Differential and unique patterns of synaptic miRNA expression in dorsolateral prefrontal cortex of depressed subjects

Altered synaptic plasticity is often associated with major depressive disorder (MDD). Disease-associated changes in synaptic functions are tightly correlated with altered microRNA (miRNA) expression. Here, we examined the role of miRNAs and their functioning at the synapse in MDD by examining miRNA processing machinery at synapse and sequencing miRNAs and analyzing their functions in synaptic and total tissue fractions obtained from dorsolateral prefrontal cortex (dlPFC) of 15 MDD and 15 matched non-psychiatric control subjects. A total of 333 miRNAs were reliably detected in the total tissue fraction. Multiple testing following the Benjamini–Hochberg false discovery rate [FDR] showed that 18 miRNAs were significantly altered (1 downregulated 4 up and 13 downregulated; p < 0.05) in MDD subjects. Out of 351 miRNAs reliably expressed in the synaptic fraction, 24 were uniquely expressed at synapse. In addition, 8 miRNAs (miR-215-5p, miR-192-5p, miR-202-5p, miR-19b-3p, miR-423-5p, miR-219a-2-3p; miR-511-5p, miR-483-5p showed significant (FDR corrected; p < 0.05) differential regulation in the synaptic fraction from dlPFC of MDD subjects. In vitro transfection studies and gene ontology revealed involvement of these altered miRNAs in synaptic plasticity, nervous system development, and neurogenesis. A shift in expression ratios (synaptic vs. total fraction) of miR-19b-3p, miR-376c-3p, miR-455-3p, and miR-337-3p were also noted in the MDD group. Moreover, an inverse relationship between the expression of precursor (pre-miR-19b-1, pre-miR-199a-1 and pre-miR-199a-2) and mature (miR-19b-3p, miR-199a-3p) miRNAs was found. Although not significantly, several miRNA processing enzymes (DROSHA [95%], DICER [17%], TARBP2 [38%]) showed increased expression patterns in MDD subjects. Our findings provide new insights into the understanding of the regulation of miRNAs at the synapse and their possible roles in MDD pathogenesis.Subject terms: Depression, Depression     

Peptide-mediated lipofection is governed by lipoplex physical properties and the density of surface-displayed amines

Peptides can potentiate lipid-mediated gene delivery by modifying lipoplex physiochemical properties to overcome rate-limiting steps to gene transfer. The objectives of this study were to determine the regimes over which cationic peptides enhance lipofection and to investigate the mechanism of action, such as increased cellular association resulting from changes in lipoplex physical properties. Short, cationic peptides were incorporated into lipoplexes by mixing peptide, lipid and DNA. Lipoplexes were characterized using gel retardation, dynamic light scattering, and fluorescent microscopy, and the amount of surface-displayed amines was quantified by fluorescamine. Size, zeta potential, and surface amines for lipoplexes were dependent on peptide/DNA ratio. Inclusion of peptides in lipoplexes resulted in up to a 13-fold increase in percentage of cells transfected, and up to a 76-fold increase in protein expression. This transfection enhancement corresponded to a small particle diameter and positive zeta potential of lipoplexes, as well as increased amount of surface-displayed amines. Relative to lipid alone, these properties of the peptide-modified lipoplexes enhanced cellular association, which has been reported as a rate-limiting step for transfection with lipoplexes. The addition of peptides is a simple method of lipofection enhancement, as direct chemical modification of lipids is not necessary for increased transfection.     

Involvement of miR-133a and miR-326 in ADM resistance of HepG2 through modulating expression of ABCC1

Recent studies have shown that a class of small, functional RNAs, named microRNAs, may regulate multidrug resistance-associated protein 1 (ABCC1). Since ABCC1 is an important efflux transporter responsible for cellular drug disposition, the discovery of microRNAs (miRNA) brings an idea that there may be some other unknown multidrug resistance (MDR) mechanisms exist. Using computational programs, we predicted that the 3′untranslated region (3′UTR) of ABCC1 contains a potential miRNA binding site for miR-133a and also two other for miR-326. These binding sites were confirmed by luciferase reporter assay. ABCC1 mRNA degradation was accelerated dramatically in cells transfected with miR-133a or miR-326 mimics using qRT-PCR, Furthermore, western blot analysis indicated that ABCC1 protein expression was significantly down-regulated in hepatocellular carcinoma cells line HepG2 after transfection with miR-133a or miR-326 mimics, suggesting the involvement of mRNA degradation and protein expression mechanism. The effects of the two miRNAs on adriamycin (ADM) sensitivity to HepG2 cells were determined by MTT assay. Compared with mock transfection, miR-133a or miR-326 mimics transfection sensitized these cells to ADM. These findings for the first time demonstrated that the involvement of miR-133a and miR-326 in MDR is mediated by ABCC1 in hepatocellular carcinoma cell line HepG2 and suggested that miR-133a and miR-326 may be efficient agents for preventing and reversing ADM resistance in cancer cells.     

Anionic polymer-coated lipoplex for safe gene delivery into tumor by systemic injection

Abstract

In this study, we developed an anionic lipoplex by coating cationic lipoplex with anionic polymers such as hyaluronan (HA), chondroitin sulfate C (CS) and poly-l-glutamic acid (PLE) to deliver the plasmid DNA efficiently into the tumor by avoiding interaction with erythrocytes. The sizes of HA-, CS- and PLE-coated lipoplexes were ～200?nm and the ζ-potentials were negative. CS- and PLE-coated lipoplexes did not induce agglutination after mixing with erythrocytes, but cationic and HA-coated lipoplexes exhibited agglutination. In terms of biodistribution and gene expression after intravenous administration, cationic and HA-coated lipoplexes largely accumulated and induced gene expression in the lung. In contrast, CS- and PLE-coated lipoplexes did not exhibit high gene expression in the lung and mainly accumulated in the liver. However, in tumor, differences in lipoplex accumulation and gene expression were not observed among the lipoplexes. In terms of toxicity after intravenous injection, CS- and PLE-coated lipoplexes did not increase tumor necrosis factor-α, aspartate aminotransferase and alanine aminotransferase concentrations in blood. From these findings, CS and PLE coatings for cationic lipoplex might produce safe systemic vectors, although they did not increase gene expression in tumor.     

miR-96在人乳腺癌中表达及其对乳腺癌细胞侵袭和迁移活性的影响

目的研究miR-96在人乳腺上皮细胞株和乳腺病变组织中表达状况及其对人乳腺癌细胞增殖、侵袭和迁移的影响。方法抽提4株人乳腺上皮细胞系和28例人乳腺病变新鲜组织总miRNA,实时定量PCR方法检测它们miR-96的表达状况;脂质体介导的转染方法将miR-96抑制物转染人乳腺癌MCF-7细胞株;通过MTS试剂盒检测细胞增殖能力,Transwell侵袭和迁移实验检测细胞侵袭及迁移能力。结果miR-96在高侵袭乳腺癌细胞株中表达较低侵袭乳腺细胞明显下调(P<0.01);人乳腺癌组织中miR-96表达较乳腺良性病变组织明显下调(P<0.01);miR-96抑制物转染乳腺癌MCF-7后,乳腺癌细胞侵袭和迁移活力较阴性对照组细胞明显增强(P<0.05),而细胞增殖活力改变无统计学意义(P>0.05)。结论 miR-96在人乳腺癌细胞株和乳腺癌组织中存在表达下调,并对人乳腺癌细胞的侵袭及迁移能力可能存在一定负性调控作用。     

Analyses of the possible anti-tumor effect of yokukansan

The Kampo medicine yokukansan (YKS) has a wide variety of properties such as anxiolytic, anti-inflammatory and analgesic effects, and is also thought to regulate tumor suppression. In this study, we investigated the anti-tumor effect of YKS. We used Lewis lung carcinoma (LLC)-bearing mice that were fed food pellets containing YKS and then performed a fecal microbiota analysis, a microarray analysis for microRNAs (miRNAs) and an in vitro anti-tumor assay. The fecal microbiota analysis revealed that treatment with YKS partly reversed changes in the microbiota composition due to LLC implantation. Furthermore, a miRNA array analysis using blood serum showed that treatment with YKS restored the levels of miR-133a-3p/133b-3p, miR-1a-3p and miR-342-3p following LLC implantation to normal levels. A TargetScan analysis revealed that the epidermal growth factor receptor 1 signaling pathway is one of the major target pathways for these miRNAs. Furthermore, treatment with YKS restored the levels of miR-200b-3p and miR-200c-3p, a recognized mediator of cancer progression and controller of emotion, in the hypothalamus of mice bearing LLC. An in vitro assay revealed that a mixture of pachymic acid, saikosaponins a and d and isoliquiritigenin, which are all contained in YKS, exerted direct and additive anti-tumor effects. The present findings constitute novel evidence that YKS may exert an anti-tumor effect by reversing changes in the fecal microbiota and miRNAs circulating in the blood serum and hypothalamus, and the compounds found in YKS could have direct and additive anti-tumor effects.

     

Combination of microRNA therapeutics with small-molecule anticancer drugs: Mechanism of action and co-delivery nanocarriers

MicroRNAs (miRNAs) regulate multiple molecular pathways vital for the hallmarks of cancer with a high degree of biochemical specificity and potency. By restoring tumor suppressive miRNAs or ablating oncomiRs, miRNA-based therapies can sensitize cancer cells to conventional cytotoxins and the molecularly targeted drugs by promoting apoptosis and autophagy, reverting epithelial-to-mesenchymal transition, suppressing tumor angiogenesis, and downregulating efflux transporters. The development of miRNA-based therapeutics in combination with small-molecule anticancer drugs provides an unprecedented opportunity to counteract chemoresistance and improve treatment outcome in a broad range of human cancers. This review summarizes the mechanisms and advantages for the combination therapies involving miRNAs and small-molecule drugs, as well as the recent advances in the co-delivery nanocarriers for these agents.     

MicroRNA-mediated suppression of P-glycoprotein by quantum dots in lung cancer cells

The interactions between adenosine triphosphate-binding cassette (ABC) transporters and nano-sized materials are attracting increasing attention, due to their great potential in overcoming the multidrug resistance (MDR) phenomena in cancer treatment. However, the inner mechanisms involved in the interactions are largely unknown. In this study, two commercial quantum dots (QDs), CdSe/ZnS-MPA and CdSe/ZnS-GSH, were tested for their interactions with P-glycoprotein (P-gp), as well as the relating mechanisms in lung cancer (A549) cells. Both QDs significantly suppressed the gene and protein expressions of P-gp in A549 cells. To explain this, the gene expressions of nine relating microRNAs (miRNAs) were evaluated. The results indicated a shared up-regulation of miR-34b and miR-185 by both QDs. Furthermore, mimics and inhibitors of miR-34b and miR-185 significantly enhanced and suppressed the gene and protein expressions of P-gp, respectively, confirming the modulatory function of these two miRNAs on P-gp. Interestingly, expressions of both miRNAs were suppressed during treatment with Cd²⁺ and doxorubicin, which induced the expression of P-gp, indicating the universality of these miRNAs-related mechanisms. Thus, as miR-34b and miR-185 participated in the suppression of P-gp functions in A549 cells they could be interesting targets for the treatment of lung cancer.     

Enhancing polysaccharide-mediated delivery of nucleic acids through functionalization with secondary and tertiary amines

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and (1)H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.     

Genomic sequence analysis of EGFR regulation by microRNAs in lung cancer

Lung cancer is known as the top cancer killer in most developed countries. Epidermal growth factor receptor (EGFR) is frequently found to be activated by mutation or amplification in lung cancer. MicroRNA (miRNA) is a new class of small molecules that has emerged as important markers of lung cancer development and therapeutic target. There are queries on which miRNAs can regulate EGFR and it is important to predict the candidate miRNAs that target EGFR by bioinformatics and to investigate on the availability of these candidate miRNA regulators in lung cancer. Systematic and rigorous searches for miRNAs targeting EGFR were performed on 10 representative databases. The identified miRNAs that target EGFR were formulated into a conditional regulation matrix and then hierarchical clustering algorithm was applied for the analysis. The systematic search came up with 138 miRNAs that potentially target EGFR. Among them, 11 miRNAs including miR-7 and miR-128b were confirmed by published experimental data or literatures. There were 14 candidate miRNAs predicted by at least 3 prediction pipelines in this study which have never been previously reported to target EGFR. Further studies of these novel identified miRNAs may provide insight on the regulation of EGFR in lung cancer. To the best of our knowledge, this is the first bioinformatic study applying genomic sequence analysis for the prediction of miRNAs that target EGFR in lung cancer. This new strategy that integrates computational and published data approaches provides a fast and effective prediction of miRNAs in specific target genes involved in various diseases.     

Enhancement of transfection activity of lipoplexes by complexation with transferrin-bearing fusogenic polymer-modified liposomes

We previously developed complexes of lipoplexes containing 3beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-chol) and succinylated poly(glycidol)-modified liposome, which becomes fusogenic under weakly acidic condition, for use as a novel gene delivery system. This study explored the effect of lipoplex structures--the type of cationic lipid and cationic lipid/DNA charge ratio--on the transfection activity of those complexes. Three types of cationic lipid with different polar groups were used for the preparation of lipoplexes: DC-chol, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), and 3,5-dipentadecyloxybenzamidine (TRX-20) with dimethylamino group, trimethylammonium group, and benzamidine group, respectively. Complexation with the SucPG-modified transferrin-bearing liposomes affected transfection activity of these lipoplexes differently. The TRX-20 lipoplexes exhibited the most marked enhancement of transfection activity upon complexation with the SucPG-modified liposomes among these lipoplexes. The cationic lipid/DNA charge ratio of the lipoplex and the amount of the transferrin-bearing SucPG-modified liposomes associated to the lipoplex also affected the transfection activity of the resultant complexes. Highly potent gene vectors were obtained by adjusting these factors.     

Anionic liposomal delivery system for DNA transfection

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.     

阳性脂质体介导基因转染及其研究进展*

基因治疗是一种很有前景的治疗模式,而阳性脂质体介导的基因转染是目前基因治疗的研究热点之一。现综述近5年来有关阳性脂质体的文献,介绍了阳性脂质体的基本组成,并从生物学、理化性质及制剂学等几个方面介绍了影响阳性脂质体/DNA复合物转染效率的主要因素,最后从新的阳性脂质成分及阳性脂质体或阳性脂质体/DNA复合物的表面修饰等方面介绍了近年来有关改善阳性脂质体介导基因转染的研究进展。     

In vivo studies of dialkynoyl analogues of DOTAP demonstrate improved gene transfer efficiency of cationic liposomes in mouse lung

A novel set of dialkynoyl analogues of the cationic, gene delivery lipid DOTAP (1) was synthesized. Structure-activity studies demonstrate that replacement of the cis-double bonds of DOTAP with triple bonds in varying positions alters both the physical properties of the resultant cationic liposome-DNA complexes and their biological functionalities, both in vitro and in vivo. Particularly, in vivo studies demonstrate that pDNA transfection of mouse lung endothelial cells with lead analogue DS(14-yne)TAP (4):cholesterol lipoplexes exhibits double the transfection level with less associated toxicity relative to the well-established DOTAP:cholesterol system. In fact, 4:cholesterol delivers up to 3 times the dose of pDNA in mice than can be tolerated by DOTAP, leading to nearly 3 times greater marker-gene expression. X-ray diffraction studies suggest that lipoplexes containing analogue 4 display increased stability at physiological temperatures. Our results thus suggest that analogue 4 is a potentially strong candidate for the gene therapy of lung tumors.     

MicroRNA-147b suppresses the proliferation and invasion of non-small-cell lung cancer cells through downregulation of Wnt/β-catenin signalling via targeting of RPS15A

Deregulation of microRNAs (miRNAs) leads to malignant growth and aggressive invasion during cancer occurrence and progression. miR-147b has emerged as one of the cancer-related miRNAs that are dysregulated in multiple cancers. Yet, the relevance of miR-147b in non-small-cell lung cancer (NSCLC) remains unclear. In the present study, we aimed to report the biological function and signalling pathways mediated by miR-147b in NSCLC. Our results demonstrate that miR-147b expression is significantly downregulated in NSCLC tissues and cell lines. Overexpression of miR-147b decreased the proliferative ability, colony-forming capability, and invasive potential of NSCLC cells. Notably, our study identified ribosomal protein S15A (RPS15A), an oncogene in NSCLC, as a target gene of miR-147b. Our results showed that miR-147b negatively modulates RPS15A expression in NSCLC cells. An inverse correlation between miR-147b and RPS15A was evidenced in NSCLC specimens. Moreover, miR-147b overexpression downregulated the activation of Wnt/β-catenin signalling via targeting of RPS15A. Overexpression of RPS15A partially reversed the miR-147b-mediated antitumour effect in NSCLC cells. Collectively, these findings reveal that miR-147b restricts the proliferation and invasion of NSCLC cells by inhibiting RPS15A-induced Wnt/β-catenin signalling and suggest that the miR-147b/RPS15A/Wnt/β-catenin axis is an important regulatory mechanism for malignant progression of NSCLC.     

Aerosolization of lipoplexes using AERx® pulmonary delivery system

The lung represents an attractive target for delivering gene therapy to achieve local and potentially systemic delivery of gene products. The objective of this study was to evaluate the feasibility of the AERx Pulmonary Delivery System for delivering nonviral gene therapy formulations to the lung. We found that “naked” DNA undergoes degradation following aerosolization through the AERx nozzle system. However, DNA formulated with a molar excess of cationic lipids (lipoplexes) showed no loss of integrity. In addition, the lipoplexes showed no significant change in particle size, zeta (ζ) potential, or degree of complexation following extrusion. The data suggest that complexation with cationic lipids had a protective effect on the formulation following extrusion. In addition, there was no significant change in the potency of the formulation as determined by a transfection study in A-549 cells in culture. We also found that DNA formulations prepared in lactose were aerosolized poorly. Significant improvements in aerosolization efficiency were seen when electrolytes such as NaCl were added to the formulation. In conclusion, the data suggest that delivery of lipoplexes using the AERx Pulmonary Delivery System may be a viable approach for pulmonary gene therapy.