Injection Of Plasmid DNA Research Articles

BMP-7 ameliorates partial epithelial-mesenchymal transition by restoring SnoN protein level via Smad1/5 pathway in diabetic kidney disease

Tubulointerstitial fibrosis (TIF) is involved in the development of diabetic kidney disease (DKD). Transforming growth factor β1 (TGF-β1) is involved in the extensive fibrosis of renal tissue by facilitating the partial epithelial-mesenchymal transition (EMT), increasing the synthesis of extracellular matrix (ECM), inhibiting degradation, inducing apoptosis of renal parenchyma cells, and activating renal interstitial fibroblasts and inflammatory cells. Recent studies indicated that bone morphogenetic protein-7 (BMP-7) upregulated the expression of endogenous SnoN against renal TIF induced by TGF-β1 or hyperglycemia. Nevertheless, the mechanisms underlying the BMP-7-mediated restoration of SnoN protein level remains elusive. The present study demonstrated the increased expression of BMP-7 in diabetic mellitus (DM) mice by hydrodynamic tail vein injection of overexpressed BMP-7 plasmid, which attenuated the effects of DM on kidney in mice. Partial tubular EMT and the accumulation of Collagen-III were resisted in DM mice that received overexpressed BMP-7 plasmid. Similar in vivo results showed that BMP-7 was competent to alleviate NRK-52E cells undergoing partial EMT in a high-glucose milieu. Furthermore, exogenous BMP-7 activated the Smad1/5 pathway to promote gene transcription of SnoN and intervened ubiquitination of SnoN; both effects repaired the SnoN protein level in renal tubular cells and kidney tissues of DM mice. Therefore, these findings suggested that BMP-7 could upregulate SnoN mRNA and protein levels by activating the classical Smad1/5 pathway to refrain from the partial EMT of renal tubular epithelial cells and the deposition of ECM in DKD-induced renal fibrosis.

PiggyBac-based transgenic RNAi of serine protease 2 results in male sterility in Hyphantria cunea

Fall webworm, Hyphantria cunea, is a global invasive forest pest that causes serious damage to the economy and ecosystem of agriculture and forestry. Due to the extent of the problem and the difficulty of conventional chemical control, new technologies must be pursued, such as genetic-based inheritable insect sterile technology (gSIT), which exhibits promise for pest control. In the present study, we established a piggyBac-based transgenic system in fall webworm and generated a dominant male-sterile strain by targeting the seminal fluid protein serine protease 2 (Hcser2), displaying an outstanding trait of gSIT. First, an RNA polymerase type III (Pol III) promoter, the HcU62 small nuclear RNA (snRNA) gene promoter, was identified and characterized through direct injection of RNAi plasmids in vivo. Quantitative real-time PCR revealed that HcU62 had the greatest knockdown efficiency of the Hcyellow gene among five short hairpin RNA (shRNA) plasmids tested, designated HcU61–HcU65. Second, subsequent application of piggyBac-based transgenic RNAi (HcU62: shHcyellow, Ysh2) significantly reduced the expression level of the Hcyellow gene, resulting in a stable yellow observable phenotype from the larval to pupal stages in Ysh2 transgenic mutants. Finally, an HcU62-driven transgenic RNAi strain targeting the Hcser2 gene was obtained, resulting in a dominant male-sterile phenotype. Significantly, this process did not affect the growth, development, mating behavior or egg laying of the mutants, and the dominant sterile trait could be inherited in the next generation through female Hcser2 mutants. Furthermore, CRISPR/Cas9-mediated disruption of the Hcser2 gene further confirmed the dominant sterile phenotype, supporting it as a generalized target for genetic control of H. cunea. This study reports the first piggyBac-mediated transgenic system in H. cunea, providing a promising genetic method for controlling this pest by targeting Hcser2 gene.

Stromal cell derived factor 1 plasmid to regenerate the anal sphincters.

Under ethics approved protocol 19 age/weight matched Sinclair mini-pigs were subjected to excision of the posterior 50% of anal sphincter muscle and left to recover for 6weeks. They were randomly allocated to receive either saline treatment (Saline 1ml, n=5), 1 injection of SDF-1 plasmid 2mg/ml (1 SDF-1, n=9) or 2 injections of SDF-1, 2mg/ml each at 2weeks intervals (2 SDF-1, n=5). Euthanasia occurred 8weeks after the last treatment. In vivo outcomes included anal resting pressures done under anesthesia pre-injury, pre-injection and before euthanasia (8weeks after treatment). Anal ultrasound was done pre injury and pre-euthanasia. Tissues were saved for histology and analyzed quantitatively. Two way ANOVA followed by Holm-Sidak test and one way ANOVA followed by the Tukey test were used for data analysis, p<0.05 was regarded as significant. Posterior anal pressures at the 3 time points were not significantly different in the saline group. In contrast, post-treatment pressures in the 1 SDF-1 group pressures were significantly higher than both pre-injury (p=0.001) and pre-treatment time points (p=0.003). At the post-treatment time point, both 1 SDF-1 (p=0.01) and 2 SDF-1 (p=0.01) groups had significantly higher mean pressures compared to the saline group. Histology showed distortion of normal anatomy with patchy regeneration in the control group while muscle was more organized in both treatment groups. Eight weeks after a single or two doses of SDF-1injected into a chronic anal sphincter injury improved resting anal pressures and regenerated muscle in the entire defect. SDF-1 plasmid is effective in treating chronic defects of the anal sphincter in a large animal and could be clinically translated.

Mitochondrial fragmentation is crucial for c-Myc-driven hepatoblastoma-like liver tumors

Hepatoblastoma is the most common liver cancer in children, and the aggressive subtype often has a poor prognosis and lacks effective targeted therapy. Although aggressive hepatoblastoma (HB) is often accompanied by abnormally high expression of the transcription factor c-Myc, the underlying mechanism remains unclear. In this study, we found that mitochondrial fragmentation was enhanced by c-Myc overexpression in human aggressive HB tissues and was associated with poor prognosis. Then, a mouse model resembling human HB was established via hydrodynamic injection of c-Myc plasmids. We observed that liver-specific knockout of the mitochondrial fusion molecule MFN1 or overexpression of mitochondrial fission molecule DRP1 promoted the occurrence of c-Myc-driven liver cancer. In contrast, when MFN1 was overexpressed in the liver, tumor formation was delayed. Invitro experiments showed that c-Myc transcriptionally upregulated the expression of DRP1 and decreased MFN1 expression through upregulation of miR-373-3p. Moreover, enhanced mitochondrial fragmentation significantly promoted aerobic glycolysis and the proliferation of HB cells by significantly increasing reactive oxygen species (ROS) production and activating the RAC-alpha serine/threonine-protein kinase (AKT)/mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathways. Taken together, our results indicate that c-Myc-mediated mitochondrial fragmentation promotes the malignant transformation and progression of HB by activating ROS-mediated multi-oncogenic signaling.

Long noncoding RNA XIST regulates brown preadipocytes differentiation and combats high-fat diet induced obesity by targeting C/EBP\u03b1

BackgroundActivation of brown adipose tissue (BAT) increases energy expenditure, which makes it an attractive therapeutic strategy for obesity. LncRNAs play an important role in adipocyte differentiation and regulation. Here we assessed the effect of lncRNA XIST on brown preadipocytes differentiation and metabolic regulation.MethodsXIST expression levels were detected in human perirenal (peri-N) and subcutaneous adipose tissues (sub-Q), brown preadipocytes and 3T3-L1 preadipocytes. XIST overexpression and knockdown experiments were performed in brown preadipocytes. XIST overexpression mouse model was established by plasmid injection through tail vein.ResultsIn human adipose tissues, XIST expression was significantly higher in female than in male individuals. In vitro, XIST expression was significantly up-regulated during brown adipocyte differentiation. XIST knockdown inhibited differentiation of brown preadipocytes, while overexpression of XIST promotes brown preadipocytes to fully differentiation. RNA Binding Protein Immunoprecipitation (RIP) experiment revealed that XIST could directly bind to C/EBPα. In vivo, XIST overexpression prevents high-fat diet induced obesity and improves metabolic dysorder in male mice.ConclusionOur results suggest that XIST combats obesity through BAT activation at least partly by combination with transcription factor C/EBPα.

Opticin Ameliorates Hypoxia-Induced Retinal Angiogenesis by Suppression of Integrin α2-I Domain-Collagen Complex Formation and RhoA/ROCK1 Signaling.

PurposeIt was previously demonstrated that opticin (OPTC) inhibits the collagen-induced promotion of bioactivities of human retinal vascular endothelial cells (hRVECs). The present in vivo study aimed to further investigate the regulatory role of opticin in vitreous collagen-mediated retinal neovascularization and to elucidate its regulatory mechanisms with regard to integrin α2-I domain–GXXGER complex formation and RhoA/ROCK1 signal change. The regulatory role of Mg2+ on integrin α2-I domain–GXXGER complex formation in the above process was also investigated.MethodsThe zebrafish model of hypoxia-induced retinopathy was established, and OPTC-overexpressing plasmids were intravitreally injected to assess the antiangiogenesis effect of opticin. The regulatory role of opticin in integrin α2-I domain–GXXGER complex formation in vivo was analyzed by mass spectrometry. The mRNA and protein expression of RhoA/ROCK1 were examined. The concentration of Mg2+ as an activator of the integrin α2-I domain–GXXGER complex was measured. Solid-phase binding assays were performed to investigate the interference of opticin in integrin α2 collagen binding and the regulatory role of Mg2+ in that process.ResultsOpticin and OPTC-overexpressing plasmid injection reduced retinal neovascularization in the zebrafish model of hypoxia-induced retinopathy. Mass spectrometry revealed that opticin could inhibit integrin α2-I domain–GXXGER complex formation. The Mg2+ concentration was also decreased by opticin, which was another indication of the complex activation. Injection of OPTC-overexpressing plasmids inhibited mRNA and the protein expression of RhoA/ROCK1 in the zebrafish model of hypoxia-induced retinopathy. The solid-phase binding assay revealed that opticin could block integrin α2–collagen I binding in the presence of Mg2+.ConclusionsOpticin exerts its antiangiogenesis effect by interfering in the Mg2+-modulated integrin α2-I domain–collagen complex formation and suppressing the downstream RhoA/ ROCK1 signaling pathway.

Noxa and Puma genes regulated by hTERT promoter can mitigate growth and induce apoptosis in hepatocellular carcinoma mouse model.

With significant high incidence and death rates, liver cancer has become one of the most common cancers all over the world. Hence, novel strategies are needed for the management of this malignancy. Apoptotic related proteins Noxa and Puma are the members of BH3-only family. In this study, human Noxa or Puma coding sequences have been inserted into plasmid pcDNA 3.1 regulated by human TERT promoter. The transfection of HepG2 cells with pcTERT-Noxa or pcTET-Puma resulted in the significant suppression of cell proliferation as well as finally led to apoptosis via mitochondrial and death receptor pathways, and also exhibited significantly reduced the ability of invasion and metastasis. Moreover, an in vivo study revealed that intratumoral injections of pcTERT-Noxa or pcTERT-Puma plasmids effectively suppressed the tumor growth and can exhibit anti-neoplastic effects by recruiting CD3, CD8, CD45 positive T lymphocytes in the tumor tissues. Overall, our findings illustrated that pcTERT-Noxa and pcTERT-Puma may exhibit significant anti-tumor effects both in vivo and in vivo.

Transthoracic ultrasound-guided percutaneous intramyocardial injection combined with ultrasound-targeted microbubble destruction-mediated angiogenin 1 gene therapy in canine myocardial infarction model.

Intravenous (IV) ultrasound-targeted microbubble destruction (UTMD) has made distinct but limited progress in gene transfected therapy. Intramyocardial (IM) injection also has some effect. In this study, IM injection could be performed under the guidance of transthoracic ultrasound without thoracotomy. We aimed to evaluate the effect of combination transthoracic ultrasound-guided percutaneous IM injection and UTMD for mediating the angiogenin 1 (Ang1) gene therapy in myocardial infarction (MI) canines. Forty-two canines were divided into four groups: IM-UTMD group (intramyocardial injection of the negative control plasmid with UTMD as the sham group); IM-Ang1 group (intramyocardial injection of the Ang1 plasmid without UTMD); IM-Ang1-UTMD group (intramyocardial injection of the Ang1 plasmid with UTMD); and IV-Ang1-UTMD group (intravenous injection of the Ang1 plasmid with UTMD). The FITC green fluorescence and Ang1 protein in myocardial tissue were used for the distribution of Ang1 gene. Left ventricular dimension and systolic function were observed by echocardiography. The Masson's and Sirius Red's staining were used for evaluating the collagen fiber. The immunohistochemistry for CD31 and alpha-smooth muscle actin (α-SMA) were as the indicators of microvasculature. Myocardial contrast echocardiography was used to reflect the microvascular perfusion. More FITC-labeled plasmid was observed in the three IM injection groups than in the IV-Ang1-UTMD group, and the Ang1 protein expression was higher in the IM-Ang1-UTMD group. One month later, no significant differences in survival rate and complications were observed among all four groups. Although there were no differences in the left ventricular end-diastolic diameter among the 4 groups, the left ventricular end-systolic diameter was lower in the IM-Ang1-UTMD group than in the other groups. Then, the left ventricular ejection fraction was increased in the IM-Ang1-UTMD group, with lower collagen fiber percentage, higher blood vessel density and myocardial reperfusion intensity than those in the other groups (P<0.05). Transthoracic ultrasound-guided percutaneous IM injection combined with UTMD is a safe and effective method for mediating Ang1 plasmid therapy in MI canines.

Unlocking loxP to Track Genome Editing In Vivo.

The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR “fingerprinting” approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.

Death-Associated Protein 6 (Daxx) Alleviates Liver Fibrosis by Modulating Smad2 Acetylation.

Transforming growth factor-β (TGF-β) has been identified as an inducer of hepatocyte epithelial–mesenchymal transition (EMT), which triggers liver fibrosis. Death-associated protein 6 (Daxx) is known to be associated with the TGF-β-induced apoptotic pathway, but the function of Daxx in liver fibrosis remains unknown. This study aimed to elucidate the role of Daxx in liver fibrosis. We used liver fibrosis tissues from humans and mice to assess Daxx expression. EMT properties and TGF-β signaling pathway activation were investigated in the Daxx-overexpressing FL83B cell line. The therapeutic effect of Daxx was investigated in a mouse model of liver fibrosis by the hydrodynamic injection of plasmids. The expression of Daxx was markedly decreased in hepatocytes from fibrotic human and mouse livers, as well as in hepatocytes treated with TGF-β in vitro. The overexpression of Daxx inhibited the EMT process by interfering with the TGF-β-induced phosphorylation of Smad2. Coimmunoprecipitation analysis confirmed that Daxx reduced the transcriptional activity of Smad2 by binding to its MH1 domain and interfering with Smad2 acetylation. In addition, the therapeutic delivery of Daxx alleviated liver fibrosis in a thioacetamide-induced fibrosis mouse model. Overall, our results indicate that Daxx could be a potential therapeutic target to modulate fibrogenesis, as well as a useful biomarker for liver fibrosis.

Neonatal stroke enhances interaction of microglia-derived extracellular vesicles with microglial cells

Microglial cells support brain homeostasis under physiological conditions and modulate brain injury in a context-dependent and brain maturation-dependent manner. Microglial cells protect neonatal brain from acute stroke. While microglial signaling via direct cell-cell interaction and release of variety of molecules is intensely studied, less is known about microglial signaling via release and uptake of extracellular vesicles (EVs). We asked whether neonatal stroke alters release of microglial EVs (MEV) and MEV communication with activated microglia. We pulled down and plated microglia from ischemic-reperfused and contralateral cortex 24 h after transient middle cerebral artery occlusion (tMCAO) in postnatal day 9 mice, isolated and characterized microglia-derived microvesicles (P3-MEV) and exosomes (P4-MEV), and determined uptake of fluorescently labeled P3-MEV and P4-MEV by plated microglia derived from ischemic-reperfused and contralateral cortex. We then examined how reducing EVs release in neonatal brain—by intra-cortical injection of CRISPR-Cas9-Smpd3/KO (Smpd3/KD) to downregulate Smpd3 gene to disrupt neutral sphingomyelinase-2 (N-SMase2)—impacts P3-MEV and P4-MEV release and stroke injury. Both size and protein composition differed between P3-MEV and P4-MEV. tMCAO further altered protein composition of P3-MEV and P4-MEV and significantly, up to 5-fold, increased uptake of both vesicle subtypes by microglia from ischemic-reperfused regions. Under physiological conditions neurons were the predominant cell type expressing N-SMase-2, an enzyme involved in lipid signaling and EVs release. After tMCAO N-SMase-2 expression was diminished in injured neurons but increased in activated microglia/macrophages, leading to overall reduced N-SMase-2 activity. Compared to intracerebral injection of control plasmid, CRISPR-Cas9-Smpd3/Ct, Smpd3/KD injection further reduced N-SMase-2 activity and significantly reduced injury. Smpd3 downregulation decreased MEV release from injured regions, reduced Smpd3/KD-P3-MEV uptake and abolished Smpd3/KD-P4-MEV uptake by microglia from ischemic-reperfused region. Cumulatively, these data demonstrate that microglial cells release both microvesicles and exosomes in naïve neonatal brain, that the state of microglial activation determines both properties of released EVs and their recognition/uptake by microglia in ischemic-reperfused and control regions, suggesting a modulatory role of MEV in neonatal stroke, and that sphingosine/N-SMase-2 signaling contributes both to EVs release and uptake (predominantly P4-MEV) after neonatal stroke.

MACF1 alleviates aging-related osteoporosis via HES1.

Ageing‐related osteoporosis is becoming an emerging threat to human health along with the ageing of human population. The decreased rate of osteogenic differentiation and bone formation is the major cause of ageing‐related osteoporosis. Microtubule actin cross‐linking factor 1 (MACF1) is an important cytoskeletal factor that promotes osteogenic differentiation and bone formation. However, the relationship between MACF1 expression and ageing‐related osteoporosis remains unclear. This study has investigated the expression pattern of MACF1 in bone tissues of ageing‐related osteoporosis patients and ageing mice. The study has further elucidated the mechanism of MACF1 promoting bone formation by inhibiting HES1 expression and activity. Moreover, the therapeutic effect of MACF1 on ageing‐related osteoporosis and post‐menopausal osteoporosis was evaluated through in situ injection of the MACF1 overexpression plasmid. The study supplemented the molecular mechanisms between ageing and bone formation, and provided novel targets and potential therapeutic strategy for ageing‐related osteoporosis.

Moderate Heat-Assisted Gene Electrotransfer for Cutaneous Delivery of a DNA Vaccine Against Hepatitis B Virus.

An estimated 350 million people are living with chronic Hepatitis B virus (HBV) worldwide. Preventative HBV vaccination in infants has reduced the disease burden; however, insufficient immunization programs and access obstacles leave vulnerable populations at risk for infection in endemic regions. Gene electrotransfer (GET) using a noninvasive multielectrode array (MEA) provides an alternative platform for DNA vaccination in the skin. DNA vaccines are nonlive and nonreplicating and temperature stable unlike their counterparts. In addition, their simple engineering allows them to be manufactured quickly at a low cost. In the current work, we present the combination of GET and moderate heating for delivery of a DNA vaccine against HBV. Our laboratory has previously shown the synergy between moderate tissue preheating at 43°C and GET with the MEA as a means to reduce both the applied voltage and pulse number to achieve similar if not higher gene expression than GET alone. In this study, we expand upon this work, by optimizing the plasmid dose to achieve the highest level of expression. Using the reporter gene luciferase, we found that an intradermal injection of 100 μL at 1 mg/mL induced the highest expression levels across all tested GET conditions. We then evaluated our moderate heat-assisted GET platform for the intradermal delivery of a plasmid encoding Hepatitis B surface antigen (pHBsAg) via a prime and prime plus boost vaccination protocol. At 18 weeks, following the prime plus boost protocol, we observed that a high-voltage low-pulse GET condition with moderate heating (45 V 36 p+heat) generated antibodies against Hepatitis B surface antigen (HBsAb) at peak measuring 230-fold over injection of plasmid DNA alone with moderate heating. HBsAbs remained robust over the 30-week observation period. These data suggest that moderate heat-assisted GET has the potential to induce strong immune responses, an attractive feature for development of an alternative vaccine delivery platform.

Biodistribution and Safety Studies of a Bicistronic Plasmid for Nerve Repair.

Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy drugs' usage became the subject of this study. To study plasmid biodistribution, toxicity, mutagenicity, and genotoxicity, we used previously described bicistronic genetic construct encoding human brain-derived neurotrophic factor (hBDNF) and human urokinase plasminogen activator (huPA) for nerve repair. Biodistribution studies were conducted in mice: a course of intramuscular plasmid injections was followed by the study of the content of the plasmid (real-time polymerase chain reaction) and recombinant proteins (enzyme-linked immunosorbent assay) in murine organs and tissues. The study of the plasmid chronic toxicity was carried out on rats with registration of their weight dynamics, neurological status, emotional state, and blood test parameters. The mutagenicity of the plasmid was studied in an in vivo DNA comet test in mice. Plasmid genotoxicity was investigated in the model of somatic recombination in Drosophila females. We have shown that plasmids can disseminate from the injection site, but do not mediate ectopic expression of growth factors upon repeated intramuscular injections. The studied plasmid also does not reveal toxic, mutagenic, or genotoxic effects. During the toxicological study on rats, we have shown that daily injections of this genetic construct, despite its ability to disseminate from the injection site, do not affect the physical, cognitive, and emotional state of experimental animals. We have demonstrated the safety of the bicistronic plasmid, encoding hBDNF and huPA, upon its repeated administration. The properties of genetic constructs strongly depend on their sequence and delivery approach, which requires conducting of their safety studies in each specific case. Impact statement Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy became the subject of this study. To our knowledge, this is a unique study that provides a thorough safety investigation of a bicistronic plasmid after its readministration.

Constitutive Expression of TERT Enhances β-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos.

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and β-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of β-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts’ development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/β-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.

EZH2 Drives Cholangiocarcinogenesis Through Downregulation of Tumor Suppressors Genes via Histone Methylation And microRNAs

EZH2 regulates gene expression through epigenetic machinery. Increased expression of EZH2 is present in cholangiocarcinoma (CCA). Importantly, EZH2 level in CCA is closely correlated with patient prognosis. The objective of this study is to further delineate the effect of EZH2 in CCA. Alb-CRE/EZH2flox/flox mice were subjected to hydrodynamic tail vein injection of transposase-based plasmids expressing AKT and Notch intracellular domain (NICD). This model was employed, along with xenograft and CCA cells, to assess the effect and mechanism of EZH2. ChIP-seq, small RNA-seq and bioinformatics analyses were performed to identify the targets of EZH2. In CCA induction by HDTV injection of NICD/AKT, we observed that the Alb-CRE/EZH2flox/flox mice exhibited significantly decreased tumor burden and prolonged overall survival. These findings reveal an important role of EZH2 in CCA development. In a CCA xenograft model with intrahepatic inoculation of human CCA cell line (CCLP1), we observed that EZH2 knockdown by shRNA significantly decreased CCA progression. Further in vitro studies showed that EZH2 depletion by shRNA significantly reduced the growth and colony formation capability of human CCA cells (CCLP1) and mouse CCA cells. RNA-seq and RT-PCR analysis revealed that depletion of EZH2 by shRNA in CCA cells restored the expression of 12 putative tumor suppressor genes (including Osgin1, Pax3, Itga7, Il1rn, Gas1, Cdkn1a, Bbc3, Wnt11, Wt1, Sulf, Sfrp1, and Ptprv). By analyzing the human CCA database GSE76297 (containing 91 CCA and 92 paired non-tumor tissues), we found that the expression levels of 12 tumor suppressor genes in CCA tissues were decreased by an average of 1.7 -folds in comparison to paired non-tumorous liver tissues. The tumor inhibitory effects of the identified genes were verified in CCA cells with altered EZH2 expression. Furthermore, we observed that intraperitoneal injection of the EZH2 inhibitor, GSK126, decreased CCA tumor burden in mice receiving HDTV injection of NICD/AKT. Treatment of human and mouse CCA cells with GSK126 restored the expression of the 12 tumor suppressor genes. These results indicate that EZH2 downregulates multiple tumor suppressor genes in CCA cells. We next carried out ChIP-seq (using anti-H3K9me2 or anit-EZH2) and small RNA-seq to determine the mechanisms underlying EZH2-mediated downregulation of tumor suppressor genes. The ChIP-seq results showed that both anti-H3K9me2 and anti-EZH2 IP led to significant enrichments of the DNA fragments of 5 out of 12 genes. The small RNA-seq analyses identified a group of EZH2-regulated microRNAs which target the 12 tumor suppressor genes. Our findings suggest that EZH2 downregulates the expression of tumor suppressor genes either directly through histone methylation or indirectly through specific microRNAs. Taken together, our findings disclose a novel mechanism by which EZH2 drives cholangiocarcinogenesis through downregulation of tumor suppressors genes via histone methylation and microRNAs. Our results provided further evidence supporting EZH2 inhibition for CCA treatment.

DNGR-1 limits Flt3L-mediated antitumor immunity by restraining tumor-infiltrating type I conventional dendritic cells

BackgroundConventional type 1 dendritic cells (cDC1s) are central to antitumor immunity and their presence in the tumor microenvironment associates with improved outcomes in patients with cancer. DNGR-1 (CLEC9A) is a...

Abstract PO-099: Overexpression of proinflammatory chemokines CCL5 and CCL17 after gene electrotransfer to murine tumors modifies their cytokine expression profile and leads to tumor cures in combination with tumor irradiation

Abstract Proinflammatory chemokines are small signaling proteins, with the ability to induce migration of different types of immune cells. The degree and type of immune cell infiltrate in the tumor microenvironment often correlates with the outcome of immunotherapies in cancer patients. Therefore, combining chemokines with classical, but immune response activating therapy, such as irradiation presents one of the potential approaches in cancer immunotherapy. Through the use of plasmid DNA encoding either proinflammatory chemokine CCL5 or CCL17 we studied their effects on cytokine expression profile in vitro and in vivo in murine breast (4T1, E0771) and colon (CT26, MC38) cancer models. While in vitro plasmid DNA was introduced in the cells using lipofection, gene electrotransfer was utilized in case of tumors in vivo. Viability of all cell lines 48 h after lipofection remained above 80%. Using qRT-PCR expression of 11 cytokines was determined 48 h after lipofection, which showed significantly increased expression of CCL5, CCL17 demonstrating successful transfection. Moreover, increased levels of IL-6 and CXCL10 were also observed in the surviving cells. Gene electrotransfer after intratumoral injection of plasmid DNA encoding either CCL5 or CCL17 in CT26 and 4T1 murine tumor model (animal license: U34401-1/2015/43) resulted in minor tumor growth delay. Concurrent expression analysis of 7 cytokines after gene electrotransfer in tumors showed overexpression of both chemokines, while levels of proinflammatory cytokines IL-6, IL-12 and IFNγ were modestly increased. Obtained altered expression profile, both in vitro and in vivo, is typical for inflammation. Furthermore, irradiation of CT26 and 4T1 tumors combined with gene electrotransfer of plasmids encoding CCL5 or CCL17 resulted in significant tumor growth delay and even tumor cures. Therefore, based on changes in tumor microenvironment after therapy future experiments will be focused towards elucidating the mechanisms of immune response and determining the optimal time window for combined tumor irradiation and gene electrotransfer of plasmids encoding chemokines CCL5 and CCL17. Citation Format: Gregor Sersa, Tim Bozic, Boštjan Markelc, Maja Cemazar, Simona Kranjc Brezar. Overexpression of proinflammatory chemokines CCL5 and CCL17 after gene electrotransfer to murine tumors modifies their cytokine expression profile and leads to tumor cures in combination with tumor irradiation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-099.

The Functional and Antiviral Activity of Interferon Alpha-Inducible IFI6 Against Hepatitis B Virus Replication and Gene Expression.

Hepatitis B virus is an enveloped DNA virus, that infects more than three hundred and sixty million people worldwide and leads to severe chronic liver diseases. Interferon-alpha inducible protein 6 (IFI6) is an IFN-stimulated gene (ISG) whose expression is highly regulated by the stimulation of type I IFN-alpha that restricts various kinds of virus infections by targeting different stages of the viral life cycle. This study aims to investigate the antiviral activity of IFI6 against HBV replication and gene expression. The IFI6 was highly induced by the stimulation of IFN-α in hepatoma cells. The overexpression of IFI6 inhibited while knockdown of IFI6 elevated replication and gene expression of HBV in HepG2 cells. Further study determined that IFI6 inhibited HBV replication by reducing EnhII/Cp of the HBV without affecting liver enriched transcription factors that have significant importance in regulating HBV enhancer activity. Furthermore, deletion mutation of EnhII/Cp and CHIP analysis revealed 100 bps (1715-1815 nt) putative sites involved in IFI6 mediated inhibition of HBV. Detailed analysis with EMSA demonstrated that 1715-1770 nt of EnhII/Cp was specifically involved in binding with IFI6 and restricted EnhII/Cp promoter activity. Moreover, IFI6 was localized mainly inside the nucleus to involve in the anti-HBV activity of IFI6. In vivo analysis based on the hydrodynamic injection of IFI6 expression plasmid along with HBV revealed significant inhibition of HBV DNA replication and gene expression. Overall, our results suggested a novel mechanism of IFI6 mediated HBV regulation that could develop potential therapeutics for efficient HBV infection treatment.

The mechanism of lncRNA‐CRNDE in regulating tumour‐associated macrophage M2 polarization and promoting tumour angiogenesis

M2 macrophages can promote liver cancer metastasis by promoting tumour angiogenesis; however, the mechanism underlying macrophage polarization has not been completely revealed. In this study, we mainly explored the mechanism underlying long non‐coding RNA‐CRNDE (lncRNA‐CRNDE) in regulating M2 macrophage polarization and promoting liver cancer angiogenesis. The expression of CRNDE was up‐regulated or down‐regulated in THP‐1 cells (CRNDE‐/‐‐THP‐1 cells and pcDNA3.1‐CRNDE‐THP‐1). THP‐1 cells were co‐cultured with liver cancer cell line H22, and M2 polarization was induced in THP‐1 by IL‐4/13 to simulate tumour‐induced macrophage polarization. As a result, after CRNDE overexpression, THP‐1 cell viability was up‐regulated, the expression of M2 membrane marker CD163 was up‐regulated, and the proportion of F4/80 + CD163+ cells was also up‐regulated. ELISA assay showed that the expression of M2 markers (including TGF‐β1 and IL‐10) and chemokines (including CCl22 and CCL22) was up‐regulated, and the expression of key signals (including STAT6, JAK‐1, p‐AKT1, and Arg‐1) was also up‐regulated, which were significantly different compared with the control group (Con). In addition, the intervention effect of CRNDE on THP‐1 was consistent between co‐culture with H22 cells and IL‐4/13 induction assay. The induced M2 THP‐1 cells were co‐cultured with HUVEC. As a result, THP‐1 cells with CRNDE overexpression can promote the migration and angiogenesis of HUVEC cells in vitro and simultaneously up‐regulate the expression of Notch1, Dll4 and VEGFR2, indicating that THP‐1 M2 polarization induced by CRNDE could further promote angiogenesis. The H22 cell tumour‐bearing mouse model was constructed, followed by injection of CRNDE anti‐oligosense nucleotides and overexpression plasmids to interfere CRNDE expression in tumour‐bearing tissues. Consequently, down‐regulation of CRNDE could down‐regulate tumour volume, simultaneously down‐regulate the expression of CD163 and CD31 in tissues, decrease the expression of key proteins (including JAK‐1, STAT‐6, p‐STAT6 and p‐AKT1), and down‐regulate the expression of key angiogenesis‐related proteins (including VEGF, Notch1, Dll4 and VEGFR2). In this study, we found that CENDE could indirectly regulate tumour angiogenesis by promoting M2 polarization of macrophages, which is also one of the mechanisms of microenvironmental immune regulation in liver cancer.