CircRNAs in Pancreatic Cancer: New Tools for Target Identification and Therapeutic Intervention.

We have reviewed the literature for circular RNAs (circRNAs) with efficacy in preclinical pancreatic-cancer related in vivo models. The identified circRNAs target chemoresistance mechanisms (n=5), secreted proteins and transmembrane receptors (n=15), transcription factors (n=9), components of the signaling- (n=11), ubiquitination- (n=2), autophagy-system (n=2), and others (n=9). In addition to identifying targets for therapeutic intervention, circRNAs are potential new entities for treatment of pancreatic cancer. Up-regulated circRNAs can be inhibited by antisense oligonucleotides (ASO), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) or clustered regularly interspaced short-palindromic repeats-CRISPR associated protein (CRISPR-CAS)-based intervention. The function of down-regulated circRNAs can be reconstituted by replacement therapy using plasmids or virus-based vector systems. Target validation experiments and the development of improved delivery systems for corresponding agents were examined.

Evaluation of the Elements of Short Hairpin RNAs in Developing shRNA-Containing CAR T Cells.

Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy.

ErbB3-Targeting Oncolytic Adenovirus Causes Potent Tumor Suppression by Induction of Apoptosis in Cancer Cells.

Cancer is a multifactorial and deadly disease. Despite major advancements in cancer therapy in the last two decades, cancer incidence is on the rise and disease prognosis still remains poor. Furthermore, molecular mechanisms of cancer invasiveness, metastasis, and drug resistance remain largely elusive. Targeted cancer therapy involving the silencing of specific cancer-enriched proteins by small interfering RNA (siRNA) offers a powerful tool. However, its application in clinic is limited by the short half-life of siRNA and warrants the development of efficient and stable siRNA delivery systems. Oncolytic adenovirus-mediated therapy offers an attractive alternative to the chemical drugs that often suffer from innate and acquired drug resistance. In continuation to our reports on the development of oncolytic adenovirus-mediated delivery of shRNA, we report here the replication-incompetent (dAd/shErbB3) and replication-competent (oAd/shErbB3) oncolytic adenovirus systems that caused efficient and persistent targeting of ErbB3. We demonstrate that the E1A coded by oAd/shErbB, in contrast to dAd/shErbB, caused downregulation of ErbB2 and ErbB3, yielding stronger downregulation of the ErbB3-oncogenic signaling axis in in vitro models of lung and breast cancer. These results were validated by in vivo antitumor efficacy of dAd/shErbB3 and oAd/shErbB3.

Effect of annexin A1 on the proliferation, migration and invasion of papillary thyroid carcinoma / 中华内分泌外科杂志

Objective:To investigate the effect of ANXA on biological behavior of papillary thyroid carcinoma (PTC) cells by interfering with the expression of annexin A1 (ANXA1) in PTC cell lines by short hairpin RNA (shRNA) .Methods:The shRNA with specific and high efficiency was designed to specifically interfere with the expression of ANXA1 in TPC-1 and BCPAP cell lines, and transfect the TPC-1 and BCPAP cell lines respectively, including specific ANXA1 interference and negative control virus transfection, and they were divided into shANXA1 group and negative control virus group. Semi-quantitative reverse transcription PCR (Q-PCR) and Western Blot were employed to verify gene expression. The shANXA1 group was used as the experimental group, the untransfected virus group and the negative control virus group were set as the control groups. The expression levels of ANXA1 in the three groups were compared and the shRNA interference efficiency was verified. The effects of ANXA1 knockdown on the proliferation, migration and invasion of TPC-1 and BCPAP cell lines were investigated by scratch, CCK8 and Transwell invasion experiments. Independent sample t test was used to compare the means between the two groups, and one-way analysis of variance was employed to compare multiple groups, with P<0.05 as statistically significant. Results:shRNA could efficiently silence the expression of ANXA1 at the transcription and translation level in PTC cell lines. Compared with the negative control cells, the cells proliferated after successful lentiviral transfection of TPC-1 and BCPAP (BCPAP, 24h: F= 25.15, P<0.001; 48h: F=6.44, P<0.001; 48h: F=46.94, P<0.001; TPC-1, 24h: F=207.50, P<0.001; 48h: F=202.45, P<0.001; 48h: F=55.89, P<0.001) , its migration (BCPAP, F=12511.10, P<0.001; TPC-1, F=3966.10, P<0.001) and invasion ability (BC-PAP: F=94.65, P<0.001; TPC-1: F=681.74, P<0.001) significantly decreased. Conclusion:After shRNA knock-down of ANXA1 gene, the proliferation, migration and invasion ability of TPC-1 and BCPAP cell lines decreased significantly, indicating that silencing this gene can reduce tumor aggressiveness, and initially reveals that ANXA1 may be an important potential in PTC biotherapy Target.

FosL1 Regulates Regional Metastasis of Head and Neck Squamous Cell Carcinoma by Promoting Cell Migration, Invasion, and Proliferation.

BACKGROUND/AIM: We evaluated the impact of FosL1, a member of the activated protein-1 family, on the pathways leading to regional metastasis of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We examined the influence of small interfering RNA (siRNA) and short heparin RNA (shRNA) mediated knockdown of FosL1 on cell migration, invasion, and proliferation in vitro as well as on regional metastasis in vivo. The prognostic significance of FosL1 was also analyzed using the Kaplan- Meier plotter using data from an HNSCC patient database. RESULTS: Down-regulation of FosL1 inhibited cell migration, invasion, and proliferation in vitro, decreased the incidence of regional metastases, and prolonged the survival of mice in vivo. We also determined that HNSCC patients with higher expression levels of FosL1 had a significantly shorter survival time than those with low expression of FosL1. CONCLUSION: FosL1 plays a crucial role in promoting cell migration, invasion, and proliferation in HNSCC.

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.

Exosomes: Structure, Biogenesis, Types and Application in Diagnosis and Gene and Drug Delivery.

In recent times, several approaches for targeted gene therapy (GT) had been studied. However, the emergence of extracellular vesicles (EVs) as a shuttle carrying genetic information between cells has gained a lot of interest in scientific communities. Owing to their higher capabilities in dealing with short sequences of nucleic acid (mRNA, miRNA), proteins, recombinant proteins, exosomes, the most popular form of EVs are viewed as reliable biological therapeutic conveyers. They have natural access through every biological membrane and can be employed for site-specific and efficient drug delivery without eliciting any immune responses hence, qualifying as an ideal delivery vehicle. Also, there are many research studies conducted in the last few decades on using exosome-mediated gene therapy into developing an effective therapy with the concept of a higher degree of precision in gene isolation, purification and delivery mechanism loading, delivery and targeting protocols. This review discusses several facets that contribute towards developing an efficient therapeutic regime for gene therapy, highlighting limitations and drawbacks associated with current GT and suggested therapeutic regimes.

An RNAi therapeutic, DFP-10825, for intraperitoneal and intrapleural malignant cancers.

RNA interference (RNAi), a potent post-transcriptional gene-silencing action, has received considerable attentions as a novel therapeutic tool to treat intractable cancers. In recent days, we have developed a novel RNAi-based therapeutic formulation, DFP-10825, for the treatment of intractable advanced cancers developed in coelomic cavities. DFP-10825 was composed of chemically synthesized short hairpin RNA (shRNA) against thymidylate synthase (TS), a key enzyme for cancer proliferation, and cationic liposomes, and achieved high therapeutic effect on the mouse models of peritoneally disseminated gastric and ovarian cancers and malignant pleural mesothelioma without severe side effects by intracoelomic direct treatment. We further designed a freeze-dried DFP-10825 formulation for mass industrial production. DFP-10825 is undergoing in pre-clinical phase and goes to clinical trials. This review introduces a DFP-10825 formulation, a potent novel RNAi-based therapeutic maximizing the benefit of RNAi molecule (shRNA).

Inhibition of encephalomyocarditis virus replication by shRNA targeting 1C and 2A genes in vitro and in vivo.

Encephalomyocarditis virus (EMCV) infects many mammalian species, causing myocarditis, encephalitis and reproductive disorders. The small interference RNA (siRNA) targeting to the virus has not been understood completely. Here, two out of six interference sequences were screened to inhibit significantly EMCV replication by using recombinant plasmids expressing small hairpin RNA (shRNA) targeting to the viral 1C or 2A genes in BHK-21 cells. And two recombinant adenoviruses expressing the shRNAs were constructed and named as rAd-1C-1 and rAd-2A-3. They inhibit EMCV replication in BHK-21 cells in protein levels, as well as the virus yields by approximately 1000 times. Furthermore, they provide high protective efficacy against the challenge with virulent EMCV NJ08 strain in mice. And the EMCV loads in the live mice in rAd-1C-1 and rAd-2A-3 groups decrease by more than 90 % compared with those in the dead mice in the challenge control groups at the same times. It indicates that the adenoviruses medicated shRNA targeting to 1C and 2A genes might provide a potential strategy for combating EMCV infection.

Genetic disruption of the small GTPase RAC1 prevents plexiform neurofibroma formation in mice with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1-/- Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) in NF1-/- Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre- littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox ;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

Knockdown of Chimeric RNA by RNAi.

Knockdown assays are widely used to study the functions of a gene of interest. RNA interference (RNAi) describes a set of well-known methods used to reduce the expression of a target gene by degrading its mRNA with short hairpin RNAs (shRNAs) or short interfering RNAs (siRNAs). Knockdown of chimeric RNAs present different challenges than standard RNAi targeting for regular genes. Most specifically, sequence homology restricts the targeting region to the chimeric junction and can result in off-target effects on the parental genes. In this chapter, we provide guidelines and procedures for RNAi design of chimeric RNAs, knockdown of chimeric RNAs, downstream experiments for chimeric RNA functional studies and necessary controls to accompany each set of experiments.

Contribution of Corticotropin-Releasing Factor Receptor 1 (CRF1) to Serotonin Receptor 5-HT2CR Function in Amygdala Neurons in a Neuropathic Pain Model.

The amygdala plays a key role in emotional-affective aspects of pain and in pain modulation. The central nucleus (CeA) serves major amygdala output functions related to emotional-affective behaviors and pain modulation. Our previous studies implicated the corticotropin-releasing factor (CRF) system in amygdala plasticity and pain behaviors in an arthritis model. We also showed that serotonin (5-HT) receptor subtype 5-HT2CR in the basolateral amygdala (BLA) contributes to increased CeA output and neuropathic pain-like behaviors. Here, we tested the novel hypothesis that 5-HT2CR in the BLA drives CRF1 receptor activation to increase CeA neuronal activity in neuropathic pain. Extracellular single-unit recordings of CeA neurons in anesthetized adult male rats detected increased activity in neuropathic rats (spinal nerve ligation model) compared to sham controls. Increased CeA activity was blocked by local knockdown or pharmacological blockade of 5-HT2CR in the BLA, using stereotaxic administration of 5-HT2CR short hairpin RNA (shRNA) viral vector or a 5-HT2CR antagonist (SB242084), respectively. Stereotaxic administration of a CRF1 receptor antagonist (NBI27914) into the BLA also decreased CeA activity in neuropathic rats and blocked the facilitatory effects of a 5-HT2CR agonist (WAY161503) administered stereotaxically into the BLA. Conversely, local (BLA) knockdown of 5-HT2CR eliminated the inhibitory effect of NBI27914 and the facilitatory effect of WAY161503 in neuropathic rats. The data suggest that 5-HT2CR activation in the BLA contributes to neuropathic pain-related amygdala (CeA) activity by engaging CRF1 receptor signaling.

A simplified method for manufacturing RNAi therapeutics for local administration.

RNA interference (RNAi) is one of the most promising strategies for cancer therapeutics. The successful translation of RNAi therapeutics to a clinic setting requires a delivery system that is efficient and simple to upscale. In this study, we devised a simple industrial method to manufacture lipoplex, which includes short hairpin RNA against the expression of thymidylate synthase (TS shRNA) - a key molecule for DNA biosynthesis. An aqueous solution of TS shRNA was gently mixed with either a precursor of cationic liposome (Presome DF-1) or a cationic lipid mixture in an o/w emulsion. This solution was subsequently lyophilized under optimal conditions to obtain either FD-lipoplex-1 or FD-lipoplex-2, respectively. With this method, a lipoplex in activated form was obtained via a simple "one-step" hydration with saline. Both forms of FD-lipoplex showed physicochemical properties comparable to those of conventional lipoplex. FD-lipoplexes stably retained TS shRNA within their formulations in the presence of tumor ascites fluid. Intraperitoneal treatment with either FD-lipoplex-1 or FD-lipoplex-2 provided a therapeutic level of efficacy comparable to that of conventional lipoplex in the treatment of a peritoneal disseminated gastric cancer mouse model. Collectively, established freeze-drying-based methods for RNAi-therapeutic preparation could realistically be used in a clinical setting for the treatment of patients with peritoneal disseminated cancer.

Cancer Immunotherapy: Silencing Intracellular Negative Immune Regulators of Dendritic Cells.

Dendritic cells (DCs) are capable of activating adaptive immune responses, or inducing immune suppression or tolerance. In the tumor microenvironment, the function of DCs is polarized into immune suppression that attenuates the effect of T cells, promoting differentiation of regulatory T cells and supporting tumor progression. Therefore, blocking negative immune regulators in DCs is considered a strategy of cancer immunotherapy. Antibodies can target molecules on the cell surface, but not intracellular molecules of DCs. The delivery of short-hairpin RNAs (shRNA) and small-interfering RNAs (siRNA) should be a strategy to silence specific intracellular targets in DCs. This review provides an overview of the known negative immune regulators of DCs. Moreover, a combination of shRNA/siRNA and DC vaccines, DNA vaccines in animal models, and clinical trials are also discussed.

IL-1ß Inflammatory Cytokine-Induced TP63 Isoform ∆NP63α Signaling Cascade Contributes to Cisplatin Resistance in Human Breast Cancer Cells.

The mechanisms behind the induction of malignancy and chemoresistance in breast cancer cells are still not completely understood. Inflammation is associated with the induction of malignancy in different types of cancer and is highlighted as an important factor for chemoresistance. In previous work, we demonstrated that the inflammatory cytokine interleukin 1ß (IL-1ß)-induced upregulation of genes was associated with chemoresistance in breast cancer cells. Here, we evaluated the participation and the expression profile of TP63 in the induction of resistance to cisplatin. By loss-of-function assays, we identified that IL-1ß particularly upregulates the expression of the tumor protein 63 (TP63) isoform ΔNP63α, through the activation of the IL-1ß/IL-1RI/ß-catenin signaling pathway. Upregulation of ΔNP63α leads to an increase in the expression of the cell survival factors epidermal growth factor receptor (EGFR) and phosphatase 1D (Wip1), and a decrease in the DNA damage sensor, ataxia-telangiectasia mutated (ATM). The participation of these processes in the increase of resistance to cisplatin was confirmed by silencing TP63 expression or by inhibition of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) activity in the IL-1ß/IL-1RI/ß-catenin signaling pathway. These data reinforced the importance of an inflammatory environment in the induction of drug resistance in cancer cells and uncovered a molecular mechanism where the IL-1ß signaling pathway potentiates the acquisition of cisplatin resistance in breast cancer cells.

Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response.

Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approved cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined antibody engineering and functional genomics screens to identify putative mechanisms of resistance or synthetic lethality with antibody-mediated anti-proliferative effects. We developed a synthetic antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization, and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 antibody therapeutics.

Study of Endogenous CRAC Channels in Human Mast Cells Using an Adenoviral Delivery System to Transduce Cells with Orai-Targeting shRNAs or with cDNAs Expressing Dominant-Negative Orai Channel Mutations.

We describe two methods to study CRAC channel function in human lung mast cells. Both methods involve suppression of endogenous channel function. In the first we use Orai-targeting shRNAs to knock down Orai channel mRNA transcripts. In the second we overexpress dominant-negative mutants of the three members of the Orai channel family. To overcome the poor transfection efficiency of mast cells, we employ an adenoviral delivery system for cell transduction. Knockdown of CRAC channel transcripts is assessed initially using quantitative RT-PCR. We describe an assay for ß-hexosaminidase release as a measure of mast cell degranulation to assess the effect of overexpression of dominant-negative mutants.

Therapeutic Potency of Nanoformulations of siRNAs and shRNAs in Animal Models of Cancers.

RNA Interference (RNAi) has brought revolutionary transformations in cancer management in the past two decades. RNAi-based therapeutics including siRNA and shRNA have immense scope to silence the expression of mutant cancer genes specifically in a therapeutic context. Although tremendous progress has been made to establish catalytic RNA as a new class of biologics for cancer management, a lot of extracellular and intracellular barriers still pose a long-lasting challenge on the way to clinical approval. A series of chemically suitable, safe and effective viral and non-viral carriers have emerged to overcome physiological barriers and ensure targeted delivery of RNAi. The newly invented carriers, delivery techniques and gene editing technology made current treatment protocols stronger to fight cancer. This review has provided a platform about the chronicle of siRNA development and challenges of RNAi therapeutics for laboratory to bedside translation focusing on recent advancement in siRNA delivery vehicles with their limitations. Furthermore, an overview of several animal model studies of siRNA- or shRNA-based cancer gene therapy over the past 15 years has been presented, highlighting the roles of genes in multiple cancers, pharmacokinetic parameters and critical evaluation. The review concludes with a future direction for the development of catalytic RNA vehicles and design strategies to make RNAi-based cancer gene therapy more promising to surmount cancer gene delivery challenges.