Lipofectamine RNAiMAX Research Articles

Hydrogel-mediated delivery of microRNA-92a inhibitor polyplex nanoparticles induces localized angiogenesis.

Localized stimulation of angiogenesis is an attractive strategy to improve the repair of ischemic or injured tissues. Several microRNAs (miRNAs) such as miRNA-92a (miR-92a) have been reported to negatively regulate angiogenesis in ischemic disease. To exploit the clinical potential of miR-92a inhibitors, safe and efficient delivery needs to be established. Here, we used deoxycholic acid-modified polyethylenimine polymeric conjugates (PEI-DA) to deliver a locked nucleic acid (LNA)-based miR-92a inhibitor (LNA-92a) in vitro and in vivo. The positively charged PEI-DA conjugates condense the negatively charged inhibitors into nano-sized polyplexes (135 ± 7.2nm) with a positive net charge (34.2 ± 10.6mV). Similar to the 25kDa-branched PEI (bPEI25) and Lipofectamine RNAiMAX, human umbilical vein endothelial cells (HUVECs) significantly internalized PEI-DA/LNA-92a polyplexes without any obvious cytotoxicity. Down-regulation of miR-92a following the polyplex-mediated delivery of LNA-92a led to a substantial increase in the integrin subunit alpha 5 (ITGA5), the sirtuin-1 (SIRT1) and Krüppel-like factors (KLF) KLF2/4 expression, formation of capillary-like structures by HUVECs, and migration rate of HUVECs in vitro. Furthermore, PEI-DA/LNA-92a resulted in significantly enhanced capillary density in a chicken chorioallantoic membrane (CAM) model. Localized angiogenesis was substantially induced in the subcutaneous tissues of mice by sustained release of PEI-DA/LNA-92a polyplexes from an in situ forming, biodegradable hydrogel based on clickable poly(ethylene glycol) (PEG) macromers. Our results indicate that PEI-DA conjugates efficiently deliver LNA-92a to improve angiogenesis. Localized delivery of RNA interference (RNAi)-based therapeutics via hydrogel-laden PEI-DA polyplex nanoparticles appears to be a safe and effective approach for different therapeutic targets.

P53.08 Analysis of Mitochondria-Mediated OCIAD2 Oncogenic Function in Lung Adenocarcinoma

Ovarian carcinoma immune-reactive antigen domain 2 (OCIAD2) has been reported as a novel tumor-specific protein (Cancer Sci, 2007). Overexpression of OCIAD2 is closely associated with the stepwise progression of early-stage lung adenocarcinoma and significantly associated with poorer outcomes in patients with ovarian and lung cancer (Pathol Int, 2012 and 2018). OCIAD2 is localized predominantly in the mitochondrial membrane in cancer cells. However, the oncogenic functions mediated by OCIAD2 are poorly understood. In this study, we explored the potential role of OCIAD2 in mitochondria and identified the OCIAD2 binding partner in lung adenocarcinoma cells. We performed most experiments after 72 hours of siRNA (scramble or siOCIAD2) transfection using lipofectamine RNAiMAX or after 24 hours of Flag-OCIAD2 plasmid transfection using Fugene HD. Alterations to mitochondrial morphology in the siRNA-transfected lung adenocarcinoma cells (A549 or HCC827) were observed using transmission electron microscopy (TEM). The siRNA-transfected cells were stained with MitoPT TMRM fluorescent dye and healthy cells were detected with electrical potential gradient by flow cytometry. To identify the OCIAD2-binding partners in lung adenocarcinoma cells, the membrane fraction including the mitochondria membrane was isolated from 3'-Flag-OCIAD2- or 5'-Flag-OCIAD2-transfected cells, followed by pull-down assay and LC-MS/MS analysis. We found that suppression of OCIAD2 led to marked destruction of morphologic features of mitochondria such as cristae in lung adenocarcinoma cells. The number of mitochondria per cell and the number of cristae per mitochondrion were significantly decreased, and there was a loss of mitochondrial electrical potential gradient in comparison with siCON. By LC-MS/MS analysis, we identified several OCIAD2-binding partner proteins that are significantly associated with apoptosis, crista formation, metabolic processes, membrane permeability, and transport. OCIAD2 is abnormally overexpressed in lung adenocarcinoma and may be associated with mitochondria-mediated oncogenic signaling such as blocking of apoptosis, active mitochondrial fitness, and maintenance of the mitochondrial electrical potential gradient through interaction with mitochondrial proteins. The fact that tumor cells are controlled by OCIAD2 and depend on its oncogenic function is of considerable interest.

Sixteen Different Types of Lipid-Conjugated siRNAs Containing Saturated and Unsaturated Fatty Acids and Exhibiting Enhanced RNAi Potency.

SiRNAs are strong gene-silencing agents that function in a target sequence-specific manner. Although siRNAs might one day be used in therapy for intractable diseases such as cancers, a number of problems with siRNAs must first be overcome. In this study, we developed 16 different types of lipid-conjugated siRNAs (lipid-siRNAs) that could effectively inhibit the expression of target genes. We determined the hybridization properties, cellular uptake efficacies, and RNAi potencies of the resulting lipid-siRNAs. The lipid-siRNAs exhibited a mild interaction with Lipofectamine RNAiMAX (LFRNAi) as a transfection reagent, and a high membrane permeability was observed in all lipid-siRNAs-LFRNAi complexes; the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed an especially good cellular uptake efficacy. The in vitro RNAi effect of lipid-siRNAs targeted to a β-catenin gene exhibited a strong RNAi potency compared with those of unmodified siRNAs. In particular, the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed excellent RNAi potencies with prolonged effectivities. Interestingly, the RNAi potencies of conjugate siRNAs containing 18 carbon chains with a trans-form (elaidic acid and trans-vaccenic acid) were inferior to those of the carbon chains with a cis-form (oleic acid and cis-vaccenic acid). These lipid-siRNAs can solve the many problems hindering the clinical application of siRNAs.

Abstract 5916: The role of YAP signaling in KRAS driven non-small cell lung cancer

Abstract Background Oncogenic KRAS mutant non-small cell lung cancer (NSCLC) remains treatment refractory. Even with the novel KRAS-G12C inhibitors in clinical development, caution should be taken because tumors will likely develop resistance. Subgroups of KRAS mutant NSCLC, defined by co-occurring genetic alterations in STK11/LKB1 (KL) and TP53 (KP) tumor suppressor genes, have distinct biology and therapeutic vulnerabilities. The Hippo pathway effector YAP has been shown to substitute oncogenic KRAS in promoting the growth of lung adenocarcinomas and confer resistance to MAPK pathway inhibitors and immunotherapy. Here we have examined the role of YAP in KL, KP and K-only (intact STK11/LKB1 and TP53) NSCLC cells. Methods All the cell lines were obtained from ATCC. Cell viability was assessed by CellTiter-Glo Luminescent Assay (Promega) and protein levels by western blot. YAP silencing was achieved by siRNA transfection (20 nM) using Lipofectamine RNAiMAX. Results YAP is phosphorylated by the Hippo/LATS kinase cascade at serine 127 (S127), resulting in cytoplasmic sequestration. YAP phosphorylation at tyrosine 357 (Y357) results in its nuclear localization and is Hippo/LATS independent. We first assessed the status of YAP phosphorylation in our KRAS mutant NSCLC cell lines. Among 5 KL (H460, H2030, A549, H23 and H2122) cells, 3 (H460, H2030 and A549) had higher YAP(Y357) and lower YAP(S127) expression compared to KP (H1792, SK-LU1, H2009 and Calu-6) and K-only (H358 and Calu-1) cells, normal fibroblasts (IMR-90) and the Hippo/LATS inactive breast cancer cell line, MDA-MB-231. These findings suggest the nuclear retention of YAP in KL cells. In all 3 KL cells we detected the expression of CTGF and/or Cyr61, the bona fide YAP target genes. H460 (KL) cells had the highest PD-L1 expression, although high PD-L1 expression was also seen in KP (H2009) and K-only (Calu-1) cells. Then we sought to explore whether YAP serves a functional role in KL cells compared to KP or K-only cells. H460 (KL), H1792 (KP), and H358 (K-only) cells were transfected with YAP siRNA or control siRNA. YAP protein expression was reduced in all cells by 70-90% compared to control siRNA-transfected cells. Transfection with YAP siRNA reduced the viability of H460 (KL) cells more profoundly compared to H1792 (KP), and H358 (K-only) cells. The effect of YAP silencing on downstream signaling and immune cell composition and the co-targeting of YAP and MEK in vitro and in vivo are ongoing. Conclusions STK11/LKB1 alterations have been identified as a major driver of primary resistance to PD-1 or PD-L1 blockade in KRAS mutant NSCLC. YAP acts as a transcriptional driver of cytokines that orchestrate the immunosuppressive microenvironment within KRAS mutant tumors. Our results corroborate the role of YAP in KRAS mutant NSCLC and allow considering the synthetic lethality of YAP suppression with MEK inhibitors or immunotherapy as a promising strategy to enhance treatment response and patient survival. Citation Format: Marina Keil, Richard Schneider, Elisabeth Trivier, Christian Dillon, Dirk Wienke, Anita Seshire, Linda Pudelko, Andree Blaukat, Niki Karachaliou. The role of YAP signaling in KRAS driven non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5916.

N-glycosylated IgG in patients with kidney transplants increases calcium/calmodulin kinase IV in podocytes and causes injury.

Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3β (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.

The Role Of Cd36 In Relieving Lipotoxicity Of Skeletal Muscle Cells In High Free Fatty Acid Environment.

PURPOSE: Lipotoxicity is closely related to the etiology and complications of type 2 diabetes mellitus (T2DM). Lipotoxicity in muscle cells induces insulin resistance, which is a key factor in the pathogenesis of T2DM. This study investigated the protective effect of Fatty Acid Translocase (FAT/CD36) against palmitic acid (PA)- induced lipoapoptosis. METHODS: Cells at ~40-60% confluence were transfected with siCtrl or siCD36 for overnight using Lipofectamine RNAiMAX. Cells were treated with PA at 200μM for 16 h. The PA-induced viability in C2C12 cells was measured by MTT assay; the PA-induced apoptosis in C2C12 cells was monitored by flow cytometry. The differences in means were analyzed by t test. RESULTS: PA treatment increased apoptosis (7.50% ± 0.21% vs. 10.40% ± 1.25%, p < 0.05) and decreased viability (100% ± 2.40% vs. 97.32% ± 3.60%, p < 0.05) in C2C12 cells in contrast to cells that were treated with PA-free media. After 16 h of PA treatment without CD36 protection, C2C12 cells had a significant increase in apoptosis when treated with siCD36 transfection, in contrast to cells that were negative control siRNA transfected (10.40% ± 1.25% vs. 16.04% ± 1.58%, p < 0.05), indicating that the existence of CD36 may protect high PA-induced lipoapoptosis by increasing cell uptake of FFA. Further statistical analysis confirmed a significant increase in the percentage of apoptotic cells both in early stage and late stage when subjected to CD36 deficiency (7.93% ± 0.57% vs. 10.70% ± 1.55%, p < 0.05; 2.47% ± 0.68% vs. 5.34% ± 0.06%, p < 0.05, respectively). CONCLUSIONS: This study demonstrated a novel function of CD36 in preventing lipotoxicity in skeletal muscle cells when subject to high FFA environment, implicating a promising target for obesity and diabetic therapy.

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.

Cationic Polymer Nanoparticles-Mediated Delivery of miR-124 Impairs Tumorigenicity of Prostate Cancer Cells.

MicroRNAs (miRNAs) play a pivotal role in regulating the expression of genes involved in tumor development, invasion, and metastasis. In particular, microRNA-124 (miR-124) modulates the expression of carnitine palmitoyltransferase 1A (CPT1A) at the post-transcriptional level, impairing the ability of androgen-independent prostate cancer (PC3) cells to completely metabolize lipid substrates. However, the clinical translation of miRNAs requires the development of effective and safe delivery systems able to protect nucleic acids from degradation. Herein, biodegradable polyethyleneimine-functionalized polyhydroxybutyrate nanoparticles (PHB-PEI NPs) were prepared by aminolysis and used as cationic non-viral vectors to complex and deliver miR-124 in PC3 cells. Notably, the PHB-PEI NPs/miRNA complex effectively protected miR-124 from RNAse degradation, resulting in a 30% increase in delivery efficiency in PC3 cells compared to a commercial transfection agent (Lipofectamine RNAiMAX). Furthermore, the NPs-delivered miR-124 successfully impaired hallmarks of tumorigenicity, such as cell proliferation, motility, and colony formation, through CPT1A modulation. These results demonstrate that the use of PHB-PEI NPs represents a suitable and convenient strategy to develop novel nanomaterials with excellent biocompatibility and high transfection efficiency for cancer therapy.

A light-triggerable formulation to control the stability of pro-angiogenic transcription factor hypoxia inducible factor-1α (HIF-1α).

The control of vascular remodeling mediated by transcription factor HIF-1α is critical in the treatment of several diseases including cancer, retinopathies, chronic wounds, and ischemic heart disease, among others. Gene silencing using a small interfering RNA (siRNA) is a promising therapeutic strategy to regulate HIF-1α; however, the delivery systems developed so far have limited endothelial targeting and efficiency. Herein, we have synthesized a light-triggerable polymeric nanoparticle (NP) library composed of 110 formulations which showed variable morphology, charge and disassembly rates after UV exposure. More than 35% of the formulations of the library were more efficient in gene knockdown than the siRNA delivered by a commercial transfection agent (lipofectamine RNAiMAX). The most efficient siRNA delivery formulations were tested against different cell types to identify one with preferential targeting to endothelial cells. Using a two-step methodology, we have identified a formulation that shows exquisite targeting to endothelial cells and is able to deliver more efficiently the siRNA that modulates HIF-1α than commercial transfection agents. Overall, the strategy reported here increases the specificity for tissue regulation and the efficiency for the intracellular delivery of siRNAs.

Cigarette Smoke Extract Promotes Human Lung Myofibroblast Differentiation by the Induction of Endoplasmic Reticulum Stress

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal fibrotic lung disease with an unknown aetiology. Persistent myofibroblast differentiation is a prominent feature of IPF. Cigarette smoking is a risk factor for IPF and an indicator of poor prognosis. Cigarette smoking induces endoplasmic reticulum (ER) stress, and it has been shown that ER stress promotes fibroblast-to-myofibroblast differentiation in lung fibrosis. In this study, we investigated whether cigarette smoke extract (CSE) promotes lung myofibroblast differentiation via the induction of ER stress. Objectives: Our study concentrates on exploring the relationship between smoking and ER stress in the differentiation of lung fibroblasts to myofibroblasts. Methods: Human embryonic lung fibroblasts (MRC-5 fibroblasts) were stimulated with various doses of CSE. Levels of α-smooth muscle actin (α-SMA) protein were evaluated by immunofluorescence and western blot analyses. ER stress was induced by thapsigargin (TG) and inhibited by 4-phenyl butyric acid (4-PBA). Protein levels of glucose-regulated protein-78 (GRP78), inositol-requiring enzyme 1 (IRE1), X box-binding protein-1 (XBP-1) and activating transcription factor 6 (ATF6) were determined by western blotting. GRP78 siRNA was transfected into MRC-5 cells using Lipofectamine RNAiMAX Reagent. Results: CSE at a concentration of 1.0% significantly increased α-SMA expression in MRC-5 cells. There was no significant cell apoptosis after cells were exposed to CSE. CSE treatment significantly increased the expression of GRP78, IRE1, XBP-1 and ATF6 at the protein level at 48 h. Pretreatment with TG enhanced, whereas pretreatment with 4-PBA inhibited, the CSE-induced expression of α-SMA, GRP78 and XBP-1. Furthermore, knockdown of GRP78 blocked α-SMA expression in MRC-5 cells exposed to CSE. Conclusion: Our data suggested that CSE promotes lung fibroblast-to-myofibroblast differentiation by the induction of ER stress.

Comparison between Lipofectamine RNAiMAX and GenMute transfection agents in two cellular models of human hepatoma.

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-trasfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR finding similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have a higher viability with respect to Lipofectamine RNAiMAXadministered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.

Interleukin-33/ST2 Signaling Promotes Hepatocellular Carcinoma Cell Stemness Expansion Through Activating c-Jun N-terminal Kinase Pathway

BackgroundInterleukin-33 (IL-33) has a variety of biological activities in different pathological models. However, the underlying effects of IL-33 on hepatocellular carcinoma (HCC) have not been fully elucidated. Therefore, we focused on investigating the biological effects of IL-33 on HCC stemness expansion. Materials and MethodsIL-33 expression in clinical tissue specimens were evaluated using immunohistochemical technology. Western blotting, flow cytometry, and quantitative real-time polymerase chain reaction were used to detect protein expressions in HCC cell lines. ST2 expression was downregulated by utilizing the synthetic siRNA sequence that specifically targets ST2. The transfection of ST2 siRNAs and control siRNAs into HCC cells was performed with Lipofectamine RNAi MAX (Life Technologies) according to the manufacturer's protocol. ResultsOur results demonstrated that IL-33 is expressed both in cancer cells and stromal cells of the HCC microenvironment, and that IL-33 expression in cancer cells, but not in stromal cells, was negatively associated with survival of HCC patients. IL-33 promotes HCC stemness expansion, including upregulating core stem cell gene expression, inducing cell sphere formation and preventing chemotherapy-induced apoptosis in vitro. Mechanistically, IL-33 binds to its receptor ST2 and induces phosphorylation of c-Jun N-terminal kinase activation (JNK), which leads to HCC stemness expansion. ConclusionsIL-33/ST2 signaling might be potential prognostic markers and therapeutic targets for HCC patients.

Abstract 4839: Double knockdown of MDM4 and MDM2 modulates the antitumor effects of cytotoxic drugs in colon and gastric cancer cells expressing high levels of MDM4

Abstract Introduction and Aim: Inactivation of the TP53 tumor suppressor gene is important for cancer development and progression. Wild-type (wt) TP53 is inactivated in various types of cancers including colon and gastric cancers by upregulated murine double minute homolog 2 (MDM2) and MDM4. We previously reported that simultaneous knockdown of MDM4 and MDM2 using synthetic DNA-modified siRNAs revived p53 activity and synergistically inhibited in vitro cell growth in cancer cells with wild-type TP53 and high MDM4 expression (wtTP53/highMDM4), whereas only MDM2 knockdown but not MDM4 knockdown suppressed that of high MDM2 expression cancer cells. In the present study, we investigated the combined activity of MDM4/MDM2 double knockdown and cytotoxic drugs (5-fluorouracil (5-FU), cisplatin, oxaliplatin) to develop a new therapy for gastric and colon cancers with wtTP53/highMDM4. Experimental procedure: Two colon (HCT116, LoVo) and two gastric cancer cell lines (NUGC-4, SNU-1) were used. Respective synthetic DNA-modified siRNAs against MDM4 (chiMDM4) and MDM2 (chiMDM2) were transfected using Lipofectamine RNAiMAX. Cell viability was determined by WST-8 assays. Expression of p53, p21, MDM2, MDM4, and β-actin were analyzed by immunoblotting. Cell cycle distribution was analyzed by flow cytometry. In vivo antitumor effects were examined using nude mice carrying HCT116 xenograft tumors. siRNAs with atelocollagen were intratumorally injected weekly and 5-FU was intraperitoneally administered three times per week. Results: MDM4/MDM2 double knockdown with the siRNAs enhanced in vitro antitumor activity of 5-FU in all four wtTP53/highMDM4 cell lines (HCT116, LoVo, SNU-1, and NUGC-4) and cisplatin and oxaliplatin in gastric (NUGC-4) and colon cancer cell lines (HCT116, LoVo), respectively. Exposure to 5-FU increased p53 and its downstream gene products (p21, PUMA, MDM2), and induced cell cycle arrest in the G1 phase. MDM4/MDM2 double knockdown suppressed MDM2, and enhanced expression of p53 and p21 and arrested G1. p53-MDM2 forms a negative feedback loop. Enhanced antitumor effects of 5-FU by MDM4/MDM2 double knockdown may be attributed to blocking this feedback loop, in addition to direct suppression of MDM4/MDM2. Since MDM2 interacts directly with molecules like RB, p21, and E2F1, other than p53, MDM2 knockdown may have some advantages over peptides or small molecular inhibitors of p53-MDM2 interaction in such combination therapies. Intratumor injection of chiMDM4/chiMDM2 suppressed in vivo tumor growth and boosted the antitumor effects of 5-FU in a xenograft model using HCT116 cells. Conclusion: Combining MDM4/MDM2 knockdown and conventional cytotoxic drugs may be a promising treatment strategy for wtTP53/highMDM4 gastrointestinal cancers. Citation Format: Mamiko Imanishi, Yoshiyuki Yamamoto, Shinji Endo, Kenji Yamato, Ichinosuke Hyodo. Double knockdown of MDM4 and MDM2 modulates the antitumor effects of cytotoxic drugs in colon and gastric cancer cells expressing high levels of MDM4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4839.

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

Optimization of miRNA delivery by using a polymeric conjugate based on deoxycholic acid-modified polyethylenimine.

Safe and efficient delivery of microRNA (miRNA) molecules is essential for their successful transition from research to the clinic setting. In the present study, we have used a bile acid, deoxycholic acid (DA), to modify 1.8 kDa branched polyethylenimine (bPEI1.8) and subsequently investigated gene delivery features of the resultant conjugates (PEI-DAn). We found significant differences between the PEI-DAn conjugates and conventional bPEIs with respect to miRNA condensation ability, buffering capacity, cellular uptake, and intracellular gene release behavior in endothelial cells (ECs) isolated from human umbilical vein (HUVECs). Changes in the conjugation degree greatly influenced the transfection performance of the PEI-DAn conjugates with respect to miRNA condensation and decondensation properties as well as cellular uptake behavior. The PEI-DA3 conjugates could significantly enhance the expression level of miRNA-210 in HUVECs. The overexpressed miRNA-210, in turn, markedly downregulated the expression levels of Efna3 and Ptp1b as well as led to a substantial rise in HUVECs' migration rate in a wound healing assay. Collectively, our results have demonstrated that PEI-DA3 conjugates facilitate the formation of stable nanocomplexes that are loose enough to release miRNAs into the cytosol. The free bioavailable miRNAs, in turn, result in efficient gene silencing comparable to bPEI25 as well as Lipofectamine RNAiMAX.

Anti-EMT properties of CoQ0 attributed to PI3K/AKT/NFKB/MMP-9 signaling pathway through ROS-mediated apoptosis

BackgroundBreast cancer is the most prevalent cancer among women. In triple-negative breast cancer (TNBC) cells, a novel quinone derivative, coenzyme Q0 (CoQ0), promotes apoptosis and cell-cycle arrest. This study explored the anti-epithelial–mesenchymal transition (EMT) and antimetastatic attributes of CoQ0 in TNBC (MDA-MB-231).MethodsInvasion, as well as MTT assays were conducted. Lipofectamine RNAiMAX was used to transfect cells with β-catenin siRNA. Through Western blotting and RT-PCR, the major signaling pathways’ protein expressions were examined, and the biopsied tumor tissues underwent immunohistochemical and hematoxylin and eosin staining as well as Western blotting.ResultsCoQ0 (0.5–2 μM) hindered tumor migration, invasion, and progression. Additionally, it caused MMP-2/− 9, uPA, uPAR, and VEGF downregulation. Furthermore, in highly metastatic MDA-MB-231 cells, TIMP-1/2 expression was subsequently upregulated and MMP-9 expression was downregulated. In addition, CoQ0 inhibited metastasis and EMT in TGF-β/TNF-α-stimulated non-tumorigenic MCF-10A cells. Bioluminescence imaging of MDA-MB-231 luciferase–injected live mice demonstrated that CoQ0 significantly inhibited metastasis of the breast cancer to the lungs and inhibited the development of tumors in MDA-MB-231 xenografted nude mice. Silencing of β-catenin with siRNA stimulated CoQ0-inhibited EMT. Western blotting as well as histological analysis established that CoQ0 reduced xenografted tumor development because apoptosis induction, cell-cycle inhibition, E-cadherin upregulation, β-catenin downregulation, and metastasis and EMT regulatory protein modulation were observed.ConclusionsCoQ0 inhibited the progression of metastasis as well as EMT (in vitro and in vivo). The described approach has potential in treating human breast cancer metastasis.

Biomimetic Dextran-Peptide Vectors for Efficient and Safe siRNA Delivery.

Small interfering RNA (siRNA)-based therapeutics have the potential to treat a series of hereditary and acquired diseases. However, one serious obstacle for siRNA therapy is the lack of an efficient strategy to transport the siRNA to the targeted organ/cell with minimal toxicity. To take advantage of the good biocompatibility and degradability of natural polymers, and to understand how the peptide sequence affects the properties of the vector, four biomimetic vectors (D10-K3H7, D10-R3H7, D20-K3H7, and D20-R3H7) were designed and synthesized by conjugating the peptide (K3H7 or R3H7) and dextran with a molecular weight of 10 or 20 kDa. Taking the commercial cellular transfection reagent Lipofectime RNAiMAX as a control, dextran-peptide/siRNA complexes exhibited smaller particle sizes, lower ζ potentials, and lower toxicity with the same value of N/P ratio. To evaluate the potential of this system for therapeutics, siRNA targeting the mRNA of the PCSK9 gene was chosen as a gene drug model to knock down the PCSK9 expression in the HepG2 cell line. Dextran-peptide/siRNA complexes exhibit a more consistent and higher knockdown efficiency than Lipofectamine RNAiMAX/PCSK9 siRNA complexes in a medium with 20% fetal bovine serum (FBS). D20-R3H7/PCSK9 siRNA complexes could knock down the level of PCSK9 mRNA by 85.2%, and they demonstrated a higher efficiency than Lipofectamine RNAiMAX, having 70.5% knockdown in the medium with 20% FBS at the PCSK9 siRNA concentration of 100 nM. These results suggest that the dextran-peptide-based vector has more efficient therapeutic agent properties for a siRNA-based drug transporter.

MiR‐590‐3p Restores Autophagy in Cultured Human Glomerular Endothelial Cells Exposed to High Glucose

BackgroundAutophagy is an important mechanism for homeostasis. Previous data mining indicated that miR‐590‐3p was involved in diabetes‐induced endothelial dysfunction. The present study was designed to investigate the role of microRNA‐590‐3p (miR‐590‐3p) in the regulation of autophagy in cultured endothelial cells incubated with high glucose.MethodsCultured human aortic endothelial cells (HAEC) and glomerular endothelial cells (HGEC) were subjected to high glucose (44.4mM). To overexpress miRNA‐590‐3p, lipofectamine RNAiMAX was used for transfection. Cells were collected for quantitative real‐time polymerase chain reaction and western blotting.ResultsAfter six hours stimulation with high glucose, expressions of miR‐590‐3p were significantly reduced in HGEC, but not in HAEC, while expressions of miR‐590‐5p were not changed in both cells. After 48 hours, high glucose treatment blunted autophagy in HGEC, which was shown as reduced protein expressions of microtubule‐associated proteins 1A/1B light chain 3B‐II (LC3B‐II), autophagy‐related genes (ATG) 5, and beclin‐1, as well as increased protein expressions of SQSTM1/p62. Overexpressing miR‐590‐3p restored the impairment of autophagy in HGEC. Incubation of HGEC with high glucose reduced phosphorylation of 5′ AMP‐activated protein kinase (AMPK) and increased phosphorylation of mammalian target of rapamycin (mTOR), two upstream signaling of autophagy. Overexpressing miR‐590‐3p increased the phosphorylation of AMPK and reduced the phosphorylation of mTOR, suggesting that miR‐590‐3p restored autophagy in HGEC through AMPK/mTOR pathway. High glucose stimulation did not alter apoptosic protein expressions in HGEC or HAEC, including bcl‐2, pBAD and caspase‐3.ConclusionHigh glucose incubation reduces autophagy in HGEC which is prevented by overexpressing MiR‐590‐3p through AMPK/mTOR signaling pathways.Support or Funding InformationThe present study was supported by the National Nature Science Foundation of China (81573418) and ZSYXRC‐014This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CIGARETTE SMOKE EXTRACT PROMOTES HUMAN LUNG MYOFIBROBLAST DIFFERENTIATION BY THE INDUCTION OF ENDOPLASMIC RETICULUM STRESS

PURPOSE: IPF is a progressive and lethal fibrotic lung disease with an unknown aetiology. Cigarette smoking is a risk factor for IPF and an indicator of poor prognosis. Cigarette smoking induces endoplasmic reticulum (ER) stress, and it has been shown that ER stress promotes fibroblast-to-myofibroblast differentiation in lung fibrosis. In this study, we investigated whether cigarette smoke extract (CSE) promotes lung myofibroblast differentiation via the induction of ER stress. METHODS: Human embryonic lung fibroblasts (MRC-5 fibroblasts) were stimulated with various doses of CSE. Levels of α-smooth muscle actin (α-SMA) protein were evaluated by immunofluorescence and western blot analyses. ER stress was induced by thapsigargin (TG) and inhibited by 4-phenyl butyric acid (4-PBA). Protein levels of GRP78, IRE1, XBP-1 and ATF6 were determined by western blotting. GRP78 siRNA was transfected into MRC-5 cells using Lipofectamine RNAiMAX Reagent. RESULTS: CSE at a concentration of 1.0% significantly increased α-SMA expression in MRC-5 cells. There was no significant cell apoptosis after cells were exposed to CSE. CSE treatment significantly increased the expression of GRP78, IRE1, XBP-1 and ATF6 at the protein level at 48 h. Pretreatment with TG enhanced, whereas pretreatment with 4-PBA inhibited, the CSE-induced expression of α-SMA, GRP78 and XBP-1. Furthermore, knockdown of GRP78 blocked α-SMA expression in MRC-5 cells exposed to CSE. CONCLUSIONS: Our data suggested that CSE promotes lung fibroblast-to-myofibroblast differentiation by the induction of ER stress. CLINICAL IMPLICATIONS: Our studies offer a new strategy to treat smoking-related lung interstitial diseases, such as in IPF patients who smoke.