Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide.

Antisense DNA oligonucleotides, short interfering RNAs (siRNAs), and CRISPR/Cas9 genetic tools are the most useful therapeutic nucleic acids regulating gene expression based on the antisense specificity towards messenger RNA. Here, we present an effective novel strategy for inhibiting translation based on the antisense-controlled formation of an RNA quadruplex-duplex hybrid (QDH) between a G-rich RNA antisense oligoribonucleotide (Q-ASO) and specific mRNA, comprising two distant G-tracts. We selected epidermal growth factor receptor (EGFR) as a well-established target protein in anticancer therapy. The chemically modified, bi-functional anti-EGFR Q-ASO and a 56-nt long EGFR mRNA fragment, in the presence of potassium ions, were shown to form in vitro very stable parallel G-quadruplex containing a 28-nt long external loop folding to two duplex-stem structure. Besides, the Q-ASOs effectively reduced EGFR mRNA levels compared to the non-modified RNA and DNA antisense oligonucleotides (rASO, dASO). In addition, the hybridization specificity of Q-ASO comprising a covalently attached fluorescent tag was confirmed in living cells by visualization of the G4 green fluorescent species in the presence of other antisense inhibitors under competitive conditions. The results presented here offer novel insights into the potential application of Q-ASOs for the detection and/or alteration of (patho)biological processes through RNA:RNA quadruplex-duplex formation in cellular systems.

Heatr9 is an infection responsive gene that affects cytokine production in alveolar epithelial cells.

During infection, viruses enter susceptible host cells in order to replicate their components for production of new virions. In the process of infection, the gene expression of infected cells undergoes changes because of the production of viral components and due to the host response from detection of viral products. In the advent of RNA sequencing, the discovery of new genes and their functions in the host response generates new avenues for interventions in the host-pathogen interaction. We have identified a novel gene, Heatr9, as a virus and cytokine inducible viral responsive gene. We confirm Heatr9's expression in vitro and in vivo during virus infection and correlate it with viral burden. Heatr9 is induced by influenza virus and RSV. Heatr9 knockdown during viral infection was shown to affect chemokine expression. Our studies identify Heatr9 as a novel inflammatory and virus infection induced gene that can regulate the induction of specific cytokines.

Exosomes released upon mitochondrial ASncmtRNA knockdown reduce tumorigenic properties of malignant breast cancer cells.

During intercellular communication, cells release extracellular vesicles such as exosomes, which contain proteins, ncRNAs and mRNAs that can influence proliferation and/or trigger apoptosis in recipient cells, and have been proposed to play an essential role in promoting invasion of tumor cells and in the preparation of metastatic niches. Our group proposed the antisense non-coding mitochondrial RNA (ASncmtRNA) as a new target for cancer therapy. ASncmtRNA knockdown using an antisense oligonucleotide (ASO-1537S) causes massive death of tumor cells but not normal cells and strongly reduces metastasis in mice. In this work, we report that exosomes derived from ASO-1537S-treated MDA-MB-231 breast cancer cells (Exo-1537S) inhibits tumorigenesis of recipient cells, in contrast to exosomes derived from control-ASO-treated cells (Exo-C) which, in contrast, enhance these properties. Furthermore, an in vivo murine peritoneal carcinomatosis model showed that Exo-1537S injection reduced tumorigenicity compared to controls. Proteomic analysis revealed the presence of Lactadherin and VE-Cadherin in exosomes derived from untreated cells (Exo-WT) and Exo-C but not in Exo-1537S, and the latter displayed enrichment of proteasomal subunits. These results suggest a role for these proteins in modulation of tumorigenic properties of exosome-recipient cells. Our results shed light on the mechanisms through which ASncmtRNA knockdown affects the preparation of breast cancer metastatic niches in a peritoneal carcinomatosis model.

Mitochondrial alarmins are tissue mediators of ventilator-induced lung injury and ARDS.

RATIONALE: Endogenous tissue mediators inducing lung inflammation in the context of ventilator-induced lung injury (VILI) and acute respiratory distress syndrome (ARDS) are ill-defined. OBJECTIVES: To test whether mitochondrial alarmins are released during VILI, and are associated with lung inflammation. METHODS: Release of mitochondrial DNA, adenosine triphosphate (ATP), and formyl-Met-Leu-Phe (fMLP) peptide-dependent neutrophil chemotaxis were measured in conditioned supernatants from human alveolar type II-like (A549) epithelial cells submitted to cyclic stretch in vitro. Similar measurements were performed in bronchoalveolar lavage fluids from rabbits submitted to an injurious ventilatory regimen, and from patients with ARDS. MEASUREMENTS AND MAIN RESULTS: Mitochondrial DNA was released by A549 cells during cell stretching, and was found elevated in BAL fluids from rabbits during VILI, and from ARDS patients. Cyclic stretch-induced interleukin-8 (IL-8) of A549 cells could be inhibited by Toll-like receptor 9 (TLR9) blockade. ATP concentrations were increased in conditioned supernatants from A549 cells, and in rabbit BAL fluids during VILI. Neutrophil chemotaxis induced by A549 cells conditioned supernatants was essentially dependent on fMLP rather than IL-8. A synergy between cyclic stretch-induced alarmins and lipopolysaccharide (LPS) was found in monocyte-derived macrophages in the production of IL-1ß. CONCLUSIONS: Mitochondrial alarmins are released during cyclic stretch of human epithelial cells, as well as in BAL fluids from rabbits ventilated with an injurious ventilatory regimen, and found in BAL fluids from ARDS patients, particularly in those with high alveolar inflammation. These alarmins are likely to represent the proximal endogenous mediators of VILI and ARDS, released by injured pulmonary cells.

BACE1 Inhibition Using 2'-OMePS Steric Blocking Antisense Oligonucleotides.

Amyloid beta-peptide is produced by the cleavage of amyloid precursor protein by two secretases, a ß-secretase, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and a γ-secretase. It has been hypothesised that partial inhibition of BACE1 in individuals with a high risk of developing Alzheimer's disease may be beneficial in preventing cognitive decline. In this study, we report the development of a novel antisense oligonucleotide (AO) that could efficiently downregulate the BACE1 transcript and partially inhibit BACE1 protein. We designed and synthesised a range of 2'-OMethyl-modified antisense oligonucleotides with a phosphorothioate backbone across various exons of the BACE1 transcript, of which AO2, targeting exon 2, efficiently downregulated BACE1 RNA expression by 90%. The sequence of AO2 was later synthesised with a phosphorodiamidate morpholino chemistry, which was found to be not as efficient at downregulating BACE1 expression as the 2'-OMethyl antisense oligonucleotides with a phosphorothioate backbone variant. AO2 also reduced BACE1 protein levels by 45%. In line with our results, we firmly believe that AO2 could be used as a potential preventative therapeutic strategy for Alzheimer's disease.

Aberrant methylation-mediated downregulation of lncRNA SSTR5-AS1 promotes progression and metastasis of laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is among the most common malignant tumors with poor prognosis. Accumulating evidences have identified the important roles of long noncoding RNAs (lncRNAs) in the initiation and progression of various cancer types; however, the global lncRNAs expression profile for metastatic LSCC is limited. RESULTS: In the present study, we screen expression profiles of lncRNAs in advanced LSCC patients with paired tumor tissues and corresponding normal tissues by microarrays. We identify numerous differentially expressed transcripts, and after the necessary verification of the transcripts expression in expanded samples, we experimentally validate the expression patterns of the remarkable low expressed gene, SSTR5, and its antisense lncRNA, SSTR5-AS1. Downregulation of SSTR5 is detected in LSCC tissues and laryngeal carcinoma cells. Aberrant DNA hypermethylation of the CpG sites clustered in the exon 1 and accumulation of inactive histone modifications at SSTR5 promoter region may be epigenetic mechanisms for its inactivation in LSCC. SSTR5-AS1 may play antitumor role in LSCC and may be regulated by the hypermethylation of the same CpG sites with SSTR5. SSTR5-AS1 inhibits laryngeal carcinoma cells proliferation, migration, and invasion. SSTR5-AS1 increases the enrichment of MLL3 and H3K4me3 at the promoter region of SSTR5 by interacting with MLL3 and further induces the transcription of SSTR5. Furthermore, SSTR5-AS1 interacts with and recruits TET1 to its target gene E-cadherin to activate its expression. CONCLUSION: These findings suggest that the identified lncRNAs and mRNAs may be potential biomarkers in metastatic LSCC, and SSTR5-AS1 may act as a tumor suppressor as well as a potential biomarker for antitumor therapy.

Long noncoding RNA MALAT1 promotes high glucose-induced human endothelial cells pyroptosis by affecting NLRP3 expression through competitively binding miR-22.

Cell death and inflammation play critical roles in atherosclerosis. Pyroptosis, a novel proinflammatory programmed cell death process, participates in atherosclerosis pathogenesis. Recently, MALAT1 was identified as a pyroptosis-related long noncoding RNA (lncRNA). Here, we investigated the potential role and underlying mechanism of lncRNA MALAT1 in endothelial cells pyroptosis. We first established an endothelial cell pyroptosis model by stimulating EA.hy926 human endothelial cells (EA.hy926 cells) with high glucose. Then, we investigated lncRNA MALAT1 expression and found that it was upregulated in high glucose-treated EA.hy926 cells. Furthermore, lncRNA MALAT1 knockdown significantly inhibited high glucose-induced pyroptosis in EA.hy926 cells, which may critically influence atherosclerosis. Moreover, miR-22 was a target of lncRNA MALAT1 and was negatively correlated with lncRNA MALAT1. NLRP3 expression was significantly suppressed by transfection with a MALAT1-targeting antisense oligonucleotide (ASO). Ultimately, miR-22 overexpression abrogated the effect of MALAT1 on high glucose-induced EA.hy926 cells pyroptosis. Together, our results suggest that lncRNA MALAT1 promotes high glucose-induced pyroptosis of endothelial cells partly by affecting NLRP3 expression through competitively binding miR-22. Our findings indicate a new regulatory mechanism for endothelial cells pyroptosis under high-glucose stress, providing a novel therapeutic target for atherosclerosis.

Current strategies for Site-Directed RNA Editing using ADARs.

Adenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A's) to inosines (I's) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I's base pair like guanosines (G's). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. This ability to re-code makes ADAR an attractive therapeutic tool to correct genetic mutations within mRNA. The main challenge in doing so is to re-direct ADAR's catalytic activity towards A's that are not naturally edited, a process termed Site-Directed RNA Editing (SDRE). Recently, a handful of labs have taken up this challenge and two basic strategies have emerged. The first involves redirecting endogenous ADAR to new sites by making editable structures using antisense RNA oligonucleotides. The second also utilizes antisense RNA oligonucleotides, but it uses them as guides to deliver the catalytic domain of engineered ADARs to new sites, much as CRISPR guides deliver Cas nucleases. In fact, despite the intense current focus on CRISPR-Cas9 genome editing, SDRE offers a number of distinct advantages. In the present review we will discuss these strategies in greater detail, focusing on the concepts on which they are based, how they were developed and tested, and their respective advantages and disadvantages. Though the precise and efficient re-direction of ADAR activity still remains a challenge, the systems that are being developed lay the foundation for SDRE as a powerful tool for transient genome editing.

A novel long noncoding RNA, LINC00483 promotes proliferation and metastasis via modulating of FMNL2 in CRC.

Long non-coding RNAs (lncRNAs) are extensively involved in multiple malignancies including colorectal cancer (CRC). In the present study, we found a novel lncRNA, long intergenic non-protein coding RNA 483 (LINC00483), which was upregulated in CRC. We also illustrated that upregulated LINC00483 was correlated with poor clinicopathological features of patients with CRC. Functionally, we displayed that a knockdown of LINC00483 suppressed LOVO and HT29 cells proliferation and metastatic ability. We further illustrated that miR-204-3p was involved in LINC00483 induced proliferation and metastasis. An overexpression of miR-204-3p could attenuate the facilitative effect which LINC00483 presented. Through a luciferase assay, we showed the direct binding effect between LINC00483 and miR-204-3p. Even further, we revealed that LINC00483 and formin like 2 (FMNL2) shared a similar miR-204-3p response elements (MREs-204-3p). FMNL2 was a direct target of miR-204-3p. FMNL2 was a downstream gene of LINC00483 and participated in LINC00483 mediated proliferation and metastasis. Lastly, we proved that LINC00483 promoted proliferation and metastasis via modulating of FMNL2 in LOVO and HT29 cells. In summary, the outcomes of this study illustrated that LINC00483 promoted CRC cells proliferation and metastasis via modulating of FMNL2 by acting as a ceRNA of miR-204-3p. LINC00483/miR-204-3p/FMNL2 axial might be a novel target in molecular treatment of CRC.

Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption.

BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney. METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice. RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis. CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.

Downregulation of hsa_circ_0007534 suppresses breast cancer cell proliferation and invasion by targeting miR-593/MUC19 signal pathway.

Circular RNAs (circRNAs) are important non-coding RNAs that are reportedly involved in the progression of diverse human cancers through their action as a microRNA (miRNA) sponge. However, the exact roles of circRNAs in breast cancer (BC) remain largely unknown. The present data demonstrate the significantly upregulated expression of hsa_circ_0007534 circRNA in BC tissues and cell lines. In contrast, miR-593 expression was significantly downregulated. Knockdown of hsa_circ_0007534 inhibited BC cell proliferation, colony formation, and invasion, and promoted apoptosis in BC cells. Moreover, hsa_circ_0007534 was demonstrated to be a sponge of miR-593, and expression of miR-593 in BC cells was negatively correlated with hsa_circ_0007534. MUC19 expression was markedly increased in BC tissues and cell lines, and the 3'-UTR of MUC19 was targeted by miR-593. The expression of MUC19 was negatively regulated by miR-593 in BC cells. Our findings suggest an oncogenic role for hsa_circ_0007534 in BC by acting as a miR-593 sponge to promote MUC19 expression.

Overexpression of circular RNA circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging miR-411-5p and miR-432-5p.

Emerging evidence indicated that abnormally expressed circular RNAs (circRNAs) are critically involved in tumorigenesis and development of several cancers. However, the study relevant to the relationship between circRNAs and hepatocellular carcinoma (HCC) is rare. In this study, the expression of circ_001569 in HCC tissue specimens and cells were determined by qRT-PCR. The clinical relevance of circ_001569 was also analyzed. In addition, loss-of-function and gain-of-function assays were conducted to explore the biological functions of circ_001569. Furthermore, tumor formation and lung metastasis studies were induced to confirm the in vitro data. Mechanistically, bioinformatics analysis and luciferase reporter assays were conducted to illustrate the underlying mechanism of circ_001569. The results indicated that circ_001569 was overexpressed in HCC tissue samples and cells. This overexpression is correlated with larger tumor size, advanced TNM stages and unfavorable prognosis in the patients with HCC. Additionally, circ_001569 significantly facilitated HCC cell growth, migration and invasion. The animal studies further confirmed thein vitroresults. More importantly, circ_001569 could directly sponge miR-411-5p and miR-432-5p. The oncogenic functions of circ_001569 is partially dependent on its regulation on miR-411-5p and miR-432-5p. In summary, circ_001569/miR-411-5p/miR-432-5p regulatory signaling might be a rational HCC-related therapeutic target.

Controlled gene and drug release from a liposomal delivery platform triggered by X-ray radiation.

Liposomes have been well established as an effective drug delivery system, due to simplicity of their preparation and unique characteristics. However conventional liposomes are unsuitable for the on-demand content release, which limits their therapeutic utility. Here we report X-ray-triggerable liposomes incorporating gold nanoparticles and photosensitizer verteporfin. The 6 MeV X-ray radiation induces verteporfin to produce singlet oxygen, which destabilises the liposomal membrane and causes the release of cargos from the liposomal cavity. This triggering strategy is demonstrated by the efficiency of gene silencing in vitro and increased effectiveness of chemotherapy in vivo. Our work indicates the feasibility of a combinatorial treatment and possible synergistic effects in the course of standard radiotherapy combined with chemotherapy delivered via X-ray-triggered liposomes. Importantly, our X-ray-mediated liposome release strategy offers prospects for deep tissue photodynamic therapy, by removing its depth limitation.

A LINE1-Nucleolin Partnership Regulates Early Development and ESC Identity.

Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.

Long noncoding RNA lncARSR promotes hepatic lipogenesis via Akt/SREBP-1c pathway and contributes to the pathogenesis of nonalcoholic steatohepatitis.

Non-alcoholic fatty liver disease and steatohepatitis (NAFLD and NASH) account for the majority of liver disease in industrialized countries. However, the pathogenesis still unclear. Long non-coding RNAs (lncRNAs) has been reported to be involved in various pathophysiological processes. Here, we reported a novel role of lncARSR in hepatic lipogenesis in NAFLD. The expression of lncARSR was induced both in NAFLD patients and mouse model, as well as in hepatocytes treated with fatty acid. Moreover, overexpression of lncARSR enhanced while knockdown of lncARSR ameliorated hepatic lipid accumulation in vivo and in vitro. Furthermore, the expression of genes related to fatty acid synthesis and oxidation increased with lncARSR overexpression in vivo. Mechanistically, we identified that lncARSR regulated hepatic lipogenesis via upregulating SREBP-1c, the key regulatory molecule involved in lipogenesis. Knockdown of SREBP-1c by shRNA blocked the effect of lncARSR on lipogenesis. Furthermore, we demonstrated that lncARSR regulated SREBP-1c expression by PI3K/Akt pathway. In conclusion, our data indicated that lncARSR potentially contributes to the hepatic steatosis in NAFLD, which may be a new therapeutic target against NAFLD.

Assessment of External Guide Sequences' (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules.

RNase P is a ribozyme consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. It was initially identified as the enzyme that mediates cleavage of precursor tRNAs at the 5'-end termini to generate the mature tRNAs. An important characteristic of RNase P is that its specificity depends on the structure rather than the sequence of the RNA substrate. Any RNA species that interacts with an antisense molecule (called external guide sequence, EGS) and forms the appropriate structure can be cleaved by RNase P. This property is the basis for EGS technology, an antisense methodology for inhibiting gene expression by eliciting RNase P-mediated cleavage of a target mRNA molecule. EGS technology is being developed to design therapies against a large variety of diseases. An essential milestone in developing EGSs as therapies is the assessment of the efficiency of antisense molecules to induce cleavage of the target mRNA and evaluate their effect in vivo. Here, we describe simple protocols to test the ability of EGSs to induce cleavage of a target mRNA in vitro and to induce a phenotypic change in growing cells.

MicroRNA-139-5p affects cisplatin sensitivity in human nasopharyngeal carcinoma cells by regulating the epithelial-to-mesenchymal transition.

Nasopharyngeal carcinoma (NPC) is a head and neck cancer associated with poor prognosis. Many studies have shown that the epithelial-to-mesenchymal transition (EMT) is important in cancer progression, metastasis, and chemotherapy resistance and that microRNAs (miRNAs) play a key role in chemotherapy resistance associated with EMT. The miRNA miR-139-5p is downregulated in many human cancers and is closely related to tumor progression. The aim of this study was to investigate the ability of miR-139-5p to influence the cisplatin resistance, apoptosis, invasion and migration in NPC cells through the regulation of the EMT. We investigated these processes in parental HNE1 and cisplatin-resistant HNE1/DDP cells transfected with miR-139-5p inhibitors and mimics, respectively. Our results suggest that the upregulation of miR-139-5p expression inhibits proliferation, invasion, migration and EMT in human NPC cells. In addition, we found that miR-139-5p expression levels and DDP-induced apoptosis positively correlate in NPC cells. In conclusion, our results demonstrate that miR-139-5p can regulate the migration, invasion and DDP resistance in human NPC by modulating the EMT. The regulation of miR-139-5p levels might be a new approach to reverse EMT and DDP resistance and counteract metastasis and chemotherapy resistance in human NPC.

Role of MiR-155 Signal Pathway in Regulating Podocyte Injury Induced by TGF-ß1.

BACKGROUND/AIMS: Transforming growth factor beta 1 (TGF-ß1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of miR-155 on podocyte injury induced by TGF-ß1 and to determine further molecular mediators involved in the effects of miR-155. METHODS: Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-ß1 treatment; TGF-ß1 with miR-155 knockdown [using antisense oligonucleotides against miR-155 (ASO-miR-155)] and TGF-ß1 with negative control antisense oligonucleotides (ASO-NC). Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. In addition, we examined the levels of miR-155, TGF-ß1, nephrin, desmin and caspase-9 in glomerular tissues of nephropathy induced by intravenous injections of adriamycin in rats. RESULTS: mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-ß1-treated podocytes, whereas nephrin was decreased as compared with the control group. Importantly, miR-155 knockdown significantly attenuated upregulation of desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-ß1. Moreover, the number of apoptotic podocytes was increased after exposure to TGF-ß1 and this was alleviated after miR-155 knockdown. Knocking down miR-155 also decreased an apoptosis rate of TGF-ß1-treated podocytes. Note that negative control antisense oligonucleotides failed to alter an increase of the apoptosis rate in TGF-ß1-treated podocytes. Consistent with in vitro results, expression of miR-155, TGF-ß1, desmin and caspase-9 was increased and nephrin was decreased in glomerular tissues with nephropathy in vivo experiments. CONCLUSIONS: TGF-ß1 impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via miR-155 signal pathway. Inhibition of miR-155 alleviates these changes in podocytes-treated with TGF-ß1 and attenuated apoptosis of podocytes. Our data suggest that miR-155 plays a role in mediating TGF-ß1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking miR-155 signal has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

Upregulation of microRNA-876 Induces Endothelial Cell Apoptosis by Suppressing Bcl-Xl in Development of Atherosclerosis.

BACKGROUND/AIMS: The injury and apoptotic cell death of endothelial cells hallmark the development of atherosclerosis (AS), characterized by dysregulation of lipid homeostasis, immune responses, and formation of coronary plaques. However, the mechanisms underlying the initiation of endothelial cell apoptosis remain ill-defined. Recent evidence suggests a role of microRNAs in the processes of AS-associated endothelial cell apoptosis. Thus, we studied this question in the current study. METHODS: AS was developed in ApoE (-/-) mice suppled with high-fat diet (HFD), compared to ApoE (-/-) mice suppled with normal diet (ND). Mouse endothelial cells were isolated from the aortic arch using flow cytometry based on their expression of Pecam-1. Oxidized low-density lipoprotein (ox-LDL) were used to treat human aortic endothelial cells (HAECs) as an in vitro model for AS. Gene expression was quantified by RT-qPCR and protein levels were analyzed by Western blotting. Apoptosis was evaluated by FITC Annexin V Apoptosis essay and by TUNEL staining. Prediction of the binding between miRNAs and 3'-UTR of mRNA from the target gene was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. RESULTS: HFD mice, but not ND mice, developed AS in 12 weeks. Significantly reduced endothelial cell marks and significantly increased mesenchymal cell marks were detected in the aortic arch of the HFD mice, compared to the ND mice. The endothelial cell apoptosis was significantly higher in HFD mice, seemingly due to functional suppression of protein translation of anti-apoptotic Bcl-Xl protein through upregulation of miR-876. Similar results were obtained from in vitro study. Inhibition of miR-876 abolished the effects of ox-LDL-induced apoptotic cell death of HAECs. CONCLUSION: AS-associated endothelial cell apoptosis may partially result from downregulation of Bcl-Xl, through upregulation of miR-876 that binds and suppresses translation of Bcl-Xl mRNA.

MicroRNA-195 prevents dendritic degeneration and neuron death in rats following chronic brain hypoperfusion.

Impaired synaptic plasticity and neuron loss are hallmarks of Alzheimer's disease and vascular dementia. Here, we found that chronic brain hypoperfusion (CBH) by bilateral common carotid artery occlusion (2VO) decreased the total length, numbers and crossings of dendrites and caused neuron death in rat hippocampi and cortices. It also led to increase in N-terminal ß-amyloid precursor protein (N-APP) and death receptor-6 (DR6) protein levels and in the activation of caspase-3 and caspase-6. Further study showed that DR6 protein was downregulated by miR-195 overexpression, upregulated by miR-195 inhibition, and unchanged by binding-site mutation and miR-masks. Knockdown of endogenous miR-195 by lentiviral vector-mediated overexpression of its antisense molecule (lenti-pre-AMO-miR-195) decreased the total length, numbers and crossings of dendrites and neuron death, upregulated N-APP and DR6 levels, and elevated cleaved caspase-3 and caspase-6 levels. Overexpression of miR-195 using lenti-pre-miR-195 prevented these changes triggered by 2VO. We conclude that miR-195 is involved in CBH-induced dendritic degeneration and neuron death through activation of the N-APP/DR6/caspase pathway.