-3p promote lung cancer progression via the NF2-mediated Hippo axis

Lung cancer is the most aggressive tumour afflicting patients on a global scale. Extracellular vesicle (EV)-delivered microRNAs (miRs) have been reported to play critical roles in cancer development. The current study aimed to investigate the role of hypoxic bone marrow mesenchymal cell (BMSC)-derived EVs containing miR-328-3p in lung cancer. miR-328-3p expression was determined in a set of lung cancer tissues by RT-qPCR. BMSCs were infected with lentivirus-mediated miR-328-3p knock-down and then cultured in normoxic or hypoxic conditions, followed by isolation of EVs. Following ectopic expression and depletion experiments in lung cancer cells, the biological functions of miR-328-3p were analysed using CCK-8 assay, flow cytometry and Transwell assay. Xenograft in nude mice was performed to test the in vivo effects of miR-328-3p delivered by hypoxic BMSC-derived EVs on tumour growth of lung cancer. Finally, the expression of circulating miR-328-3p was detected in the serum of lung cancer patients. miR-328-3p was highly expressed in EVs derived from hypoxic BMSCs. miR-328-3p was delivered to lung cancer cells by hypoxic BMSC-derived EVs, thereby promoting lung cancer cell proliferation, invasion, migration and epithelial-mesenchymal transition. miR-328-3p targeted NF2 to inactivate the Hippo pathway. Moreover, EV-delivered miR-328-3p increased tumour growth in vivo. Additionally, circulating miR-328-3p was bioactive in the serum of lung cancer patients. Taken together, our results demonstrated that hypoxic BMSC-derived EVs could deliver miR-328-3p to lung cancer cells and that miR-328-3p targets the NF2 gene, thereby inhibiting the Hippo pathway to ultimately promote the occurrence and progression of lung cancer.     

The miR-223-3p/MAP1B axis aggravates TGF-β-induced proliferation and migration of BPH-1 cells

Uncontrolled proliferation and migration of benign prostatic hyperplasia (BPH) epithelial cells play a critical role in the pathogenesis of BPH. The regulatory roles of microRNAs (miRNAs) in multiple human diseases have been observed. This study was dedicated to investigating the regulatory effects of the miR-223-3p on the proliferation and migration of BPH progress. In the present study, the aberrant upregulation of miR-223-3p in BPH samples and BPH-1 cells was determined. TGF-β stimulation induced miR-223-3p expression, promoted BPH-1 cell viability and DNA synthesis, inhibited BPH-1 cell apoptosis, and decreased pro-apoptotic Bax/caspase 3. These changes induced by TGF-β stimulation were further enhanced the overexpression of miR-223-3p and attenuated via the inhibition of miR-223-3p. Under TGF-β stimulation, the overexpression of miR-223-3p enhanced, whereas the inhibition of miR-223-3p inhibited the EMT and MAPK signaling pathways. By targeting the MAP1B 3’UTR, miR-223-3p repressed MAP1B expression. In contrast to miR-223-3p overexpression, MAP1B overexpression attenuated TGF-β-induced changes in BPH-1 cell phenotypes, pro-apoptotic Bax/caspase 3, and the EMT and MAPK signaling pathways; more importantly, MAP1B overexpression significantly attenuated the roles of miR-223-3p overexpression in BPH-1 cell phenotypes, pro-apoptotic Bax/caspase 3, and the EMT and MAPK signaling pathways under TGF-β stimulation. In conclusion, miR-223-3p aggravates the uncontrolled proliferation and migration of BPH-1 cells through targeting MAP1B. The EMT and MAPK signaling pathways might be involved.     

miR-126-3p is essential for CXCL12-induced angiogenesis

Atherosclerosis, in the ultimate stage of cardiovascular diseases, causes an obstruction of vessels leading to ischemia and finally to necrosis. To restore vascularization and tissue regeneration, stimulation of angiogenesis is necessary. Chemokines and microRNAs (miR) were studied as pro-angiogenic agents. We analysed the miR-126/CXCL12 axis and compared impacts of both miR-126-3p and miR-126-5p strands effects in CXCL12-induced angiogenesis. Indeed, the two strands of miR-126 were previously shown to be active but were never compared together in the same experimental conditions regarding their differential functions in angiogenesis. In this study, we analysed the 2D-angiogenesis and the migration assays in HUVEC in vitro and in rat's aortic rings ex vivo, both transfected with premiR-126-3p/-5p or antimiR-126-3p/-5p strands and stimulated with CXCL12. First, we showed that CXCL12 had pro-angiogenic effects in vitro and ex vivo associated with overexpression of miR-126-3p in HUVEC and rat's aortas. Second, we showed that 2D-angiogenesis and migration induced by CXCL12 was abolished in vitro and ex vivo after miR-126-3p inhibition. Finally, we observed that SPRED-1 (one of miR-126-3p targets) was inhibited after CXCL12 treatment in HUVEC leading to improvement of CXCL12 pro-angiogenic potential in vitro. Our results proved for the first time: 1-the role of CXCL12 in modulation of miR-126 expression; 2-the involvement of miR-126 in CXCL12 pro-angiogenic effects; 3-the involvement of SPRED-1 in angiogenesis induced by miR-126/CXCL12 axis.     

miR-145-5p targets paxillin to attenuate angiotensin II-induced pathological cardiac hypertrophy via downregulation of Rac 1, pJNK,p-c-Jun,NFATc3, ANP and by Sirt-1 upregulation

Pathological cardiac hypertrophy is associated with many diseases including hypertension. Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-145-5p in the cardiac setting is still unclear. In this study, H9C2 cells were overexpressed with microRNA-145-5p, and then treated with Ang-II for 24 h, to study the effect of miR-145-5p on Ang-II-induced myocardial hypertrophy in vitro. Results showed that Ang-II treatment down-regulated miR-145-5p expression were revered after miR-145-5p overexpression. Based on results of bioinformatics algorithms, paxillin was predicted as a candidate target gene of miR-145-5p, luciferase activity assay revealed that the luciferase activity of cells was substantial downregulated the following co-transfection with wild paxillin 3′UTR and miR-145-5p compared to that in scramble control, while the inhibitory effect of miR-145-5p was abolished after transfection of mutant paxillin 3′UTR. Additionally, overexpression of miR-145-5p markedly inhibited activation of Rac-1/ JNK /c-jun/ NFATc3 and ANP expression and induced SIRT1 expression in Ang-II treated H9c2 cells. Jointly, our study suggested that miR-145-5p inhibited cardiac hypertrophy by targeting paxillin and through modulating Rac-1/ JNK /c-jun/ NFATc3/ ANP / Sirt1 signaling, therefore proving novel downstream molecular pathway of miR-145-5p in cardiac hypertrophy

     

miR-497-5p inhibits tumor cell growth and invasion by targeting SOX5 in non–small-cell lung cancer

MicroRNAs plays an important role in the ccurrence and development of non–small-cell lung cancer (NSCLC). miR-497-5p has been reported to function as a tumor suppressor in various cancers. However, the role of miR-497-5p in NSCLC remains poorly understood. In this study, we aimed to investigate the biological role and potential molecular mechanism of miR-497-5p in NSCLC. Our results showed that the messenger RNA (mRNA) expression level of miR-497-5p was notably downregulated in human NSCLC tissues and cell lines. miR-497-5p overexpression remarkably inhibited NSCLC cell proliferation and increased cell apoptosis in A549 and H460 cells, whereas inhibition of miR-497-5p had an opposite effect. The ability of cell migration and invasion was inhibited by miR-497-5p overexpression but was increased by miR-497-5p inhibition. Moreover, our findings indicated that SOX5 was a direct target of miR-497-5p. The protein and mRNA expression levels of SOX5 in A549 cells were remarkably inhibited by miR-497-5p overexpression but was upregulated by miR-497-5p inhibition. Furthermore, SOX5 overexpression notably reversed the effect of miR-497-5p mimic on NSCLC cell proliferation, cell apoptosis, cell migration, and invasion. Taken together, these results indicated that miR-497-5p overexpression inhibited NSCLC cell proliferation, migration and invasion, and induced cell apoptosis through inhibiting SOX5 gene expression. It was conceivable that miR-497-5p might serve as a potential molecular target for NSCLC treatment.     

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.     

miR-103a-2-5p/miR-30c-1-3p inhibits the progression of prostate cancer resistance to androgen ablation therapy via targeting androgen receptor variant 7

Androgens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly used to treat advanced prostate cancer. However, the acquisition of androgen ablation therapy resistance remains a challenge. Recently, androgen receptor splicing variants lacking the ligand-binding domain have been reported to play a critical role in the acquisition of androgen ablation therapy resistance. In the present study, we revealed that the messenger RNA expression and the protein levels of an androgen receptor variant 7 (AR-V7) were higher in prostate cancer tissue samples and in the AR-positive prostate cancer cell line, VCaP. In contrast, microRNA (miR)-30c-1-3p/miR-103a-2-5p expression was significantly downregulated in tumor tissues and cells. miR-30c-1-3p/miR-103a-2-5p overexpression could inhibit AR-V7 expression, suppress VCaP cell growth, and inhibit AR-V7 downstream factor expression by directly targeting the 3′-untranslated region of AR-V7. Under enzalutamide (Enza) treatment, the effects of AR-V7 overexpression were the opposite of those of miR-103a-2-5p/miR-30c-1-3p overexpression; more importantly, the effects of miR-103a-2-5p/miR-30c-1-3p overexpression could be significantly reversed by AR-V7 overexpression under Enza. In summary, we demonstrated a novel mechanism of the miR-30c-1-3p/miR-103a-2-5p/AR-V7 axis modulating the cell proliferation of AR-positive prostate cancer cells via AR downstream targets. The clinical application of miR-30c-1-3p/miR-103a-2-5p needs further in vivo validation.     

Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.