Total Flavones Isolated from Lycium Barbarum L. Inhibit the Proliferation, Migration and Invasion of GlioblastomaU-87MG Cells by Decreasing Formyl Peptide Receptor 1 Expression

Background: To study the role and mechanisms of total flavones from Lycium barbarum L. (TFL) and FPR1 in the growth of glioblastoma U-87MG cells.Main Methods: CCK-8, wound-healing and Transwell were used for investigating proliferation, motility and invasion of U87 cells after treating with total flavones. RT-qPCR and Western blot were used to study the effect of total flavones on proliferating cell nuclear antigen (PCNA), matrix metalloproteinase2 (MMP2) and FPR1. The short hairpin RNA and FPR agonist fMLP were used to delineate the role of FPR1.Results: TFL was successfully isolated, and its concentration was determined to be 6.205 mg/l. TFL inhibited the proliferation, migration and invasion of U-87MG cells in a time and dose-dependent manner compared to controls. Decreasing FPR1 expression using short hairpin RNA significantly inhibited the migration and invasion of U-87MG cells. Notably, increased expression of FPR1 and treatment with FPR-agonist peptides such as N-formylmethionyl-leucyl-phenylalanine induced the migration and invasion of U-87MG cells, which was significantly decreased when the cells were treated with TFL. Conclusion: TFL inhibits the proliferation, migration and invasion of human glioblastoma U-87MG cells through decreasing the expression of FPR1. These findings provide valuable evidence for the development of antitumor drugs.

Hepatitis B Virus-Like Particle: Targeted Delivery of Plasmid Expressing Short Hairpin RNA for Silencing the Bcl-2 Gene in Cervical Cancer Cells

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells’ viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.

Intratumoral spatial heterogeneity of BTK kinomic activity dictates distinct therapeutic response within a single glioblastoma tumor

OBJECTIVEDespite significant recent efforts applied toward the development of efficacious therapies for glioblastoma (GBM) through exploration of GBM’s genome and transcriptome, curative therapeutic strategies remain highly elusive. As such, novel and effective therapeutics are urgently required. In this study, the authors sought to explore the kinomic landscape of GBM from a previously underutilized approach (i.e., spatial heterogeneity), followed by validation of Bruton’s tyrosine kinase (BTK) targeting according to this stepwise kinomic-based novel approach.METHODSTwelve GBM tumor samples were obtained and characterized histopathologically from 2 patients with GBM. PamStation peptide-array analysis of these tissues was performed to measure the kinomic activity of each sample. The Ivy GBM database was then utilized to determine the intratumoral spatial localization of BTK activity by investigating the expression of BTK-related transcription factors (TFs) within tumors. Genetic inhibition of BTK family members through lentiviral short hairpin RNA (shRNA) knockdown was performed to determine their function in the core-like and edge-like GBM neurosphere models. Finally, the small-molecule inhibitor of BTK, ONO/GS-4059, which is currently under clinical investigation in nonbrain cancers, was applied for pharmacological inhibition of regionally specified newly established GBM edge and core neurosphere models.RESULTSKinomic investigation identified two major subclusters of GBM tissues from both patients exhibiting distinct profiles of kinase activity. Comparatively, in these spatially defined subgroups, BTK was the centric kinase differentially expressed. According to the Ivy GBM database, BTK-related TFs were highly expressed in the tumor core, but not in edge counterparts. Short hairpin RNA–mediated gene silencing of BTK in previously established edge- and core-like GBM neurospheres demonstrated increased apoptotic activity with predominance of the sub-G1 phase of core-like neurospheres compared to edge-like neurospheres. Lastly, pharmacological inhibition of BTK by ONO/GS-4059 resulted in growth inhibition of regionally derived GBM core cells and, to a lesser extent, their edge counterparts.CONCLUSIONSThis study identifies significant heterogeneity in kinase activity both within and across distinct GBM tumors. The study findings indicate that BTK activity is elevated in the classically therapy-resistant GBM tumor core. Given these findings, targeting GBM’s resistant core through BTK may potentially provide therapeutic benefit for patients with GBM.

Are Viral Vectors Any Good for RNAi Antiviral Therapy?

RNA interference (RNAi) represents a novel approach for alternative antiviral therapy. However, issues related to RNA delivery and stability have presented serious obstacles for obtaining good therapeutic efficacy. Viral vectors are capable of efficient delivery of RNAi as short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA). Efficacy in gene silencing for therapeutic applications against viral diseases has been demonstrated in various animal models. Rotavirus (RV) miR-7 can inhibit rotavirus replication by targeting the RV nonstructural protein 5. Viral gene silencing by targeting the RNAi pathway showed efficient suppression of hepatitis B virus replication by adeno-associated virus (AAV)-based delivery of RNAi hepatitis B virus (HBV) cassettes. Hepatitis C virus replication has been targeted by short hairpin RNA molecules expressed from lentivirus vectors. Potentially, RNAi-based approaches could be suitable for antiviral drugs against COVID-19.

Attenuation of Oxidative Injury With Targeted Expression of NADPH Oxidase 2 Short Hairpin RNA Prevents Onset and Maintenance of Electrical Remodeling in the Canine Atrium

Background: Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K + current ( I KACh ), called I KH ; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. Methods: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining. Results: First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating I KACh by a mechanism involving frequency-dependent activation of PKC ε (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA–treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of I KACh . Attenuated membrane translocation of PKC ε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. Conclusions: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.

The Expression Profile of mRNA and tRNA Genes in Splenocytes and Neutrophils after In Vivo Delivery of Antitumor Short Hairpin RNA of Indoleamine 2,3- Dioxygenase

RNA-based therapeutics are considered as novel treatments for human diseases. Our previous study demonstrated that treatment with short-hairpin RNA against Ido1 (IDO shRNA) suppresses tumor growth, detects Th1-bias immune responses, and elevates expression of tryptophan transfer RNA (tRNATrp) in total splenocytes. In addition, depletion of Ly6g+ neutrophils attenuates the effect of IDO shRNA. The aim of this study was to investigate the regulatory network and the expression profile of tRNAs and other non-coding RNAs in IDO shRNA-treated spleens. The total splenocytes and magnetic bead-enriched splenic neutrophils were collected from the lung tumor bearing mice, which were treated with IDO shRNA or scramble IDO shRNA, and the collected cells were subsequently subjected to RNA sequencing. The gene ontology analysis revealed the different enrichment pathways in total splenocytes and splenic neutrophils. Furthermore, the expression of tRNA genes was identified and validated. Six isoacceptors of tRNA, with different expression patterns between total splenocytes and splenic neutrophils, were observed. In summary, our findings not only revealed novel biological processes in IDO shRNA-treated total splenocytes and splenic neutrophils, but the identified tRNAs and other non-coding RNAs may contribute to developing a novel biomarker gene set for evaluating the clinical efficiency of RNA-based cancer immunotherapies.