Exosome-Mediated miR-21 Was Involved in the Promotion of Structural and Functional Recovery Effect Produced by Electroacupuncture in Sciatic Nerve Injury.

PURPOSE: Our study is aimed at investigating the mechanism by which electroacupuncture (EA) promoted nerve regeneration by regulating the release of exosomes and exosome-mediated miRNA-21 (miR-21) transmission. Furthermore, the effects of Schwann cells- (SC-) derived exosomes on the overexpression of miR-21 for the treatment of PNI were investigated. METHODS: A sciatic nerve injury model of rat was constructed, and the expression of miR-21 in serum exosomes and damaged local nerves was detected using RT-qPCR after EA treatment. The exosomes were identified under a transmission electron microscope and using western blotting analysis. Then, the exosome release inhibitor, GW4869, and the miR-21-5p-sponge used for the knockdown of miR-21 were used to clarify the effects of exosomal miR-21 on nerve regeneration promoted by EA. The nerve conduction velocity recovery rate, sciatic nerve function index, and wet weight ratio of gastrocnemius muscle were determined to evaluate sciatic nerve function recovery. SC proliferation and the level of neurotrophic factors were assessed using immunofluorescence staining, and the expression levels of SPRY2 and miR-21 were detected using RT-qPCR analysis. Subsequently, the transmission of exosomal miR-21 from SC to the axon was verified in vitro. Finally, the exosomes derived from the SC infected with the miR-21 overexpression lentivirus were collected and used to treat the rat SNI model to explore the therapeutic role of SC-derived exosomes overexpressing miR-21. RESULTS: We found that EA inhibited the release of serum exosomal miR-21 in a PNI model of rats during the early stage of PNI, while it promoted its release during later stages. EA enhanced the accumulation of miR-21 in the injured nerve and effectively promoted the recovery of nerve function after PNI. The treatment effect of EA was attenuated when the release of circulating exosomes was inhibited or when miR-21 was downregulated in local injury tissue via the miR-21-5p-sponge. Normal exosomes secreted by SC exhibited the ability to promote the recovery of nerve function, while the overexpression of miR-21 enhanced the effects of the exosomes. In addition, exosomal miR-21 secreted by SC could promote neurite outgrowth in vitro. CONCLUSION: Our results demonstrated the mechanism of EA on PNI from the perspective of exosome-mediated miR-21 transport and provided a theoretical basis for the use of exosomal miR-21 as a novel strategy for the treatment of PNI.

FTO (Fat-Mass and Obesity-Associated Protein) Participates in Hemorrhage-Induced Thalamic Pain by Stabilizing Toll-Like Receptor 4 Expression in Thalamic Neurons.

Background and Purpose: Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N6-methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N6-methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown. Methods: Expression of Fto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus of Ftofl/fl mice on the Coll IV microinjection­induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressing Fto (AAV5-Fto) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N6-methyladenosine in Tlr4 mRNA and its binding to FTO or YTHDF2 (YTH N6-methyladenosine RNA binding protein 2) were observed. Results: FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection into Ftofl/fl mouse thalamus attenuated the Coll IV microinjection­induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5-Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of N6-methyladenosine sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus. Conclusions: Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.

Silencing of Gasdermin D by siRNA-Loaded PEI-Chol Lipopolymers Potently Relieves Acute Gouty Arthritis through Inhibiting Pyroptosis.

Gasdermin D (GSDMD) plays a causal role in NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis eruption, which has been regarded as a potential therapeutic target for pyroptosis-related diseases including acute gouty arthritis. In the present study, the synthesized PEI-Chol (cholesterol grafted polyethylenimine) was assembled with GSDMD small interfering RNA (siRNA) to form PEI-Chol/siGSDMD polyplexes, which provided high transfection efficiency for siRNA-mediated GSDMD knockdown. Then we evaluated the effect of GSDMD siRNA-loaded PEI-Chol on inflammatory cascades in bone-marrow-derived macrophages (BMDMs) and acute gouty arthritis animal models under MSU exposure. When accompanied by pyroptosis blockade and decreased release of interleukin-1 beta (IL-1ß), NLRP3 inflammasome activation was also suppressed by GSDMD knockdown in vivo and in vitro. Moreover, in MSU-induced acute gouty arthritis mice, blocking GSDMD with siRNA significantly improved ankle swelling and inflammatory infiltration observed in histopathological analysis. Furthermore, investigation using a mouse air pouch model verified the effect of siGSDMD-loaded PEI-Chol on pyroptosis of recruited macrophages and related signaling pathways in response to MSU. These novel findings exhibited that GSDMD knockdown relieved acute gouty arthritis through inhibiting pyroptosis, providing a possible therapeutic approach for MSU-induced acute gouty arthritis molecular therapy using PEI-Chol as a nucleic acid delivery carrier.

Generation of self-compatible diploid potato by knockout of S-RNase.

Re-domestication of potato into an inbred line-based diploid crop propagated by seed represents a promising alternative to traditional clonal propagation of tetraploid potato, but self-incompatibility has hindered the development of inbred lines. To address this problem, we created self-compatible diploid potatoes by knocking out the self-incompatibility gene S-RNase using the CRISPR-Cas9 system. This strategy opens new avenues for diploid potato breeding and will also be useful for studying other self-incompatible crops.

High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue.

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 µM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.

Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide-mediated appetite control in amphetamine-treated rats.

BACKGROUND AND PURPOSE: Amphetamine is a releaser of dopamine stored in synaptic terminals, which can suppress appetite by changing the expression levels of neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamus. This study explored whether ERKs are involved in appetite control mediated by cAMP response element binding protein (CREB), NPY and POMC in amphetamine-treated rats. EXPERIMENTAL APPROACH: Rats were given amphetamine for 4 days, and changes in feeding behaviour and expression levels of phosphorylated-ERK (pERK), pCREB, NPY and melanocortin MC3 receptors were examined and compared. KEY RESULTS: Following amphetamine treatment, food intake, body weight and NPY expression decreased, whereas the expression of pERK, pCREB, MC3 receptors and pCREB/DNA binding activity increased. In amphetamine-treated rats, both cerebral ERK knockdown and pretreatment with a peripheral dopamine receptor antagonist decreased NPY but increased pERK, pCREB and MC3 receptor expression. Moreover, the immunofluorescence of hypothalamic pERK increased following amphetamine treatment. CONCLUSIONS AND IMPLICATIONS: These results suggest that ERK/CREB signalling participates in the effects mediated by dopamine receptor/NPY/POMC on appetite control in rats treated with amphetamine. These findings advance the knowledge on the involvement of ERK/CREB signalling in the reciprocal regulation by NPY and POMC of appetite after amphetamine treatment.

Evodiamine Exerts an Anti-Hepatocellular Carcinoma Activity through a WWOX-Dependent Pathway.

Evodiamine is one of the main components isolated from Evodia rutaecarpa, and it has been reported to exert inhibitory effects on cancers by anti-proliferative and apoptosis-inducing activities. Although the anti-cancer activity of evodiamine has been identified, the precise mechanisms of this action remain obscure. While previous studies indicated that evodiamine exerts anti-tumor effects through inhibiting ß-catenin activity, and WW domain-containing oxidoreductase (WWOX) regulates ß-catenin accumulation in cytoplasm, the effects of evodiamine on the expression of WWOX are still unknown. In this study, we provide evidence that evodiamine dose- and time-dependently inhibits both Mus musculus and Homo sapiens hepatocellular carcinoma (HCC) cells, as well as Hepa1-6 and HepG2 cell proliferation. We further tested the therapeutic effects of evodiamine in Hepa1-6 hepatoma-bearing mice, and we found that treatment of evodiamine by oral gavage significantly decreased the tumor size of the mice. Moreover, the expressions of WWOX were dose-dependently increased in HCC cell lines as well as in Hepa1-6 hepatoma-bearing mice after the treatment with evodiamine. Knockdown of WWOX in HepG2 and Hepa1-6 cells diminished the effects of evodiamine on the inhibitory effect of cancer cell growth, indicating that evodiamine induced anti-cancer activity through a WWOX-dependent pathway. As such, evodiamine activated WWOX to exert an anti-HCC activity, and might be a potential therapeutic or preventive candidate for HCC treatment.

Suppression of protein tyrosine phosphatase PTPN22 gene induces apoptosis in T-cell leukemia cell line (Jurkat) through the AKT and ERK pathways.

The aim of this study was to investigate the effect of specific PTPN22 small interfering RNAs (siRNAs) on the viability and induction of apoptosis in Jurkat cells and to evaluate apoptosis signaling pathways. In this study, Jurkat cells were transfected with specific PTPN22 siRNA. Relative PTPN22 mRNA expression was measured by Quantitative Real-time PCR. Western blotting was performed to determine the protein levels of PTPN22, AKT, P-AKT, ERK, and P-ERK. The cytotoxic effects of PTPN22 siRNA were determined using the MTT assay. Apoptosis was quantified using TUNEL assay and flow cytometry. Results showed that in Jurkat cells after transfection with PTPN22 siRNA, the expression of PTPN22 in both mRNA and protein levels was effectively reduced. Moreover, siRNA transfection induced apoptosis on the viability of T-cell acute leukemia cells. More importantly, PTPN22 positively regulated the anti-apoptotic AKT kinase, which provides a powerful survival signal to T-ALL cells as well as the suppression of PTPN22 down regulated ERK activity. Our results suggest that the PTPN22 specific siRNA effectively decreases the viability of T-cell acute leukemia cells, induces apoptosis in this cell line, and therefore could be considered as a potent adjuvant in T-ALL therapy.

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-µM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

Lentiviral vector-mediated doxycycline-inducible USP39 shRNA or cDNA expression in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC), characterized by distinct biological and clinicopathological features, has a poor prognosis due to lack of effective therapeutic targets. Our previous data revealed that high levels of USP39 were selectively present in TNBC samples compared with their normal breast tissue samples and USP39 was also expressed at different levels in cultured TNBC cells and normal breast cells. Yet, the underlying cellular and molecular mechanisms of USP39 remain unclear. In the present study, we describe a doxycycline (DOX)-regulated lentiviral vector system expressing shRNA or cDNA of the USP39 gene in the TNBC cell line MDA-MB-231. USP39 expression was knocked down by the miR-30-based inducible lentiviral short hairpin RNA (shRNA) delivery system or overexpressed by the inducible cDNA system. The inducible shRNA-mediated downregulation of USP39 expression markedly reduced the proliferation and colony-forming ability of MDA-MB-231 cells, while overexpression of USP39 by the inducible system did not promote cancer cell proliferation. The lentiviral vector-mediated Tet-on system demonstrated efficient and inducible knockdown of USP39 or overexpression of USP39 in TNBC cells, facilitating a wide variety of applications for gene knockdown and overexpression experiments in gene functional studies in vitro and in vivo.

Vitamin D counteracts fibrogenic TGF-ß signalling in human hepatic stellate cells both receptor-dependently and independently.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity and constitutes part of the metabolic syndrome, which have been associated with low serum vitamin D (VD). Due to known crosstalk between VD and transforming growth factor (TGF)-ß signalling, VD has been proposed as an antifibrotic treatment. DESIGN: We evaluated the association between VD, the vitamin D receptor (VDR) and liver fibrosis in primary human hepatic stellate cells (phHSC) and 106 morbidly obese patients with NAFLD. RESULTS: Treating phHSC with VD ameliorated TGF-ß-induced fibrogenesis via both VDR-dependent and VDR-independent mechanisms. Reduction of fibrogenic response was abolished in cells homozygous for GG at the A1012G single nucleotide polymorphisms within the VDR gene. Compared with healthy livers, NAFLD livers expressed higher levels of VDR mRNA and VDR fragments. VDR mRNA was lower in patients homozygous for GG at A1012G and expression of pro-fibrogenic genes was higher in patients carrying the G allele. CONCLUSIONS: VD may be an antifibrotic treatment option early in the onset of fibrosis in specific genotypes for VDR. Known polymorphisms of the VDR may influence the response to VD treatment.

Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).

Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium.

Virus-aided gene expression and silencing using TRV for functional analysis of floral scent-related genes.

Flower scent is a composite character determined by a complex mixture of low-molecular-weight volatile molecules. Despite the importance of floral fragrance, our knowledge on factors regulating these pathways remains sketchy. Virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE) are characterized by a simple inoculation procedure and rapid results as compared to transgenesis, allowing screening and characterization of scent-related genes. Here, we describe methods using TRV as a VIGS/VAGE vector for the characterization of scent-related genes, protein compartmentalization studies, and protein subcellular targeting.

Hydrogen peroxide-mediated oxidative stress and collagen synthesis in cardiac fibroblasts: blockade by tanshinone IIA.

ETHNOPHARMACOLOGICAL RELEVANCE: We have recently reported that tanshinone IIA attenuated cardiac fibrosis in two-kidney, two-clip renovascular hypertensive rats via inhibiting NAD(P)H oxidase. However, little is known about the cellular and molecular mechanisms of tanshinone IIA mediated anti-fibrotic effects in cardiac fibroblasts after H(2)O(2) stimulation. The present study was performed to investigate whether H(2)O(2) may increase collagen synthesis in cardiac fibroblasts by affecting the expression and activity of NAD(P)H oxidase and whether the effects of H(2)O(2) on cardiac fibroblasts can be blocked by treatment of tanshinone IIA. MATERIALS AND METHODS: Cardiac fibroblasts were treated with H(2)O(2) (100 µmol/L) in the presence or absence of tanshinone IIA (1 µmol/L), NAD(P)H oxidase inhibitors diphenyleneiodonium (10 µmol/L), siRNA-p47phox, siRNA-Nox2 and siRNA-Nox4. Collagen synthesis was measured by [(3)H]proline incorporation, O(2)(-) production were determined by flow cytometry and DHE fluorescence microscopy. NAD(P)H oxidase activity was measured by lucigenin-enhanced chemiluminescence. RESULTS: H(2)O(2) induced the activity of NAD(P)H oxidase, O(2)(-) production, collagen synthesis and fibronectin expression in cardiac fibroblasts, and DPI abolished this induction. Exposure of adult rat cardiac fibroblasts to H(2)O(2) had time-dependent increase in the expression of p47phox, Nox2 and Nox4 oxidases. In addition, tanshinone IIA significantly inhibited H(2)O(2)-induced collagen synthesis via attenuation of O(2)(-) generation and NAD(P)H oxidase activity. Moreover, siRNA-mediated knockdown of p47phox, Nox2 and Nox4 inhibited H(2)O(2)-induced NADPH oxidase activity. H(2)O(2)-induced collagen synthesis and fibronectin expression were also inhibited by p47phox, Nox2 and Nox4 knock down. CONCLUSIONS: Our data show that NAD(P)H oxidase plays a significant role in regulating collagen synthesis in H(2)O(2)-stimulated cardiac fibroblasts. Inhibition of NAD(P)H oxidase with tanshinone IIA completely blocked the H(2)O(2)-stimulated collagen production, which will raise the experimental basis for using tanshinone IIA to cardiac fibrosis in clinic.

Systemic delivery of therapeutic small interfering RNA using a pH-triggered amphiphilic poly-L-lysine nanocarrier to suppress prostate cancer growth in mice.

Prostate cancer is associated with high mortality and new therapeutic strategies are necessary for improved patient outcome. The utilisation of potent, sequence-specific small interfering RNA (siRNA) to facilitate down-regulation of complementary mRNA sequences in vitro and in vivo has stimulated the development of siRNA-based cancer therapies. However, the lack of an effective siRNA delivery system significantly retards clinical application. Amphiphilic polycations with 'stealth' capacity have previously been synthesised by PEGylation of poly-l-lysine-cholic acid (PLL-CA). The benzoic imine linker between PEG and PLL-CA was designed to be stable at physiological pH but cleavable at lower pHs, consistent with the extracellular environment of tumours and the interior of endosomes/lysosomes. The selective hydrolysis of the PEG linker at these targeted sites should provide enhanced cellular uptake and endosomal escape while simultaneously ensuring prolonged blood circulation times. In this study, physicochemical profiling demonstrated nano-complex formation between the PLL derivatives and siRNA (200-280 nm in diameter). At physiological pH only a slight cationic surface charge (<20 mV) was detected, due to the masking effect of the PEG. In contrast, significantly higher positive charges (∼20 to 30 mV and >40 mV) were detected upon hydrolysis of the PEG linker at acidic pHs (pH=6.8 and 5.5, respectively). The PEGylated complexes were stable in serum without significant aggregation or decomplexation of siRNA for up to 48 h. At the cellular level, PEG-PLLs were comparable with the commercial carrier INTERFRin, in terms of cellular uptake, endosomal escape and in vitro reporter gene knockdown. In vivo, utilising a mouse model grafted with prostate carcinoma, significant tumour suppression was achieved using PEGylated complexes without marked toxicity or undesirable immunological response, this was accompanied by a simultaneous reduction in target mRNA levels. In summary, the advantages of these vectors include: the in vitro and in vivo silencing efficiency, and the low toxicity and immunogenicity.

Effect of dietary polyunsaturated fatty acids on castration-resistant Pten-null prostate cancer.

A common treatment of advanced prostate cancer involves the deprivation of androgens. Despite the initial response to hormonal therapy, eventually all the patients relapse. In the present study, we sought to determine whether dietary polyunsaturated fatty acid (PUFA) affects the development of castration-resistant prostate cancer. Cell culture, patient tissue microarray, allograft, xenograft, prostate-specific Pten knockout and omega-3 desaturase transgenic mouse models in conjunction with dietary manipulation, gene knockdown and knockout approaches were used to determine the effect of dietary PUFA on castration-resistant Pten-null prostate cancer. We found that deletion of Pten increased androgen receptor (AR) expression and Pten-null prostate cells were castration resistant. Omega-3 PUFA slowed down the growth of castration-resistant tumors as compared with omega-6 PUFA. Omega-3 PUFA decreased AR protein to a similar extent in tumor cell cytosolic and nuclear fractions but had no effect on AR messenger RNA level. Omega-3 PUFA treatment appeared to accelerate AR protein degradation, which could be blocked by proteasome inhibitor MG132. Knockdown of AR significantly slowed down prostate cancer cell proliferation in the absence of androgens. Our data suggest that omega-3 PUFA inhibits castration-resistant prostate cancer in part by accelerating proteasome-dependent degradation of the AR protein. Dietary omega-3 PUFA supplementation in conjunction with androgen ablation may significantly delay the development of castration-resistant prostate cancer in patients compared with androgen ablation alone.

Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes.

Regulation of the human HBA genes by KLF4 in erythroid cell lines.

KLF1/EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the HBB-like globin genes. Prompted by the observation that four KLF sites are distributed in the human alpha-globin gene (HBA) promoter, we investigated if KLFs could also act to modulate the expression of the HBA genes. Among the KLFs tested, only KLF4/GKLF bound specifically to three out of four alpha-globin KLF sites. The occupancy of the same sites by KLF4 in vivo was confirmed by chromatin immunoprecipitation assays with KLF4-specific antibodies. In luciferase reporter assays in MEL cells, high levels of the wild type HBA promoter, but not mutated promoters bearing point mutations that disrupted KLF4-DNA binding, were transactivated by over-expression of KLF4. In K562 cells, induced KLF4 expression with a Tet-off regulated cassette stimulated the expression of the endogenous HBA genes. In a complementary assay in the same cell line, knocking down KLF4 with lentiviral delivered sh-RNAs caused a parallel decrease in the transcription of the HBA genes. All experiments combined support a regulatory role of KLF4 in the control of HBA gene expression.