Delivery Of Short Hairpin RNA Research Articles

Chaperone-mediated autophagy (CMA) confers neuroprotection of HBO preconditioning against stroke

Previous attempts to identify neuroprotective targets for acute ischemic stroke by studying ischemic cascades and devising ways to suppress these pathways have failed in translational research. We hypothesized that studying the molecular determinants of endogenous neuroprotection, namely, the tolerance against ischemic stroke conferred by hyperbaric oxygen (HBO) preconditioning, via a well-established paradigm would reveal new neuroprotective targets. By a combination of proteomics, KEGG pathway analysis, lysosome fraction and western blot analysis, we found that chaperone-mediated autophagy (CMA) was activated by HBO preconditioning. In addition, LAMP2A is uniquely decreased in cortical neurons in the early stage of stroke. Suppression of CMA with recombinant adeno-associated viral vector (rAAV)-mediated delivery of short hairpin RNAs (shRNAs) targeting the LAMP2A transcript increased the neuronal susceptibility of apoptosis and abolished the neuroprotection induced by HBO preconditioning. Administration of the clinically utilized FDA-approved drug mycophenolate mofetil induced long-term neuroprotection post-stroke in a CMA-dependent manner. In summary, HBO preconditioning confers neuroprotection against ischemia by inducing CMA, which is a promising translational treatment target for stroke.

Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions

BackgroundExosomes released by cardiomyocytes are essential mediators of intercellular communications within the heart, and various exosomal proteins and miRNAs are associated with cardiovascular diseases. However, whether the endosomal sorting complex required for transport (ESCRT) and its key component Alix is required for exosome biogenesis within cardiomyocyte remains poorly understood. MethodsSuper-resolution imaging was performed to investigate the subcellular location of Alix and multivesicular body (MVB) in primary cardiomyocytes. Cardiomyocyte-specific Alix-knockout mice were generated using AAV9/CRISPR/Cas9-mediated in vivo gene editing. A stable Alix-knockdown H9c2 cardiomyocyte line was constructed through lentiviral-mediated delivery of short hairpin RNA. In order to determine the role of Alix in controlling exosome biogenesis, exosomes from cardiomyocyte-specific Alix-knockout mice plasma and Alix-knockdown H9c2 culture medium were isolated and examined by western blot, NTA analysis and transmission electron microscopy. Biochemical and immunofluorescence analysis were performed to determine the role of ESCRT machinery in regulating MVB formation. Lastly, transverse aortic constriction (TAC)-induced cardiac pressure overload model was established to further explore the role of Alix-mediated exosome biogenesis under stress conditions. ResultsA significant proportion of Alix localized to the MVB membrane within cardiomyocytes. Genetic deletion of Alix in murine heart resulted in a reduction of plasma exosome content without affecting cardiac structure or contractile function. Consistently, the downregulation of Alix in H9c2 cardiomyocyte line also suppressed the biogenesis of exosomes. We found the defective ESCRT machinery and suppressed MVB formation upon Alix depletion caused compromised exosome biogenesis. Remarkably, TAC-induced cardiac pressure overload led to increased Alix, MVB levels, and elevated plasma exosome content, which could be totally abolished by Alix deletion. ConclusionThese results establish Alix as an essential and stress-sensitive regulator of cardiac exosome biogenesis and the findings may yield valuable therapeutic implications.

Nucleolin-targeted cationic nanoparticle for delivery of survivin shRNA against colorectal cancer in vitro and in vivo

In this study, synthesis of Polylactic acid (PLA) polymer was carried out through ring-opening polymerization (ROP) method. Then, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as the CTA molecule was attached to the PLA, and subsequently, amphiphilic copolymer, with estimated structure of Poly(lactic acid)121-b-Poly(N-(3-aminopropyl)methacrylamide)103 (PLA121-b-PAPMA103) was obtained through the Reversible Addition Fragmentation chain Transfer (RAFT) reaction. The synthesized copolymer was self-assembled into spherical nanoparticles with size, Poly Dispersity Index (PDI) and zeta potential of 181.5 ± 21.3, 0.643 and of 44.9 ± 2.5 mV, respectively. The optimal copolymer/plasmid ratio was 4 with size, PDI and zeta potential of 161.7 ± 10.2, 0.277 and 36.5 ± 0.7, respectively for the delivery of short hairpin RNA (shRNA) plasmid against survivin. Additionally, the AS1411 aptamer was electrostatically decorated on the surface of the polyplex. In vitro studies on C26 cells revealed that the aptamer rendered nanoparticle transfer into the cell, dependent solely on the aptamer-receptor interactions. Lastly, in vivo studies involving tumor size and body weight change, survival and histological analysis indicated potential therapeutic index of the targeted nanoparticles with good safety profile.

Peroxiredoxin 1 regulates crosstalk between pyroptosis and autophagy in oral squamous cell carcinoma leading to a potential pro-survival

Peroxiredoxin 1 (Prdx1), a vital antioxidant enzyme, has been proven to play an important role in the occurrence and development of cancers, but its effects on oral squamous cell carcinoma (OSCC) remain unclear. Here, we performed bioinformatics analysis and immunohistochemical (IHC) staining to confirm that Prdx1 was higher in OSCC tissues than in normal tissues. Consistently, RT-PCR and Western blot showed elevated Prdx1 expression in OSCC cell lines compared to human oral keratinocytes (HOK), which could be knockdown by small interfering RNA (siRNA) and Lentiviral vector delivery of short hairpin RNA (shRNA). Prdx1 silencing significantly blocked OSCC cell proliferation and metastasis, as evidenced by the CCK8, colony formation, in vivo tumorigenesis experiment, wound healing, transwell assays, and changes in migration-related factors. siPrdx1 transfection increased intracellular reactive oxygen species (ROS) levels and provoked pyroptosis, proved by the upregulation of pyroptotic factors and LDH release. Prdx1 silencing ROS-independently blocked autophagy. Mature autophagosome failed to form in the siPrdx1 group. Up-regulated autophagy limited pyroptosis triggered by Prdx1 deficiency, and down-regulated pyroptosis partly reversed siPrdx1-induced autophagy defect. Collectively, Prdx1 regulated pyroptosis in a ROS-dependent way and modulated autophagy in a ROS-independent way, involving the crosstalk between pyroptosis and autophagy.

Oct4 and Hypoxia Dual-Regulated Oncolytic Adenovirus Armed with shRNA-Targeting Dendritic Cell Immunoreceptor Exerts Potent Antitumor Activity against Bladder Cancer.

Immunotherapy has emerged as a promising modality for cancer treatment. Dendritic cell immunoreceptor (DCIR), a C-type lectin receptor, is expressed mainly by dendritic cells (DCs) and mediates inhibitory intracellular signaling. Inhibition of DCIR activation may enhance antitumor activity. DCIR is encoded by CLEC4A in humans and by Clec4a2 in mice. Gene gun-mediated delivery of short hairpin RNA (shRNA) targeting Clec4a2 into mice bearing bladder tumors reduces DCIR expression in DCs, inhibiting tumor growth and inducing CD8+ T cell immune responses. Various oncolytic adenoviruses have been developed in clinical trials. Previously, we have developed Ad.LCY, an oncolytic adenovirus regulated by Oct4 and hypoxia, and demonstrated its antitumor efficacy. Here, we generated a Clec4a2 shRNA-expressing oncolytic adenovirus derived from Ad.LCY, designated Ad.shDCIR, aimed at inducing more robust antitumor immune responses. Our results show that treatment with Ad.shDCIR reduced Clec4a expression in DCs in cell culture. Furthermore, Ad.shDCIR exerted cytolytic effects solely on MBT-2 bladder cancer cells but not on normal NIH 3T3 mouse fibroblasts, confirming the tumor selectivity of Ad.shDCIR. Compared to Ad.LCY, Ad.shDCIR induced higher cytotoxic T lymphocyte (CTL) activity in MBT-2 tumor-bearing immunocompetent mice. In addition, Ad.shDCIR and Ad.LCY exhibited similar antitumor effects on inhibiting tumor growth. Notably, Ad.shDCIR was superior to Ad.LCY in prolonging the survival of tumor-bearing mice. In conclusion, Ad.shDCIR may be further explored as a combination therapy of virotherapy and immunotherapy for bladder cancer and likely other types of cancer.

Adeno-Associated Virus-Mediated Knockdown of Agmatinase Attenuates Inflammation and Tumorigenesis in a Mouse Model of Colitis-Associated Colorectal Cancer.

Agmatinase (AGMAT) is an enzyme that hydrolyzes agmatine to putrescine and urea. Here, we explored the functions of AGMAT in colorectal cancer (CRC). By performing gain-of-function and loss-of-function experiments, we investigated the roles of AGMAT in proliferation, cell cycle progression, and apoptosis of CRC cells. We also established a colitis-associated colorectal cancer (CAC) model by challenging mice with azoxymethane (AOM) and dextran sodium sulfate (DSS), and we subsequently silenced AGMAT expression in mice by adeno-associated virus 9 (AAV9)-mediated delivery of short hairpin RNA (shRNA). In vitro experiments showed that overexpression of AGAMT accelerated the proliferation and inhibited the apoptosis of CRC cells, and AGMAT knockdown exhibited the opposite effects. Interestingly, the oncogenic transcription factor c-Myc could bind to the AGMAT promoter and transcriptionally increase AGMAT expression in CRC cells. Additionally, c-Myc and AGMAT were upregulated in the colon of AOM/DSS-treated mice, and AGMAT silencing significantly mitigated colitis in AOM/DSS-treated mice, as evidenced by the increased colon length, attenuated crypt damage, and reduced levels of inflammatory indicators (myeloperoxidase, interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, and phosphorylated p65) in colon tissues. Notably, AGMAT silencing decreased both the number and size of tumors, reduced expression of proliferating cell nuclear antigen (PCNA), and inhibited the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in the colon of AOM/DSS-treated mice. Overall, we determined that AGMAT facilitates tumor progression in CRC. Our findings will be helpful in the search for potential therapeutic targets for CRC.

Silencing TLR4 using an ultrasound-targeted microbubble destruction-based shRNA system reduces ischemia-induced seizures in hyperglycemic rats.

The toll-like receptor 4 (TLR4) pathway is involved in seizures. We investigated whether ultrasound-targeted microbubble destruction (UTMD)-mediated delivery of short hairpin RNA (shRNA) targeting the TLR4 gene (shRNA-TLR4) can reduce ischemia-induced seizures in rats with hyperglycemia. A total of 100 male Wistar rats were randomly assigned to five groups: (1) Sham; (2) normal saline (NS); (3) shRNA-TLR4, where rats were injected with shRNA-TLR4; (4) shRNA-TLR4 + US, where rats were injected with shRNA-TLR4 followed by ultrasound (US) irradiation; and (5) shRNA-TLR4 + microbubbles (MBs) + US, where rats were injected with shRNA-TLR4 mixed with MBs followed by US irradiation. Western blot and immunohistochemical staining were used to measure TLR4-positive cells. Half of the rats in the NS group developed tonic-clonic seizures, and TLR4 expression in the CA3 region of the hippocampus was increased in these rats. In addition, the NS group showed an increased number of TLR4-positive cells compared with the Sham group, while there was a decreased number of TLR4-positive cells in the shRNA, shRNA + US, and shRNA + MBs + US groups. Our findings indicate that the TLR4 pathway is involved in the pathogenesis of ischemia-induced seizures in hyperglycemic rats and that UTMD technology may be a promising strategy to treat brain diseases.

Rifaximin stimulates nitrogen detoxification by PXR-independent mechanisms in human small intestinal organoids.

Recurrent hepatic encephalopathy (HE) is characterized by hyperammonaemia in combination with neuropsychiatric abnormalities and is treated with lactulose and rifaximin. Rifaximin is a pregnane X receptor (PXR) agonist with low systemic and high intestinal bioavailability. The mechanisms by which it alleviates HE are unclear. We used human small intestinal (hSI) organoids to study whether rifaximin, via PXR activation, affects the epithelial biotransformation machinery, and to gain understanding of its low systemic availability. We generated PXR knockdown hSI organoids via lentiviral delivery of short hairpin RNAs. Organoids were cultured for 24 h with rifaximin or rifampicin. RNA-sequencing and metabolomics were performed to analyse gene expression and amino acid metabolism. Luminal rifaximin was quantified by photospectrometry. Treatment of wild-type hSI organoids with rifaximin resulted in >twofold differential expression of 131 genes compared to DMSO. These effects were largely PXR independent and related to amino acid metabolism. Rifaximin decreased expression of glutaminase-2 and increased expression of asparagine synthetase and solute carrier 7A11, thereby increasing intracellular glutamine and asparagine concentrations, indicating active ammonia detoxification. Rifaximin was apically excreted into the lumen in an ATP binding cassette B1 (ABCB1)-dependent manner. Rifaximin-after uptake into enterocytes-stimulates intracellular nitrogen detoxification by PXR-independent mechanisms. Active apical excretion of rifaximin by ABCB1 into the intestinal lumen explains its low systemic bioavailability. Our study implies that rifaximin, next to modulation of the microbiome, has direct effects on ammonia scavenging in the human small intestinal epithelium.

SiRNA-mediated gene knockdown via electroporation in hydrozoan jellyfish embryos

As the sister group to bilaterians, cnidarians stand in a unique phylogenetic position that provides insight into evolutionary aspects of animal development, physiology, and behavior. While cnidarians are classified into two types, sessile polyps and free-swimming medusae, most studies at the cellular and molecular levels have been conducted on representative polyp-type cnidarians and have focused on establishing techniques of genetic manipulation. Recently, gene knockdown by delivery of short hairpin RNAs into eggs via electroporation has been introduced in two polyp-type cnidarians, Nematostella vectensis and Hydractinia symbiolongicarpus, enabling systematic loss-of-function experiments. By contrast, current methods of genetic manipulation for most medusa-type cnidarians, or jellyfish, are quite limited, except for Clytia hemisphaerica, and reliable techniques are required to interrogate function of specific genes in different jellyfish species. Here, we present a method to knock down target genes by delivering small interfering RNA (siRNA) into fertilized eggs via electroporation, using the hydrozoan jellyfish, Clytia hemisphaerica and Cladonema paciificum. We show that siRNAs targeting endogenous GFP1 and Wnt3 in Clytia efficiently knock down gene expression and result in known planula phenotypes: loss of green fluorescence and defects in axial patterning, respectively. We also successfully knock down endogenous Wnt3 in Cladonema by siRNA electroporation, which circumvents the technical difficulty of microinjecting small eggs. Wnt3 knockdown in Cladonema causes gene expression changes in axial markers, suggesting a conserved Wnt/β-catenin-mediated pathway that controls axial polarity during embryogenesis. Our gene-targeting siRNA electroporation method is applicable to other animals, including and beyond jellyfish species, and will facilitate the investigation and understanding of myriad aspects of animal development.

Fibroblast expression of CD248 may contribute to exacerbation of microvascular damage during systemic sclerosis.

CD248 is a glycoprotein, highly expressed on pericytes and fibroblasts (FBs), that is implicated in the fibrotic process. During angiogenesis, CD248 can promote vessel regression, binding multimerin-2 (MMRN-2). Thus, we investigated the expression of MMRN-2 in systemic sclerosis (SSc)-skin and of CD248 in isolated SSc-FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by co-culturing these cells with healthy control endothelial cells (HC-ECs). The apoptotic effect of CD248 on HC-ECs was evaluated. Finally, the ability of CD248 to prevent activation of VEGF receptor 2 (VEGFR2) was assessed. By IF, MMRN-2 was investigated in SSc-skin and CD248 in SSc FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by HC-ECs/SSc-FBs co-cultures. Lentiviral-induced CD248 short-hairpin RNA delivery was employed for loss-of-function studies in SSc-FBs. HC-ECs were cultured in the presence of CD248 to assess apoptosis by IF and VEGFR2 phosphorylation by western blot. MMRN-2 expression was increased in skin SSc-ECs, whereas CD248 expression was increased in SSc-FBs. Functionally, CD248+-SSc-FBs suppressed angiogenesis in the organotypic model, as assessed by the reduction in total tube length of HC-ECs. This anti-angiogenetic behaviour was reversed by CD248 silencing. Furthermore, the presence of CD248 promoted the apoptosis of HC-ECs. Finally, CD248 prevented activation of VEGFR2 by reducing its phosphorylation after VEGF stimulation. CD248 was anti-angiogenic in vitro due to a reduction in tube formation and to induction of apoptosis of ECs. Increased expression of CD248 in SSc could contribute to the microvascular rarefaction observed at the tissue level in SSc. Our results suggest a pathogenic role for CD248-MMRN-2 in SSc.

Mitofusin 2 confers the suppression of microglial activation by cannabidiol: Insights from in vitro and in vivo models

Currently, there is increasing attention on the regulatory effects of cannabidiol (CBD) on the inflammatory response and the immune system. However, the mechanisms have not yet been completely revealed. Mitofusin 2 (Mfn2) is a mitochondrial fusion protein involved in the inflammatory response. Here, we investigated whether Mfn2 confers the anti-inflammatory effects of CBD. We found that treatment with CBD decreased the levels of tumor necrosis factor α, interleukin 6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and ionized calcium-binding adaptor molecule-1 (Iba1) in lipopolysaccharide (LPS)-challenged microglia. CBD also significantly suppressed the increase in reactive oxygen species (ROS) and the decline of mitochondrial membrane potential in BV-2 cells subjected to LPS. Interestingly, CBD treatment increased the expression of Mfn2, while knockdown of Mfn2 blocked the effect of CBD. By contrast, overexpression of Mfn2 reversed the increase in the levels of iNOS, COX-2, and Iba1 induced by Mfn2 small interfering RNA. In mice challenged with LPS, we found that CBD ameliorated the anxiety responses and cognitive deficits, increased the level of Mfn2, and decreased the expression of Iba1. Since neuro-inflammation and microglial activation are the common events that are observed in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, we treated EAE mice with CBD. Mice that received CBD showed amelioration of clinical signs, reduced inflammatory response, and increased myelin basic protein level. Most importantly, the adeno-associated virus delivery of short hairpin RNA against Mfn2 reversed the protective effects of CBD. Altogether, these results indicate that Mfn2 is an essential immunomodulator conferring the anti-inflammatory effects of CBD. Our results also shed new light on the mechanisms underlying the protective effects of CBD against inflammatory diseases including multiple sclerosis.

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI).

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) as well as neural progenitor cells (NPCs) derived from hESCs at high efficiency. Lentiviral particles were generated by co-transfecting HEK293T cells using entry vectors (carrying shRNAs) along with packaging plasmids (pAX and pMD2.G) using the low-cost cationic polymer polyethylenimine (PEI). Viral particles were concentrated using ultracentrifugation, which resulted in average titers above 5 x 107. Both hESCs and NPCs could be infected at high efficiencies using these lentiviral particles, as shown by puromycin selection and stable expression in hESCs, as well as transient GFP expression in NPCs. Furthermore, western blot analysis showed a significant reduction in the expression of genes targeted by shRNAs. In addition, the cells retained their pluripotency as well as differentiation potential, as evidenced by their subsequent differentiation into different lineages of CNS. The current protocol deals with the delivery of shRNAs; however, the same approach could be used for the ectopic expression of cDNAs for overexpression studies.

High Mobility Group A1 (HMGA1) Epigenetic Regulators Induce ETV5 Networks in Relapsed B-Cell Leukemia and Provide Novel Therapeutic Targets

Introduction: Despite advances in therapy for B-cell acute lymphoblastic leukemia (B-ALL), relapsed disease remains the leading cause of death in children with cancer. The gene encoding the High Mobility Group A1 (HMGA1) chromatin regulator is highly expressed in stem cells and diverse malignancies where high levels portend poor outcomes. We discovered that transgenic mice misexpressing Hmga1 in lymphoid cells develop leukemic transformation by amplifying transcriptional networks involved in stem cell function, proliferation, and inflammation (Hillion et al, Cancer Res 2008, Schuldenfrei et al, BMC Genomics 2011, Xian et al, Nature Commun 2017). In pediatric B-ALL (pB-ALL), HMGA1 is overexpressed with highest levels in blasts from early relapse (Roy et al, Leuk Lymphoma 2013). Together, these findings suggest that HMGA1 is required for leukemogenesis and drives relapse through epigenetic reprogramming. We therefore sought to: 1) test the hypothesis that HMGA1 is required for leukemogenesis and relapse in pB-ALL, and, 2) elucidate targetable mechanisms mediated by HMGA1.Methods: To elucidate the function of HMGA1 and downstream targets, we employed CRISPR/Cas9 gene inactivation and lentiviral-mediated gene silencing via delivery of short hairpin RNA (shRNA) targeting 2 sequences per gene in cell lines from relapsed pB-ALL, including REH, which harbor the TEL-AML1 fusion, and 697, which harbor the E2A-PBX1 fusion. We assessed leukemia phenotypes in vitro and leukemic engraftment in vivo. To dissect molecular mechanisms, we performed RNA sequencing (RNAseq) and applied in silico pathway analysis. To validate these pathways in human pB-ALL, we assessed gene expression and clinical outcomes in independent cohorts. The Broad Institute Connectivity Map (CMAP) was applied to identify drugs to target HMGA1 networks.Results:HMGA1 is overexpressed in pB-ALL in independent cohorts with highest levels at relapse. Decreasing HMGA1 expression via CRISPR/Cas9 inactivation or shRNA-mediated gene silencing in relapsed pB-ALL cell lines (REH, 697) disrupts proliferation, decreases the frequency of cells in S phase concurrent with increases in G0/G1, enhances apoptosis, and impairs clonogenicity. To assess HMGA1 function in vivo, we compared leukemogenesis following tail vein injection of pB-ALL cell lines with or without HMGA1 depletion in immunodeficient mice (NOD/SCID/IL2 receptor gamma null). Survival was prolonged in mice injected with either pB-ALL cell line (REH, 697) after HMGA1 depletion. Further, leukemic cells that ultimately engraft show increased HMGA1 expression relative to the pool of injected cells with HMGA1 silencing, suggesting that escape from HMGA1 silencing was required for engraftment. RNAseq revealed transcriptional networks governed by HMGA1 that regulate proliferation (G2M checkpoint, E2F), RAS/ERK signaling, hematopoietic stem cells, and ETV5 (ETS variant 5 transcription factor) targets. Given its association with aggressive ALL harboring the BCR-ABL fusion, we focused on the ETV5 gene. CRISPR/Cas9 inactivation or gene silencing of ETV5 in relapsed pB-ALL cell lines (REH, 697) decreases proliferation and clonogenicity in vitro, while delaying leukemogenesis in vivo. Further, restoring ETV5 expression in pB-ALL cell lines with HMGA1 silencing partially rescues anti-leukemogenic effects of HMGA1 depletion. Mechanistically, HMGA1 binds to AT-rich regions within the ETV5 promoter (-0.7 kb and -0.2 kb) and recruits active histone marks (H3K27Ac, H3K4me3, H3K4me1) to induce ETV5. Epigenetic drugs predicted to target HMGA1-ETV5 networks synergize with HMGA1 silencing in cytotoxicity assays with pB-ALL cell lines. Most importantly, HMGA1 and ETV5 are co-expressed and up-regulated in primary blasts from children with pB-ALL with highest levels at relapse, thus underscoring the significance of this pathway in relapsed pediatric B-ALL.Conclusions: We discovered a previously unknown epigenetic program whereby HMGA1 up-regulates ETV5 networks by binding to chromatin and recruiting active histone marks to the ETV5 promoter. Both HMGA1 and ETV5 are up-regulated at relapse. Finally, the HMGA1-ETV5 axis can be targeted by epigenetic drugs (HDAC inhibitors) that synergize with HMGA1 depletion. Our findings reveal the HMGA1-ETV5 axis as a key molecular switch in relapsed pB-ALL and rational therapeutic target to treat or prevent relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TSPAN-7 as a key regulator of glucose-stimulated Ca2+ influx and insulin secretion.

TSPAN-7 as a key regulator of glucose-stimulated Ca2+ influx and insulin secretion.

Efficient, Non-Viral and Reproducible Protocol for Stable Knockdown of Genes in Mantle Cell Lymphoma Cell Lines

Introduction The biological role of specific genes can be investigated using gene silencing by cell transfection with small interfering RNA (siRNA), which leads to degradation of the corresponding mRNA and reduced target protein expression. The study of biological functions in modified cell cultures often requires a long-lasting knockdown (KD) of gene expression, which can be obtained by viral delivery of short hairpin RNA (shRNA) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 systems. However, these methods are laborious and require high level of laboratory safety. Hematopoietic cells, typically grown in suspension, are known to be difficult to transfect. Previous efforts to transfect lymphocytes using liposomes (H Guven et al, ExpHem2005) showed limited transfection efficiencies of only 10-30%. Transfection by electroporation leads to decreased viability, and therefore poses a risk of unintentional selection of cell subsets. Transfection of leukemia cells with siRNAs using Accell siRNA Delivery Media showed KD efficiencies of 56-65% and a viability of 57-95% (H Larsen et al,ExpHem2011). However, this was only investigated 24-72h post-transfection. By whole transcriptome sequencing, we previously identified a number of candidate genes, includingLILRA4, PTPRJ and SYNE2, which were significantly upregulated in mantle cell lymphoma (MCL) cells compared to B cells from healthy donors (MH Hansen et al, ExpHem 2020). To investigate the biological function of these genes, an efficient and stable KD was required. Aim The aim of this study was to identify the most effective non-viral method for siRNA-mediated knockdown in MCL cells. Methods The MCL cell lines Mino, Jeko-1 and Granta 519, were transfected using three different methodologies; Lipofectamine 2000 (Invitrogen), Electroporation with Amaxa Nucleofector (Lonza), and Accell siRNA Delivery Media (Dharmacon). For Accell delivery a pool of 4 siRNAs targeting each gene was used (SMARTpool siRNA, Dharmacon). For the others, 2 pre-designed siRNAs (Ambion) targeting each gene were used. For all, a positive control siRNA targetingGAPDHwas included and different conditions tested e.g. siRNA concentration, incubation time and addition of serum. KD efficiency was evaluated by qPCR usingABLandGUSreference genes. Viability of cells was measured either by flow cytometry staining with a live/dead marker or by trypan blue. Optimization of the Accell protocol was performed in Granta 519 cells transfected with SMARTpool siRNAs targeting eitherSYNE2,LILRA4,orGAPDH. KD efficiency was evaluated with qPCR and viability was counted with trypan blue. Results Using Lipofectamine, at 24h a non-reproducible and non-efficient KD (<40%) was achieved forLILRA4in the Jeko-1 cell line, while only 5-22% gene silencing was achieved for Mino. Using electroporation, a KD efficiency of 45-70% was achieved in Mino at 24h forGAPDH,LILRA4andPTPRJ, while inferior efficiency (<15%) and viability were observed in Jeko-1 cells. However, in the Mino cell line,PTPRJwas almost fully re-expressed (80% expression) after 48h. Using transfection with Accell delivery medium,GAPDH,LILRA4,andPTPRJwere successfully knocked down in all tested cell lines with a median efficiency of 75% (range 50%-98%) after 72h. When performing KD in Accell delivery medium, the viability of the cells decreased after 72h and dropped to 26-39% at day 5 and 6, and a decrease was found in the total number of cells, suggesting that proliferation may also be affected. By adding 10% fetal bovine serum (FBS) to this medium after 48h, the viability was kept above 66.7% for up to 6 days, and the total number of cells was comparable to that observed in normal culture medium, while the KD efficiency was still preserved. In Ganta 519 cells with addition of FBS, an average KD efficiency of 67% was achieved forLILRA4(range 56-72%) at 72h-5 days, while the average KD efficiency ofSYNE2was 77% (range: 67-95%) at 72h-5 days. Conclusion This study propose that transfection with SMARTpool siRNAs in Accell Delivery Medium with addition of 10% FBS 48h post transfection is an applicable procedure for siRNA-mediated KD in MCL suspension cell lines. This protocol permits efficient, non-viral, and stable KD across different MCL cell lines without affecting the cells' ability to proliferate and survive, and is therefore suitable for investigating biological processes. Disclosures No relevant conflicts of interest to declare.

Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons.

Piezo channels are mechanosensitive ion channels. Piezo1 is primarily expressed in nonsensory tissues, whereas Piezo2 is predominantly found in sensory tissues, including dorsal root ganglion (DRG) neurons. However, a recent study demonstrated the intracellular calcium response to Yoda1, a selective Piezo1 agonist, in trigeminal ganglion (TG) neurons. Herein, we investigate the expression of Piezo1 mRNA and protein in mouse and human DRG neurons and the activation of Piezo1 via calcium influx by Yoda1. Yoda1 induces inward currents mainly in small- (<25 μm) and medium-sized (25–35 μm) mouse DRG neurons. The Yoda1-induced Ca2+ response is inhibited by cationic channel blocker, ruthenium red and cationic mechanosensitive channel blocker, GsMTx4. To confirm the specific inhibition of Piezo1, we performed an adeno-associated virus serotype 2/5 (AAV2/5)-mediated delivery of short hairpin RNA (shRNA) into mouse DRG neurons. AAV2/5 transfection downregulates piezo1 mRNA expression and reduces Ca2+ response by Yoda1. Piezo1 also shows physiological functions with transient receptor potential vanilloid 1 (TRPV1) in the same DRG neurons and is regulated by the activation of TRPV1 in mouse DRG sensory neurons. Overall, we found that Piezo1 has physiological functions in DRG neurons and that TRPV1 activation inhibits an inward current induced by Yoda1.

ER stress-induced upregulation of NNMT contributes to alcohol-related fatty liver development

N-nicotinamide methyltransferase (NNMT) is emerging as an important enzyme in the regulation of metabolism. NNMT is highly expressed in the liver. However, the exact regulatory mechanism(s) underlying NNMT expression remains unclear and its potential involvement in alcohol-related liver disease (ALD) is completely unknown. Both traditional Lieber-De Carli and the NIAAA mouse models of ALD were employed. A small-scale chemical screening assay and a chromatin immunoprecipitation assay were performed. NNMT inhibition was achieved via both genetic (adenoviral short hairpin RNA delivery) and pharmacological approaches. Chronic alcohol consumption induces endoplasmic reticulum (ER) stress and upregulates NNMT expression in the liver. ER stress inducers upregulated NNMT expression in both AML12 hepatocytes and mice. PERK-ATF4 pathway activation is the main contributor to ER stress-mediated NNMT upregulation in the liver. Alcohol consumption fails to upregulate NNMT in liver-specific Atf4 knockout mice. Both adenoviral NNMT knockdown and NNMT inhibitor administration prevented fatty liver development in response to chronic alcohol feeding; this was also associated with the downregulation of an array of genes involved in de novo lipogenesis, including Srebf1, Acaca, Acacb and Fasn. Further investigations revealed that activation of the lipogenic pathway by NNMT was independent of its NAD+-enhancing action; however, increased cellular NAD+, resulting from NNMT inhibition, was associated with marked liver AMPK activation. ER stress, specifically PERK-ATF4 pathway activation, is mechanistically involved in hepatic NNMT upregulation in response to chronic alcohol exposure. Overexpression of NNMT in the liver plays an important role in the pathogenesis of ALD. In this study, we show that nicotinamide methyltransferase (NNMT) - the enzyme that catalyzes nicotinamide degradation - is a pathological regulator of alcohol-related fatty liver development. NNMT inhibition protects against alcohol-induced fatty liver development and is associated with suppressed de novo lipogenic activity and enhanced AMPK activation. Thus, our data suggest that NNMT may be a potential therapeutic target for the treatment of alcohol-related liver disease.

Isoform-specific functions of c-Jun N-terminal kinase 1 and 2 in lung ischemia-reperfusion injury through the c-Jun/activator protein-1 pathway

Background: c-Jun N-terminal kinase 1 (JNK1) and JNK2 regulate distinct pathological processes in lung diseases. Here we discriminated the respective roles of these kinases in lung transplantation-induced ischemia-reperfusion injury (IRI). MethodsRat pulmonary microvascular endothelial cells were transfected with JNK1 small-interfering RNA (siRNA) and JNK2 siRNA and then subjected to in vitro IRI. For the isoform confirmed to aggravate IRI, the delivery of short-hairpin RNA (shRNA) plasmid was performed by intratracheal administration 48 hours before transplantation into donor rats. After a 3-hour reperfusion, the samples were collected. ResultsJNK1 siRNA decreased but JNK2 siRNA increased JNK phosphorylation and activity, phosphorylated and total c-Jun, and activator protein-1 activity. Although JNK1 siRNA decreased apoptosis and the levels of malondialdehyde, interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF-α), it increased the levels of superoxide dismutase, S-phase percentage, and cyclin D1; JNK2 siRNA had a converse effect. JNK1 siRNA decreased the level of lactate dehydrogenase and increased the levels of VE-cadherin, nitric oxide, phosphorylated nitric oxide synthase, and cell viability; JNK2 si RNA had a converse effect. Compared with the control group, the JNK1 shRNA group exhibited a higher lung oxygenation index and lower lung apoptosis index, injury score, wet weight:dry weight ratio, and levels of IL-1, IL-6, and TNF-α. ConclusionsJNK1 aggravated, but JNK2 alleviated, IRI through differential regulation of the JNK1 pathway in in vitro ischemia-reperfusion. JNK1 silence attenuated lung graft dysfunction by inhibiting inflammation and apoptosis. These findings provide a theoretical basis for devising therapeutic strategies against IRI after lung transplantation.

High Mobility Group A1 Chromatin Regulators Function As Critical Drivers of Leukemogenesis in Preclinical Models of Relapsed, Pediatric B-Cell ALL

Introduction: Acute B-cell lymphoblastic leukemia (B-ALL) is the most common form of childhood leukemia and the leading cause of death in children with cancer. While therapy is often curative, about 10-15% of children will relapse with recurrent disease and abysmal outcomes. Actionable mechanisms that mediate relapse remain largely unknown. The gene encoding the High Mobility Group A1(HMGA1) chromatin regulator is overexpressed in diverse malignancies where high levels portend poor outcomes. In murine models, we discovered thatHmga1 overexpression is sufficient for clonal expansion and progression to aggressive acute lymphoid leukemia (Cancer Res 2008,68:10121, 2018,78:1890; Nature Comm 2017,8:15008). Further, HMGA1 is overexpressed in pediatric B-ALL (pB-ALL) blasts with highest levels in children who relapse early compared to those who achieve chronic remissions. Together, these findings suggest that HMGA1 is required for leukemogenesis and may foster relapse in B-ALL. We therefore sought to: 1) test the hypothesis that HMGA1 is a key epigenetic regulator required for leukemogenesis and relapse in pB-ALL, and, 2) elucidate targetable mechanisms mediated by HMGA1 in leukemogenesis. Methods: We silenced HMGA1 via lentiviral delivery of short hairpin RNAs targeting 2 different sequences in cell lines derived from relapsed pB-ALL (REH, 697). REH cells harbor the TEL-AML1 fusion; 697 cells express BCL2, BCL3, and cMYC. Next, we assessed leukemogenic phenotypes in vitro (proliferation, cell cycle progression, apoptosis, and clonogenicity) and leukemogenesis invivo. To dissect molecular mechanisms underlying HMGA1, we performed RNA-Seq and applied in silico pathway analysis. Results: There is abundant HMGA1 mRNA and protein in both pB-ALL cell lines and HMGA1 was effectively silenced by short hairpin RNA. Further, silencing HMGA1 dramatically halts proliferation in both cell lines, leading to a decrease in cells in S phase with a concurrent increase in G0/S1. Apoptosis also increased by 5-10% after HMGA1 silencing based on flow cytometry for Annexin V. In colony forming assays, silencing HMGA1 impaired clonogenicity in both pB-ALL cell lines. To assess HMGA1 function in leukemogenesis in vivo, we implanted control pB-ALL cells (transduced with control lentivirus) or those with HMGA1 silencing via tail vein injection into immunosuppressed mice (NOD/SCID/IL2 receptor γ). All mice receiving control REH cells succumbed to leukemia with a median survival of only 29 days. At the time of death, mice had marked splenomegaly along with leukemic cells circulating in the peripheral blood and infiltrating both the spleen and bone marrow. In contrast, mice injected with REH cells with HMGA1 silencing survived for &gt;40 days (P&lt;0.001) and had a significant decrease in tumor burden in the peripheral blood, spleen, and bone marrow. Similar results were obtained with 697 cells, although this model was more fulminant with control mice surviving for a median of only 17 days. To determine whether the leukemic blasts found in mice injected with ALL cells after HMGA1 silencing represented a clone that expanded because it escaped HMGA1 silencing, we assessed HMGA1 levels and found that cells capable of establishing leukemia had high HMGA1 expression, with levels similar to those observed in control cells without HMGA1 silencing. RNA-Seq analyses from REH and 697 cell lines with and without HMGA1 silencing revealed that HMGA1 up-regulates transcriptional networks involved in RAS/MAPK/ERK signaling while repressing the IDH1 metabolic gene, the latter of which functions in DNA and histone methylation. Studies are currently underway to identify effective agents to target HMGA1 pathways. Conclusions: Silencing HMGA1 dramatically disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression and epigenetic modifications. Our findings highlight the critical role for HMGA1 as a molecular switch required for leukemic transformation in pB-ALL and a rational therapeutic target that may be particularly relevant for relapsed B-ALL. Disclosures No relevant conflicts of interest to declare.

Evaluation of AAV-mediated delivery of shRNA to target basal-like breast cancer genetic vulnerabilities

Adeno-associated viral vectors (AAV) for gene therapy applications are gaining momentum, with more therapies moving into later stages of clinical development and towards market approval, namely for cancer therapy. The development of cytotoxic vectors is often hampered by side effects arising when non-target cells are infected, and their production can be hindered by toxic effects of the transgene on the producing cell lines. In this study, we evaluated the potential of rAAV-mediated delivery of short hairpin RNAs (shRNA) to target basal-like breast cancer genetic vulnerabilities. Our results show that by optimizing the stoichiometry of the plasmids upon transfection and time of harvest, it is possible to increase the viral titers and quality. All rAAV-shRNA vectors obtained efficiently transduced the BLBC cell lines MDA-MB-468 and HCC1954. In MDA-MB-468, transduction with rAAV-shRNA vector targeting PSMA2 was associated with significant decrease in cell viability and apoptosis induction. Importantly, rAAV2-PSMA2 also slowed tumor growth in a BLBC mouse xenograft model, thus potentially representing a therapeutic strategy against this type of cancer.