Scramble siRNA Research Articles

Microneedle-Mediated Delivery of siRNA via Liposomal-Based Transfection for Inner Ear Gene Therapy.

Microneedle-mediated intracochlear injection of siRNA-Lipofectamine through the round window membrane (RWM) can be used to transfect cells within the cochlea. Our laboratory has developed 100-μm diameter hollow microneedles for intracochlear injection through the guinea pig RWM. In this study, we test the feasibility of microneedle-mediated injection of siRNA and Lipofectamine, a commonly used reagent with known cellular toxicity, through the RWM for cochlear transfection. Fluorescently labeled scramble siRNA was diluted into Lipofectamine RNAiMax and OptiMEM. One microliter of 5 μM siRNA was injected through the RWM of Hartley guinea pigs at a rate of 1 μl/min (n = 22). In a control group, 1.0 μl of Lipofectamine, with no siRNA, was diluted into OptiMEM and injected in a similar fashion (n = 5). Hearing tests were performed before and either at 24 hours, 48 hours, or 5 days after injection. Afterward, animals were euthanized, and cochleae were harvested for imaging. Control cochleae were processed in parallel to untreated guinea pigs. Fluorescence, indicating successful transfection, was observed within the basal and middle turns of the cochlea with limited distribution in the apex at 24 and 48 hours. Signal was most intense in the organ of Corti, spiral ligament, and spiral ganglion. Little to no fluorescence was observed at 5 days post-injection. No significant changes in auditory brainstem response (ABR) were noted post-perforation at 5 days, suggesting that siRNA-Lipofectamine at low doses does not cause cochlear toxicity. Small volumes of siRNA and Lipofectamine can be effectively delivered to cochlear structures using microneedles, paving the way for atraumatic cochlear gene therapy.

Normalization of Muc5b ameliorates airway mucus plugging during persistent Pseudomonas aeruginosa infection in the CFTR-/- rat.

In cystic fibrosis, the airway gel-forming mucin MUC5B accumulates in the airways, preventing clearance of pathogens like Pseudomonas aeruginosa (PA). The cystic fibrosis transmembrane conductance regulator (CFTR)-/- (KO) rat model exhibits a similar accumulation of Muc5b. Our lab has shown that increased Muc5b precipitates the development of chronic PA infection. We hypothesized that reducing Muc5b in the KO rat airway would prevent occlusive mucus plugs and development of persistent PA infection. Six-month-old KO rats received Muc5b or scramble siRNA via intratracheal instillation. Rats were then inoculated with 106 colony-forming units of mucoid P. aeruginosa isolate PAM57-15 and euthanized at 3- or 14-days post infection (dpi) to assess acute and persistent infection. At 14 dpi, Muc5b siRNA-treated KO rats had increased weight, decreased neutrophilic inflammation, and reduced mucus plugging in the small airways compared with scramble-treated KO and WT rats. These results indicate that pharmacological intervention of Muc5b reduces mucus plugging during persistent PA infection.NEW & NOTEWORTHY Although highly effective modulator therapies for cystic fibrosis (CF) have improved mucus-related outcomes of disease for people with CF, eradication of Pseudomonas aeruginosa (PA) infection has not been achieved in this population. In addition, current therapies for CF do not target mucin hypersecretion directly. Here, we show that a novel approach of normalizing airway Muc5b hypersecretion ameliorates infection-induced mucus plugging and neutrophilic inflammation during persistent PA infection in CFTR-/- rats.

Abstract 12: Deficiency of Progranulin Decreases Mitochondrial Function and Induces a Whitening Phenotype in Perivascular Adipose Tissue, Suppressing Its Anti-Contractile Effects

Perivascular adipose tissue (PVAT) is recognized as an endocrine organ that secretes a variety of adipokines, which negatively regulate vascular contraction. Progranulin (PGRN), a glycoprotein encoded by the GRN gene, is highly expressed in adipocytes, and is implicated in inflammation, cellular proliferation, and repair mechanisms. The relationship between PGRN and PVAT remains unclear. We hypothesized that the lack of PGRN negatively affects PVAT quality. We used male C57BL/6J wild-type (WT) and global PGRN knockout (KO) mice, aged 10-12 weeks. Energy expenditure was analyzed via the Comprehensive Lab Animal Monitoring System (CLAMS). Vascular function was studied in endothelium-intact aortae with PVAT [PVAT(+)] and without PVAT [PVAT(-)]. Additionally, 3T3-L1 cells were used to evaluate the molecular mechanisms. The KO mice showed a decreased respiratory exchange ratio (RER) and a whitening phenotype of PVAT, which was accompanied by a loss of anti-contractile effects [(mN) WT_PVAT(-): 5.2±0.3 vs PVAT(+): 3.4±0.5*, *P<0.05 and KO_PVAT(-): 6.1±0.7 vs KO_PVAT(+): 5.7±0.4]. Additionally, PVAT from KO displayed a significant reduction in nitric oxide production [(RFU) WT_PVAT: 223.1± 12.3 vs KO_PVAT: 67.3±9.1*, *P<0.05] and endothelial NO synthase phosphorylation. Additionally, the lack of PGRN attenuated the expression of mitochondrial complexes [(AU) WT_PVAT: 1.3± 0.03 vs KO_PVAT: 0.6±0.06*, *P<0.05] measured by the OXPHOS cocktail antibody. Via high-resolution respirometry, we found that isolated mitochondria from KO_PVAT showed impaired succinate-driven respiration or complex II activity. To understand if the whitening phenotype of PVAT was leading to its dysfunction, we induced a browning phenotype of PVAT by treating the mice with a β3-agonist for 7 days (CL 316243, 1mg/kg/day). This treatment increased RER restored the PVAT phenotype, and improved PVAT anti-contractile effect and mitochondrial respiration. Using 3T3-L1 cells, we engineered the overexpression and knockdown of PGRN via lentivirus (LV) infection and siRNA, respectively. Suppression of PGRN increased mRNA of whitening markers (1.5-fold increase vs scramble siRNA), while overexpression elevated mRNA of browning and mitochondrial markers (1.3-fold increase vs scramble LV). In summary, our findings reveal that PGRN is crucial for maintaining the phenotype and anti-contractile function of PVAT and highlight PGRN as a pharmacological target for vascular outcomes in cardiometabolic conditions.

AURKA blockage as a strategy to overcome resistance to olaparib and platinum in high-grade ovarian cancer (HGOC).

e15198 Background: PARP inhibitors (PARPi) as olaparib (OLA) have become the new standard in maintenance treatment in high grade ovarian carcinoma (HGOC). Thus, there is an important need to identify the mechanisms for PARPi resistance and the strategies to overcome them. Our aim is to assess if in vitro inhibition of AURKA, a protein that plays a relevant role in centrosome spindle formation and mitotic regulation, might overcome olaparib resistance mechanisms in HGOC. Methods: Three HGOC cell lines (PEO1, Kuramochi and SKOV3) were selected, and isogenic resistant pairs generated in vitro, by pulsed exposure to OLA (PEO1-R) or by exposure to growing concentrations of OLA (Kuramochi-R and SKOV-R). AURKA expression was assessed by PCR and Western blot (WB). Cells were OLA-treated before and after the downregulation of AURKA expression by a specific small inhibitor RNA (siRNA) and by pharmacologic inhibition with alisertib. Cell viability was assessed by MTT assay and DNA damage by the evaluation of markers by WB. Combination index of the combo OLA+alisertib was assessed by Chou-Talalay method. Results: AURKA expression was lower at gene and protein levels in resistant cell lines compared to parental. Upon AURKA silencing (Si) of parental (OLA-sensitive) cell lines, no differences in the OLA IC50 values between AURKA-Si and control cells were seen. However AURKA-Si of the OLA-resistant cell lines was associated with a decreased in OLA IC50 as follows: 1) In PEO1R control scramble siRNA (sc) was 226 mM vs SI 128 mM, p < 0.0001; 2) In Kuramochi-R scRNA 268.4 mM vs si 130.2 mM p < 0.0001 and 3) in SKOV3-R scRNA 187 mM vs si 74.7 mM, p < 0.0001 suggesting a role of AURKA-si in overcoming OLA-resistance. Pharmacologic AURKA targeting with the inhibitor alisertib in all 3 sensitive and resistant cell lines showed an increase of DNA damage measured by the expression of H2AX protein by WB. Combination index of the OLA+alisertib in all resistant cell lines showed a synergistic effect of the combination with PEO1-R FA50 = 0.25 (FA25-75 0.2-0.31), Kuramochi-R FA50 = 0.29 (0.19-9.36) and SKOV3-R FA50 = 0.24 (0.19-0.36). Conclusions: AURKA targeting either by silencing or pharmacologic inhibition increased OLA-induced apoptosis or had a synergistic effect in OLA-resistant cell lines suggesting AURKA as a potential target for overcoming OLA resistance.

Phosphoinositide 3-Kinase C2alpha controls cardiac contractility through regulation of beta2-adrenergic receptor recycling

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral fellowship Introduction Phosphoinositide 3-kinase C2α (PI3KC2α) is a ubiquitously expressed class II PI3K isoform, which has been previously shown to be involved in the control of vesicular trafficking. However, its role in the myocardium has not been investigated. The primary aim of this study is to elucidate the mechanism by which mechanism PI3KC2α controls cardiac pathophysiology. Methods Cardiomyocyte-specific PI3KC2α knockout (PI3KC2α KO) animals were generated by crossing mice expressing a tamoxifen-inducible Cre recombinase under the control of the αMHC promoter with PI3KC2αflox/flox mice. Zebrafish embryos were injected with a PI3KC2α (PI3KC2α morphants) or a control morpholino (control morphants) at 1-cell stage. HEK293 with stable overexpression of GFP-tagged β2-AR were generated and transfected with either scramble or PI3KC2α siRNAs. Results Our findings indicate that PI3KC2α KO mice display a reduced cardiac contractility compared to wildtype animals. Similarly, PI3KC2α morphants zebrafish exhibit lower heart rate and fractional shortening compared to controls, both at baseline and after isoproterenol (ISO) stimulation. A similar unresponsiveness to ISO was found in vivo in PI3KC2α KO mice where chronic treatment with the β-adrenergic receptor (β-AR) agonist failed to induce the classical β-AR-mediated remodeling, characterized by an increase of the left ventricular mass and of cardiomyocyte area. These findings suggest that PI3KC2α plays a critical role in the regulation of β-AR signaling. In agreement, cAMP levels failed to increase in response to ISO treatment in PI3KC2α morphants. Furthermore, silencing of PI3KC2α in HEK293-GFP-β2-AR cells resulted in an increased GFP-β2-AR internalization compared to control cells, both at baseline and after ISO stimulation. Interestingly, preliminary findings indicate that overexpression of constitutively active Rab11Q70L induced a redistribution of GFP-β2-AR at the plasma membrane in PI3KC2α-silenced cells. Conclusion Overall, we identify a key role for PI3KC2α in the control of cardiac contractility through the regulation of β2-AR trafficking to the plasma membrane through a Rab11-dependent mechanism.

Establishment and characterization of cell line from fin of Chinense bahaba (Bahaba taipingensis) and transcriptome analysis of its response to megalocytivirus SKIV-SD strain infection

The Chinese bahaba (Bahaba taipingensis) is an endangered fish species with high economic value, found in the southeastern sea of China. In this study, we established the first Chinese bahaba cell line, named BTF, derived from fin tissue. The primary cells were cultured for over 55 passages in L-15 medium with 15% FBS at 27 °C. These cells exhibited a fibroblast-like morphology with multipolar shapes. Karyotype testing revealed a chromosome number of 2n = 48. After thawing the cells that had been stored in liquid nitrogen for 10 months, their viability was >80%. The cell was separately transfected with EGFP-N3 plasmid and cy3 labeled scramble siRNA and clear fluorescence were observed. Moreover, the cells displayed sensitivity to megalocytivirus SKIV-SD strain, demonstrating a pronounced cytopathic effect (CPE) 24 h after infection. Transcriptome sequencing analysis of SKIV-SD infected cells revealed highly expressed genes significantly enriched in nucleic acid replication and protein translation-related pathways, such as Ribosome biogenesis, RNA polymerase, Nucleocytoplasmic transport, and DNA replication. In addition, the infected cells showed differential expression of immune-related genes such as cxcr4 and dr5, which are involved in virus infection. In conclusion, the BTF cell line, derived from the fin tissue of the Chinese bahaba, exhibits stable growth and can be stored in liquid nitrogen for extended periods. This cell line plays a crucial role in conserving the germplasm resources of Chinese bahaba and will serve as a valuable reference for establishing cell lines from other tissues of the Chinese bahaba. Furthermore, our findings suggest that this cell line holds significant potential for virology research, foreign protein expression, and in vitro biological studies of Chinese bahaba.

Smooth Muscle Cofilin Confers Elasticity to Coronary Resistance Microvessels in Diabetes

Coronary resistance microvessels (CRMs) from type 2 diabetic (T2D) mice and pigs are less stiff compared to normal, a finding that is dictated by less stiff coronary vascular smooth muscle cells (VSMCs) Cofilin is an endogenous actin regulatory protein that depolymerizes filamentous F-actin, and recent studies have shown that portions of F-actin bound to cofilin are less stiff compared to their unbound F-actin counterparts. In this study, we tested the hypothesis that augmented cofilin confers elasticity to diabetic CRMs. CRMs were isolated and pooled (n=6 pooled CRMs per biological replicate) for proteomic data collection from both normal (n=3) and diabetic (n=5) mice. In separate experiments, we utilized deidentified normal and T2D human primary coronary VSMCs obtained from commercial vendors (Lonza and ATCC; n=4 per group) and experiments were performed on passage 5-8 VSMCs. Using these cells, we determined cofilin expression by Western blot, and cofilin siRNA (Cell Signaling Technology) was used 48 hours prior to experimentation to knock down cofilin expression. Elastic modulus, a measure of cell stiffness, was assessed by atomic force microscopy (AFM). Globular G-actin was labeled with fluorescein-deoxyribonuclease I (DNAse I) and F-actin was labeled with phalloidin. Images were taken in blinded manner and Image J was used to assess expression. In the proteomics screen, we observed zero cofilin spectral hits in the normal CRMs and 3-4 spectral hits in db/db CRMs (0.00 ± 0.0 vs. 3.40 ± 0.25, p<0.0001). Cofilin protein was increased in the coronary VSMCs of diabetic patients compared to normal coronary VSMCs (1.00 ± 0.16 vs. 1.837± 0.35, p=0.052). In human coronary VSMCs, silencing cofilin caused a significant increase in the F/G actin ratio when normal and diabetic data were pooled (scramble 3.029 ± 0.49 vs. siRNA 4.029 ± 0.68, p= 0.018, n=8). This increased F/G ratio was significant in the diabetic coronary VSMCs (scramble 2.636 ± 0.46 vs. siRNA 3.685 ± 0.41, p=0.049, n=4), and trended in the normal cells but did not reach statistical significance (scramble 3.423 ± 0.89 vs. cofilin siRNA 4.373 ± 1.37, p=0.224, n=4). Cofilin knockdown also caused a significant increase in elastic modulus by AFM when normal and diabetic data were pooled (scramble siRNA 1.814 ± 0.25 vs. cofilin siRNA 3.074 ± 0.35, p=0.0057, n=8). This trend persisted in the normal VMSCs (scramble siRNA 2.038 ± 0.31 vs. cofilin siRNA 3.445± 0.30, p=0.075, n=4) and in the diabetic VSMCs (scramble siRNA 1.589 ± 0.40 vs. cofilin siRNA 2.702 ± 0.62, p=0.085, n=4) but did not reach significance due to low available sample number. Overall, we demonstrate increased cofilin in human diabetic coronary VSMCs and in CRMs from diabetic mice, and silencing cofilin increased F/G actin ratio and increased cellular stiffness. These data support the notion that increased cofilin in T2D VSMCs may be the cause of decreased diabetic CRM stiffness. Support: NIH R00 HL116769, R01 HL165124, and S10 OD023438 and Nationwide Children’s Hospital to AJT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2084: Endothelial Cells With Silenced Flrt2 Expression Exhibit Enhanced Retention On Vascular Graft Materials Under Fluid Shear Stress In Vitro

Myocardial infraction is a leading cause of death, and surgical intervention is often required to treat severe coronary artery disease. Autologous vessel grafts are the only option for bypass graft procedures. Synthetic vascular grafts less than 6 mm diameter fail due to unacceptable patency rates (~60% at 1 year). The loss of patency is accredited to platelet activation, thrombosis, and neointimal hyperplasia. A promising strategy to improve patency is to endothelialize synthetic vascular grafts ex-vivo, but previous attempts have met with modest success due to detachment of cells upon exposure to fluid shear stress. We previously used RNA-seq to characterized gene expression of endothelial cells (ECs) which remained adherent to synthetic grafts post shear stress. We found that fibronectin leucine-rich transmembrane protein 2 (FLRT2) was significantly downregulated in adherent subpopulations. We therefore hypothesize that suppressing FLRT2 expression in ECs will result in better retention on synthetic vascular grafts upon exposure to shear stress. We used silencing RNA approach using lipofectamine and confirmed successful knockdown of FLRT2 expression (Si-FLRT2). We then seeded Si-FLRT2 cells onto a commercially available Goretex graft material and subjected them to fluid shear stress of for 20 min. We then stained the samples with DAPI. We observed increased retention of Si-FLRT2 ECs on Goretex graft material in comparison to non-specifically targeted scramble siRNA containing ECs. In addition, we compared both vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) levels to assess endothelial cell activation and found that silencing of FLRT2 lead to decreased levels of both VCAM-1 (by 49.2%±3.5%) and ICAM-1 (by 57.1%±3.4%) suggestive of decreased endothelial cell activation. In conclusion, we show that FLRT2 plays an important role in adhesion of ECs onto a commercially available synthetic graft material and silencing its expression ex-vivo may have beneficial clinical outcomes. In addition, we found that FLRT2 silencing may lead to decreased endothelial cell activation and we would like to further investigate the molecular mechanisms underlying the role of FLRT2 in both EC adhesion and activation.

AURKA Kinase Controls Erythroblast Enucleation Via Regulation of Centrosome Localization and ECT2 Degradation

Mature red blood cells (RBCs) in the peripheral circulation of mammals are nuclear-free. Erythroblast enucleation is proposed to be analogous to asymmetric cell division based on cellular progress. the molecular mechanisms of how spherical erythroblasts determine the direction of nuclear polarization and accomplish the final separation of the nucleus from the nascent reticulocytes remain largely unclear, until now. Aurora kinases are highly conserved serine/threonine kinases required for regulation of mitosis (AURKA and AURKB). However, up to now, the role of Aurora kinases in erythroid cells, especially in erythroblast enucleation has been fully undefined. Here, we found that AURKA and AURKB messenger RNA (mRNA) expression levels were similar early in terminal differentiation, but followed by a prominent increase in AURKA levels in polychromatic and orthochromatic erythroblasts, whereas AURKB levels were dramatically downregulated. The expression patterns of Auraro kinases were further confirmed by western blotting analysis. More notably, the expression of Aurora kinases is low or absent in most adult tissues or cells due to their lower proliferation rates. However, AURKA and AURKB are still highly expressed in orthochromatic erythroblasts compared to other precursor erythroblasts. The unconventional expression profile of Aurora kinases, especially AURKA, during terminal erythroid differentiation suggests an extraordinary role in erythroblast enucleation. To explore the role of Aurora kinases in erythroblast enucleation, we first tested whether pharmacological inhibition of these kinases blocked the enucleation of human erythroblasts. Treatment of erythroblasts with MLN8237 (Aliseritib, AURKA selective inhibitor), AZD1152(Barasertib, AURKB selective inhibitor), or AT9283 (AURKA and AURKB inhibitor) dramatically blocked enucleation in a dose-dependent manner. Furthermore, we found that knockdown of Aurora kinases by siRNA indeed resulted in impaired enucleation. We next examined whether Aurora kinases regulated nuclear polarization, which is considered the onset of erythroblast enucleation, by measuring the △centroid distribution (distance between centers of the cytoplasm and nucleus). We found that erythroblast nuclear polarization is AURKA dependent, but not AURKB dependent. Intriguingly, high-resolution confocal microscopy analyses revealed that AURKA colocalized with the centrosome on the side of the nucleus opposite its membrane point of contact during polarization and subsequently solely translocated to the anterior end of the protrusive nucleus upon nuclear exit, whereas AURKB consistently localized to the actin contraction ring during enucleation. Colocalization of AURKA and centrosome during nuclear polarization raised the question whether AURKA controls polarization by regulating centrosome organization and maturation. Co-immunoprecipitation (co-IP) verified a direct interaction between AURKA and γ-tubulin. We found that AURKA loss did not alter the expression levels of γ-tubulin in erythroblasts. However, immunofluorescence showed that MLN8237 treatment obviously impaired the proper organization of centrosome. These phenotypes of centrinone-B, an inhibitor of the master centriole biogenesis regulator PLK4 treated erythroblasts mimic the results of AUKRA-deficient erythroblasts, suggesting a close connection between AUKRA and centrosome in polarization during erythroblast enucleation. We intriguingly found that ECT2 (Epithelial Cell Transforming 2), was only constricted at the rear of the thanslocating nucleus in enucleating erythroblasts. Inhibition of AURKA obviously impaired this transformation, exhibiting persistent cytoplasmic localization. MLN8237 treatment resulted in a significant increase of ECT2 proteins in K562 cells.In contrast to control scramble siRNA, ECT2 siRNA significantly increased the enucleation of MLN8237-treated erythroblast. The translocation of AURKA, which is originally restricted to centrosome relied on γ-tubulin interaction during nuclear polarization, was to directly degrade ECT2 at the anterior end of the protrusive nucleus for final nuclear expulsion. Our findings reveal a previously unrecognized localization and role of Aurora kinases in terminal erythroid differentiation and provide new mechanistic insights into erythroblast enucleation.

Establishment and identification of a skin cell line from Chinese tongue sole (Cynoglossus semilaevis) and analysis of the changes in its transcriptome upon LPS stimulation

The Chinese tongue sole (Cynoglossus semilaevis) holds significant economic importance within the fishing industry along the eastern coasts of China. In recent years, the frequent outbreaks of bacterial diseases have become a common concern as the aquaculture scale expands. The majority of the diseased fish exhibit symptoms such as skin congestion, damage and skin ulceration. As the skin serves as the first line of defense against bacterial infections, establishing a skin cell line for immunological research on Chinese tongue sole's response to bacterial infection is of utmost importance. In this study, a cell line named CSS (derived from the skin of the Chinese tongue sole) was successfully established. The cells have demonstrated stability during passages and exhibit a multipolar fibroblast-like morphology. They were cultured in L-15 medium with 20% serum and have been successfully passed through 60 passages over a period of 20 months. The identification of the mitochondrial CO1 gene confirmed that the cell originated from Chinese tongue sole. The karyotype detection revealed that the cell had a chromosome number of 2n = 42. After being stored in liquid nitrogen for 15 months, the cells can maintain more than 75% viability upon recovery. After transfecting with cy3-labeled scramble siRNA and pEGFP-N3 plasmid, clear fluorescence was observed in the transfected cells. We observed that lipopolysaccharide (LPS) from Escherichia coli significantly upregulate the gene expression of various immune-related pathways at 2 h in the CSS cell line. Additionally, the differentially expressed genes showed a higher enrichment in immune-related pathways at 2 and 6 h after stimulation compared to the 24 h point. Moreover, we identified 347 genes that exhibited a gradual increase in expression during the 0–24 h stimulation period. These genes were primarily enriched in pathways related to Autophagy, GABAergic synapse, Apelin signaling and Ferroptosis. In general, the CSS cell line established in this study exhibits stable growth and can serve as a valuable tool for in vitro studies of immunology and other basic biologies of Chinese tongue sole.

Establishment, characterization and application in germplasm conservation and disease resistance: An embryonic cell line from Yangtze sturgeon (Acipenser dabryanus)

Yangtze sturgeon (Acipenser dabryanus), is a particularly valuable and endangered fish species distributed in the Yangtze River of China. Bacterial diseases outbreaks have caused catastrophic losses in sturgeon farming. To conserve Yangtze sturgeon germplasm resources and facilitate the research of sturgeon etiology without harming individual, the first A. dabryanus embryonic cell line-ADEc was successfully established from its neural plate embryo at 72 h after fertilization. ADEc cells were cultured in Leibovitz's L-15 (L-15) medium containing 20% fetal bovine serum at 25 °C and were stably passaged up to 50 generations. Karyotyping analysis has indicated that the chromosome number for most ADEc cells was 2n = 234. The successful transfection of Cy3-labeled scramble siRNA and GFP reporter gene into ADEc cells were observed. The alkaline phosphatase (AP) staining showed that there were few cells showing pluripotency. The differentiation of ADEc cells toward to neuronal cell type could be observed after all-trans retinoic acid (RA) induction. In addition, embryonic stem cell marker-wdr43 and neural stem cell markers such as bmi1, nes, and ocln were expressed in ADEc cells. Transcriptome sequencing revealed that 1562 differentially expressed genes were identified at 2 h after lipopolysaccharide (LPS) stimulation, which significantly enriched into the Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and NF-kappa B signaling pathway. The establishment of ADEc cell line will help to conserve germplasm resources and provide useful in vitro research tools for immunological, biological, and genomic studies of Yangtze sturgeon.

Proteomic analysis of STEAP1 knockdown in human LNCaP prostate cancer cells

Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. The six transmembrane epithelial antigen of the prostate 1 (STEAP1) protein is overexpressed in several types of human tumors, particularly in PCa. Our research group has demonstrated that STEAP1 overexpression is associated with PCa progression and aggressiveness. Therefore, understanding the cellular and molecular mechanisms triggered by STEAP1 overexpression will provide important insights to delineate new strategies for PCa treatment. In the present work, a proteomic strategy was used to characterize the intracellular signaling pathways and the molecular targets downstream of STEAP1 in PCa cells. A label-free approach was applied using an Orbitrap LC-MS/MS system to characterize the proteome of STEAP1-knockdown PCa cells. More than 6700 proteins were identified, of which a total of 526 proteins were found differentially expressed in scramble siRNA versus STEAP1 siRNA (234 proteins up-regulated and 292 proteins down-regulated). Bioinformatics analysis allowed us to explore the mechanism through which STEAP1 exerts influence on PCa, revealing that endocytosis, RNA transport, apoptosis, aminoacyl-tRNA biosynthesis, and metabolic pathways are the main biological processes where STEAP1 is involved. By immunoblotting, it was confirmed that STEAP1 silencing induced the up-regulation of cathepsin B, intersectin-1, and syntaxin 4, and the down-regulation of HRas, PIK3C2A, and DIS3. These findings suggested that blocking STEAP1 might be a suitable strategy to activate apoptosis and endocytosis, and diminish cellular metabolism and intercellular communication, leading to inhibition of PCa progression.

Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells as an Efficient Nanocarrier to Deliver siRNA or Drug to Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Treatment of PDAC remains a major challenge. This study aims to evaluate, in vitro, the use of human umbilical cord mesenchymal stromal cell (UC-MSC)-derived EVs to specifically target pancreatic cancer cells. EVs were isolated from the FBS-free supernatants of the cultured UC-MSCs by ultracentrifugation and characterized by several methods. EVs were loaded with scramble or KRASG12D-targeting siRNA by electroporation. The effects of control and loaded EVs on different cell types were evaluated by assessing cell proliferation, viability, apoptosis and migration. Later, the ability of EVs to function as a drug delivery system for doxorubicin (DOXO), a chemotherapeutic drug, was also evaluated. Loaded EVs exhibited different kinetic rates of uptake by three cell lines, namely, BxPC-3 cells (pancreatic cancer cell line expressing KRASwt), LS180 cells (colorectal cell line expressing KRASG12D) and PANC-1 cells (pancreatic cell line expressing KRASG12D). A significant decrease in the relative expression of the KRASG12D gene after incubation with KRAS siRNA EVs was observed by real-time PCR. KRASG12D siRNA EVs significantly reduced the proliferation, viability and migration of the KRASG12D cell lines compared to scramble siRNA EVs. An endogenous EV production method was applied to obtain DOXO-loaded EVs. Briefly, UC-MSCs were treated with DOXO. After 24 h, UC-MSCs released DOXO-loaded EVs. DOXO-loaded EVs were rapidly taken up by PANC-1 cells and induced apoptotic cell death more efficiently than free DOXO. In conclusion, the use of UC-MSC-derived EVs as a drug delivery system for siRNAs or drugs could be a promising approach for the targeted treatment of PDAC.

Btg1 and Btg2 regulate neonatal cardiomyocyte cell cycle arrest

Rodent cardiomyocytes undergo mitotic arrest in the first postnatal week. Here, we investigate the role of transcriptional co-regulator Btg2 (B-cell translocation gene 2) and functionally-similar homolog Btg1 in postnatal cardiomyocyte cell cycling and maturation. Btg1 and Btg2 (Btg1/2) are expressed in neonatal C57BL/6 mouse left ventricles coincident with cardiomyocyte cell cycle arrest. Btg1/2 constitutive double knockout (DKO) mouse hearts exhibit increased pHH3+ mitotic cardiomyocytes compared to Wildtype at postnatal day (P)7, but not at P30. Similarly, neonatal AAV9-mediated Btg1/2 double knockdown (DKD) mouse hearts exhibit increased EdU+ mitotic cardiomyocytes compared to Scramble AAV9-shRNA controls at P7, but not at P14. In neonatal rat ventricular myocyte (NRVM) cultures, siRNA-mediated Btg1/2 single and double knockdown cohorts showed increased EdU+ cardiomyocytes compared to Scramble siRNA controls, without increase in binucleation or nuclear DNA content. RNAseq analyses of Btg1/2-depleted NRVMs support a role for Btg1/2 in inhibiting cell proliferation, and in modulating reactive oxygen species response pathways, implicated in neonatal cardiomyocyte cell cycle arrest. Together, these data identify Btg1 and Btg2 as novel contributing factors in mammalian cardiomyocyte cell cycle arrest after birth.

G protein-coupled lactate receptor GPR81 control of ventrolateral ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and 5'-AMP-activated protein kinase expression.

Astrocytes store glycogen as energy and promote neurometabolic stability through supply of oxidizable l-lactate. Whether lactate regulates ventromedial hypothalamic nucleus (VMN) glucostatic function as a metabolic volume transmitter is unknown. Current research investigated whether G protein-coupled lactate receptor GPR81 controls astrocyte glycogen metabolism and glucose-regulatory neurotransmission in the ventrolateral VMN (VMNvl), where glucose-regulatory neurons reside. Female rats were pretreated by intra-VMN GPR81 or scramble siRNA infusion before insulin or vehicle injection. VMNvl cell or tissue samples were acquired by laser-catapult- or micropunch microdissection for Western blot protein or uHPLC-electrospray ionization-mass spectrometric glycogen analyses. Data show that GPR81 regulates eu- and/or hypoglycemic patterns of VMNvl astrocyte glycogen metabolic enzyme and 5'-AMP-activated protein kinase (AMPK) protein expression according to VMNvl segment. GPR81 stimulates baseline rostral and caudal VMNvl glycogen accumulation but mediates glycogen breakdown in the former site during hypoglycemia. During euglycemia, GPR81 suppresses the transmitter marker neuronal nitric oxide synthase (nNOS) in rostral and caudal VMNvl nitrergic neurons, but stimulates (rostral VMNvl) or inhibits (caudal VMNvl) GABAergic neuron glutamate decarboxylase65/67 (GAD)protein. During hypoglycemia, GPR81 regulates AMPK activation in nitrergic and GABAergic neurons located in the rostral, but not caudal VMNvl. VMN GPR81 knockdown amplified hypoglycemic hypercorticosteronemia, but not hyperglucagonemia. Results provide novel evidence that VMNvl astrocyte and glucose-regulatory neurons express GPR81 protein. Data identify neuroanatomical subpopulations of VMNvl astrocytes and glucose-regulatory neurons that exhibit differential reactivity to GPR81 input. Heterogeneous GPR81 effects during eu- versus hypoglycemia infer that energy state may affect cellular sensitivity to or postreceptor processing of lactate transmitter signaling.

A β-Cyclodextrin-Based Nanoparticle with Very High Transfection Efficiency Unveils siRNA-Activated TLR3 Responses in Human Prostate Cancer Cells

Synthetic double-stranded small interfering RNAs (siRNAs) mimic interference RNAs (RNAi) and can bind target mRNAs with a high degree of specificity, leading to selective knockdown of the proteins they encode. However, siRNAs are very labile and must be both protected and transported by nanoparticles to be efficiently delivered into cells. In this work, we used a Janus-type polycationic amphiphilic β-cyclodextrin derivative to efficiently transfect siRNAs targeting mRNAs encoding mitogen-activated protein kinase (p42-MAPK) or Ras homolog enriched in brain (Rheb) into different cancer cell lines as well as astrocytes. We took advantage of this high transfection efficiency to simultaneously knock down p42-MAPK and Rheb to boost docetaxel (DTX)-mediated toxicity in two human prostate cancer cell lines (LNCaP and PC3). We found that double knockdown of p42-MAPK and Rheb increased DTX-toxicity in LNCaP but not in PC3 cells. However, we also observed the same effect when scramble siRNA was used, therefore pointing to an off-target effect. Indeed, we found that the siRNA we used in this work induced toll-like receptor 3 activation, leading to β-interferon production and caspase activation. We believe that this mechanism could be very useful as a general strategy to elicit an immune response against prostate cancer cells.

The human batokine EPDR1 regulates β-cell metabolism and function

ObjectiveEpendymin-Related Protein 1 (EPDR1) was recently identified as a secreted human batokine regulating mitochondrial respiration linked to thermogenesis in brown fat. Despite that EPDR1 is expressed in human pancreatic β-cells and that glucose-stimulated mitochondrial metabolism is critical for stimulus-secretion coupling in β-cells, the role of EPDR1 in β-cell metabolism and function has not been investigated. MethodsEPDR1 mRNA levels in human pancreatic islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects were assessed. Human islets, EndoC-βH1 and INS1 832/13 cells were transfected with scramble (control) and EPDR1 siRNAs (EPDR1-KD) or treated with human EPDR1 protein, and glucose-stimulated insulin secretion (GSIS) assessed by ELISA. Mitochondrial metabolism was investigated by extracellular flux analyzer, confocal microscopy and mass spectrometry-based metabolomics analysis. ResultsEPDR1 mRNA expression was upregulated in human islets from T2D and obese donors and positively correlated to BMI of donors. In T2D donors, EPDR1 mRNA levels negatively correlated with HbA1c and positively correlated with GSIS. EPDR1 silencing in human islets and β-cell lines reduced GSIS whereas treatment with human EPDR1 protein increased GSIS. Epdr1 silencing in INS1 832/13 cells reduced glucose- and pyruvate- but not K+-stimulated insulin secretion. Metabolomics analysis in Epdr1-KD INS1 832/13 cells suggests diversion of glucose-derived pyruvate to lactate production and decreased malate-aspartate shuttle and the tricarboxylic acid (TCA) cycle activity. The glucose-stimulated rise in mitochondrial respiration and ATP/ADP-ratio was impaired in Epdr1-deficient cells. ConclusionThese results suggests that to maintain glucose homeostasis in obese people, upregulation of EPDR1 may improve β-cell function via channelling glycolysis-derived pyruvate to the mitochondrial TCA cycle.

H19 inhibition increases HDAC6 and regulates IRS1 levels and insulin signaling in the skeletal muscle during diabetes

BackgroundHistone deacetylases (HDACs) that catalyze removal of acetyl groups from histone proteins, are strongly associated with several diseases including diabetes, yet the precise regulatory events that control the levels and activity of the HDACs are not yet well elucidated.MethodsLevels of H19 and HDACs were evaluated in skeletal muscles of normal and diabetic db/db mice by Western Blot analysis. C2C12 cells were differentiated and transfected with either the scramble or H19 siRNA and the levels of HDACs and Prkab2, Pfkfb3, Srebf1, Socs2, Irs1 and Ppp2r5b were assessed by Western Blot analysis and qRT-PCR, respectively. Levels of H9, HDAC6 and IRS1 were evaluated in skeletal muscles of scramble/ H19 siRNA injected mice and chow/HFD-fed mice.ResultsOur data show that the lncRNA H19 and HDAC6 exhibit inverse patterns of expression in the skeletal muscle of diabetic db/db mice and in C2C12 cells, H19 inhibition led to significant increase in HDAC activity and in the levels of HDAC6, both at the transcript and protein levels. This was associated with downregulation of IRS1 levels that were prevented in the presence of the HDAC inhibitor, SAHA, and HDAC6 siRNA suggesting the lncRNA H19-HDAC6 axis possibly regulates cellular IRS1 levels. Such patterns of H19, HDAC6 and IRS1 expression were also validated and confirmed in high fat diet-fed mice where as compared to normal chow-fed mice, H19 levels were significantly inhibited in the skeletal muscle of these mice and this was accompanied with elevated HDAC6 levels and decreased IRS1 levels. In-vivo inhibition of H19 led to significant increase in HDAC6 levels and this was associated with a decrease in IRS1 levels in the skeletal muscle.ConclusionsOur results suggest a critical role for the lncRNA H19-HDAC6 axis in regulating IRS1 levels in the skeletal muscle during diabetes and therefore restoring normal H19 levels might hold a therapeutic potential for the management of aberrant skeletal muscle physiology during insulin resistance and type 2 diabetes.

Protective effect of pentraxin 3 on pathological retinal angiogenesis in an in vitro model of diabetic retinopathy

BackgroundDiabetic retinopathy (DR) is the most common retinal microvascular disease caused by diabetes. Previous studies indicated that Pentraxin 3 (PTX3), an acute phase reactant, was closely related to the development of DR. But the exact effect of PTX3 in diabetic retinopathy needs more investigations. MethodsReal-time fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis and western blot (WB) were used to detect the expression of PTX3 in vitro. The Ki67 immunofluorescent staining, scratch-wound migration assay, and tube formation experiments were performed to detect the effect of PTX3 knockdown and overexpression on the fibroblast growth factor (FGF)-induced proliferation, migration and tube-forming ability of human retinal microvascular endothelial cells (HRMECs). The phosphorylation levels of extracellular regulated protein kinases (ERK) and fibroblast growth factor receptor (FGFR) in HRMECs were detected by WB. ResultsIn vitro, the mRNA and protein expressions of PTX3 in the high-concentration glucose condition group were upregulated compared with the normal group (p < 0.05). The proliferation, migration and tube-forming abilities of HRMECs exposed to high-concentration glucose were enhanced (p < 0.01, p < 0.01, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.01, p < 0.05 respectively) compared with the normal condition group. Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 siRNA condition group were further strengthened (p < 0.001, p < 0.0001, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.001, p < 0.01 respectively). Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 overexpression condition group were compromised (p < 0.001, p < 0.05, p < 0.01 respectively), and the phosphorylation of FGFR and ERK1/2 were inhibited (p < 0.001, p < 0.0001 respectively). Neither the scramble siRNA condition group nor the blank plasmid condition group showed significant difference on the proliferation, migration and tube-forming abilities of HRMECs compared with the high glucose condition group (p > 0.05). ConclusionsThe upregulated expression of PTX3 may play a protective role on pathological angiogenesis in DR. PTX3 may serve as a new target for the treatment of DR.

Systemically Silencing Long Non-coding RNAs Maclpil With Short Interfering RNA Nanoparticles Alleviates Experimental Ischemic Stroke by Promoting Macrophage Apoptosis and Anti-inflammatory Activation.

BackgroundMaclpil is a proinflammatory long non-coding RNA highly expressed on monocyte-derived macrophages in the ischemic brain. This study investigated the impact and the mechanisms of systemically delivering nanoparticle Maclpil short interfering RNA (siRNA) on experimental ischemic stroke in a mouse model.MethodsIschemic stroke (focal cerebral ischemia) was induced in male C57BL/6 mice through the middle cerebral artery occlusion. Three hours thereafter, mice were intravenously injected with Maclpil siRNA or scramble siRNA nanoparticles. Bone marrow cell-derived macrophages were transfected with Maclpil or scramble siRNA and subjected to oxygen glucose deprivation culture. The influence of silencing Maclpil on stroke outcomes, neuroinflammation, and macrophage fates was assessed via histology, flow cytometry, Western blotting, and quantitative PCR analysis.ResultsThree days following stroke induction, siRNA silencing Maclpil substantially reduced ischemic infarction size and improved neurological behaviors. Silencing Maclpil also markedly attenuated the accumulation of monocyte-derived macrophages, CD4+ T cells, and CD8+ T cells in the ischemic hemisphere without affecting microglia cellularity. Reciprocally, myeloid cells and both subsets of T cells were elevated in mouse peripheral blood following Maclpil siRNA treatment. Under oxygen glucose deprivation conditions that mimicked hypoxia and hypoglycemia in vitro, Maclpil siRNA silencing augmented macrophage apoptosis in conjunction with upregulation of proapoptotic Bax and caspase 3 expressions. siRNA knocking down Maclpil skewed macrophages from proinflammatory classical toward anti-inflammatory alternative activation as evidenced by increased arginase 1, Ym1, and Fizz1 and reduced inducible nitric oxide synthase, IL-1β, and TNF-α mRNA levels. Consistent with macrophage phenotype switching, silencing Maclpil by siRNA enhanced fatty acid oxidation as indicated by increased mRNA levels of 3 key metabolic enzymes (ACADM, ACADVL, and HADHA).ConclusionSystemically silencing Maclpil by siRNA nanoparticles attenuated experimental ischemic stroke by promoting macrophage apoptosis and anti-inflammatory alternative activation. Identifying and targeting Maclpil human homolog(s) may help develop a novel therapy for stroke clinical management.