Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression

MicroRNAs (miRNAs), small noncoding RNAs with a length of about 22 nucleotides, harbor the potential to be powerful tools for the genetic engineering of production cell lines like Chinese hamster ovary (CHO) cells. Their ability to regulate multiple targets at once and their potential to fine-tune effect strengths contrast withclassical engineering approaches. However, most studies of miRNAs rely on transiently flooding the cells with miRNA mimics. Since this approach is not suitable for long-term cultivation in a bioprocess, stable overexpression of miRNAs becomes more and more important for the biotech industry. Here, the user might be confronted with insufficient overexpression of the miRNA of interest. In this chapter, we present a method for the generation of stable CHO cell lines expressing a miRNA from a plasmid-based system containing multiple copies of the miRNA, allowing tuning of overexpression and regulation.

Background: Neuroblastoma (NB) is a tumor of the sympathetic nervous system that is the most common extracranial solid tumor of childhood. NBs show remarkable clinical heterogeneity, and we, along with others, have identified different patterns of genomic change that underlie these diverse clinical behaviors. We first identified 1p deletion as a characteristic change in advanced stage NBs. CHD5, a tumor suppressor gene, was first identified because of its location on 1p36. However, mutation of the remaining allele of CHD5 is rare in these tumors. Therefore, it is likely that epigenetic mechanisms play important roles in CHD5 downregulation. MicroRNAs (miRNAs) are small RNAs that bind to the 3′ UTR to mediate downregulation of target gene expression by translational repression or cleavage of the target gene message. Dysregulation of CHD5 via miR-211 has been reported in colon cancer. In order to understand the role of miRNA regulation of CHD5 in NBs, we wanted to assess the function of all miRNAs that are predicted to target CHD5. Method/approach: We used TargetScan, miRanda, MirTarget2 and other prediction algorithms to identify miRNAs that were predicted to bind to the CHD5-3′UTR. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204-5p, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We used a renilla-luciferase reporter plasmid that contains 103 bp of the 3′UTR of CHD5 targeted by all miRNAs except miR-204, -211, - 219b, and -3666. We also created control plasmids with no CHD5 3′UTR insert and a mutated CHD5 3′UTR with mutated miRNA binding sites. Allstar siRNA served as an additional negative control. We performed transient transfections in two NB cell lines, NLF (a MYCN amplified cell line) and SY5Y (a MYCN nonamplified cell line), with the reporter plasmid and miRNA mimic. We measured luciferase (internal control) and renilla (reporter) activity. Results: Our results from the luciferase reporter assay indicate that four miRNAs are strong candidates for CHD5 downregulation in NB: miR-17, -93, -20b, and miR-106b. Each exhibited a significant decrease of fluorescence activity in cells transfected with CHD5 3′UTR in comparison to cells transfected with either of the control plasmids. Interestingly, MYCN upregulates three of the four miRNA candidates: miR-17, -93, & -20b. We further confirmed consistent downregulation of the CHD5 by all four miRNAs. Conclusion: CHD5 expression is downregulated by miR-17, -93, -2b, and 106b. MYCN amplification and overexpression leads to upregulation of miR-17, -93, and -20b, and this may lead to down regulation of CHD5. Citation Format: Koumudi Naraparaju, Venkatadri Kolla, Tiangang Zhuang, Mayumi Higashi, Gerd A. Blobel, Garrett M. Brodeur. The role of microRNAs in the epigenetic silencing of CHD5, a tumor suppressor in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2015-191

Kaposi's sarcoma-associated herpesvirus (KSHV), a γ-herpesvirus, is predominantly associated with Kaposi's sarcoma (KS) as well as two lymphoproliferative disorders: primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other herpesviruses, KSHV employs two distinct life cycles: latency and lytic replication. To establish a lifelong persistent infection, KSHV has evolved various strategies to manipulate the epigenetic machinery of the host. In latently infected cells, most viral genes are epigenetically silenced by components of cellular chromatin, DNA methylation and histone post-translational modifications. However, some specific latent genes are preserved and actively expressed to maintain the virus's latent state within the host cell. Latency is not a dead end, but the virus has the ability to reactivate. This reactivation is a complex process that involves the removal of repressive chromatin modifications and increased accessibility for both viral and cellular factors, allowing the activation of the full transcriptional program necessary for the subsequent lytic replication. This review will introduce the roles of epigenetic modifications in KSHV latent and lytic life cycles, including DNA methylation, histone methylation and acetylation modifications, chromatin remodeling, genome conformation, and non-coding RNA expression. Additionally, we will also review the transcriptional regulation of viral genes and host factors in KSHV infection. This review aims to enhance our understanding of the molecular mechanisms of epigenetic modifications and transcriptional regulation in the KSHV life cycle, providing insights for future research.

Background and Aims Mitophagy activation is crucial for hypoxia signaling in acute kidney injury (AKI). Selective removal of damaged mitochondria is mediated through a concerted function of coding and non-coding RNA expressions. Insights on HIF-1α signaling in lncRNA-regulated mitophagy mechanism are not known. The study aims to investigate the role of hypoxia-inducible factor 1α (HIF-1α) regulated long non-coding RNA (lncRNA) ATP6V0E2-AS1 in acute kidney injury (AKI) by examining its effect on mitophagy. Method Mitophagy proteins (PINK1, p-PARKIN, LC3) were determined through western blot analysis. Immunofluorescence studies of PINK1 mitochondrial translocation and LC3/TOMM20 localization were determined using confocal analysis. HIF-1α interaction with lncRNA-ATP6V0E2-AS1 through promoter binding activity was evaluated through chromatin immunoprecipitation analysis. RNA interaction between lncRNA-ATP6V0E2-AS1 and PINK1 was analyzed by dual-luciferase reporter assay. Results Hypoxia upregulated PINK1, p-PARKIN, LC3 expressions, and mitophagosome in HK-2 cells. Overexpression and knockdown studies of hypoxia-regulated lncRNA-ATP6V0E2-AS1 significantly regulated PINK1/p-PARKIN expressions, subsequent PINK1 mitochondrial co-localization, and mitophagosome formation. shHIF-1α knockdown in HK-2 cells reveals that HIF-1α mediates PINK1/p-PARKIN mitophagy regulation under hypoxic conditions. In detail, HIF-1α binds to the promoter of lncRNA-ATP6V0E2-AS1 and down-regulates its expression under hypoxia. Deficiency of both HIF-1α and lncRNA-ATP6V0E2-AS1 reverted shHIF-1α mediated suppression of PINK1 expression and mitophagosome formation. Further, ATP6V0E2-AS1 post-transcriptionally regulates PINK1 expression by RNA-RNA interaction. Conclusion Altogether, our study shows novel findings on the HIF-1α mediated lncRNA-ATP6V0E2-AS1 in hypoxia-induced mitophagy regulation in the renal tubular cells. Thus, downregulated ATP6V0E2-AS1 expression with subsequent mitophagy activation is critical for hypoxia-induced mitophagy regulation through HIF-1α/ATP6V0E2-AS1/PINK1 axis.

Expansion of GAA/TTC repeats is the causative event in Friedreich's ataxia. GAA repeats have been shown to hinder replication in model systems, but the mechanisms of replication interference and expansion in human cells remained elusive. To study in vivo replication structures at GAA repeats, we designed a new plasmid-based system that permits the analysis of human replication intermediates by two-dimensional gel electrophoresis and EM. We found that replication forks transiently pause and reverse at long GAA/TTC tracts in both orientations. Furthermore, we identified replication-associated intramolecular junctions, located between GAA/TTC repeats and other homopurine-homopyrimidine tracts, that were associated with breakage of the plasmid fork not traversing the repeats. Finally, we detected postreplicative, sister-chromatid hemicatenanes on control plasmids, which were replaced by persistent homology-driven junctions at GAA/TTC repeats. These data prove that GAA/TTC tracts interfere with replication in humans and implicate postreplicative mechanisms in trinucleotide repeat expansion.

Clinical application of gene therapy depends on the development of suitable gene transfer vehicles (vectors). Although generally not as efficient as viral vectors, nonviral systems such as lipidic vectors have the potential advantages of being less toxic, nonrestrictive in cargo DNA size, potentially targetable, and easy to produce in relatively large amounts. More important, lipidic vectors generally lack immunogenicity, allowing repeated in vivo transfection using the same vector. In this paper, we will attempt to summarize some of the recent advances in lipidic gene delivery vectors. Three types of lipidic gene transfer vectors are described: 1) DNA/cationic liposome complexes, 2) DNA encapsulated in neutral or anionic liposomes, and 3) liposome-entrapped, polycation-condensed DNA (LPDI and LPDII). We review the various factors affecting vector structure and gene delivery efficiency, and we discuss the possible mechanisms of gene transfer and their implications in vector design.

Unlike viral vectors with their undesired safety or immunogenicity concerns, biocompatible polymeric nonviral vectors as gene delivery systems are more promising in gene or cell therapy. Evidence has demonstrated that the rational design of polymeric nonviral gene vectors with optimal structure, charge density, biocompatibility, and stimulus responsiveness can deliver therapeutic genes or gene vaccines (in terms of deoxyribonucleic acid DNA or ribonucleic acid RNA) into tumor‐associated immunocytes (i.e., macrophages, T cells, or dendritic cells) in an effective and controllable manner for enhanced cancer immunotherapy. However, a timely and systematic summary of this subject is lacking. This review presents an overview of polymeric nonviral carriers with immune cell transfection ability and their potential applications in cancer immunotherapy; it also provides a tutorial for designing polymeric immune‐cell genetic modification vector, although there are still few vectors in the product pipeline. With the rapid growth of immunotherapy in cancer treatment and knowledge accumulation in vector structure design, polymeric nonviral gene delivery systems may provide new hope for tumor therapy.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotes, and it participates in the regulation of pathophysiological processes in various diseases, including malignant tumors, by regulating the expression and function of both coding and non-coding RNAs (ncRNAs). More and more studies demonstrated that m6A modification regulates the production, stability, and degradation of ncRNAs and that ncRNAs also regulate the expression of m6A-related proteins. Tumor microenvironment (TME) refers to the internal and external environment of tumor cells, which is composed of numerous tumor stromal cells, immune cells, immune factors, and inflammatory factors that are closely related to tumors occurrence and development. Recent studies have suggested that crosstalk between m6A modifications and ncRNAs plays an important role in the biological regulation of TME. In this review, we summarized and analyzed the effects of m6A modification-associated ncRNAs on TME from various perspectives, including tumor proliferation, angiogenesis, invasion and metastasis, and immune escape. Herein, we showed that m6A-related ncRNAs can not only be expected to become detection markers of tumor tissue samples, but can also be wrapped into exosomes and secreted into body fluids, thus exhibiting potential as markers for liquid biopsy. This review provides a deeper understanding of the relationship between m6A-related ncRNAs and TME, which is of great significance to the development of a new strategy for precise tumor therapy.

Cancer has been associated with epigenetic abnormalities leading to deregulated expression of both coding and non-coding RNAs (ncRNAs) involved in oncogenesis. Among these ncRNAs, microRNAs (miRNAs) were identified acting as oncogenes or tumor suppressors by repressing the expression of cancer related genes. In addition, other studies suggest that other ncRNAs are similarly involved in tumorigenesis, including long non-coding RNAs (lncRNAs) and transcribed Ultraconserved Regions (t-UCRs) by different mechanisms. Recent studies have shown the impact of small nucleolar RNAs (snoRNAs) in the onset or progression of cancer. SnoRNAs encoded by the ncRNA GAS5 that is repressed in breast cancer or the snoRNA U50, which is mutated in breast and prostate cancer, are examples of the connection between snoRNAs and cancer. It is also known that some snoRNAs are differentially expressed in cancer in comparison with the correspondent matched tissue, indicating their possible role in this disease. This study focuses in the analysis of the DNA methylation profile associated with the differentially expression of snoRNAs in cancer considering a host gene dependent or independent regulation. To select snoRNAs regulated by DNA methylation we used two different epigenetic approaches: snoRNA expression arrays upon DNA methylation inhibition and global genomic DNA methylation arrays. Herein, we were able to indentify snoRNAs with different DNA methylation that might be involved in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 106. doi:10.1158/1538-7445.AM2011-106

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

BackgroundAbnormal expression of long non-coding RNA (lncRNAs) often facilitates unrestricted growth of cancer cells. Long intergenic non-protein coding RNA 1510, an enhancer lncRNA (LINC01510), a lncRNA enhancer is upregulated in colorectal cancer (CRC), and its expression might relate to MET as revealed by lncRNA microarray data. However, the potential biological role of LINC01510 and its regulatory mechanism in CRC remain unclear. Therefore, we investigated the involvement of LINC01510 in the proliferation of CRC cells.MethodsMicroarray analysis, In situ hybridization, colony formation assay, MTT assay, Western blotting, quantitative RT-PCR and flow cytometry were applied. The two-tailed Student’s t test and analysis of variance or general linear model of single factor variable was used for statistical analyse.ResultsIn the present study, we found that LINC01510 was significantly upregulated in CRC tissues and cell lines. The LINC01510 expression level were associated with the clinicopathological grade and stage. Meanwhile, gain- and loss-of-function assays demonstrated that LINC01510 overexpression increased CRC cell proliferation, and promoted cell cycle progression from the G1 phase to the S phase. Further study indicated that LINC01510 was positively correlated with the expression of MET, and its effects were most likely at the transcriptional level.ConclusionsTaken together, our findings suggested that upregulation of LINC01510 contributes to the proliferation of CRC cells, at least in part, through the regulation of MET protein. LINC01510 could be a candidate prognostic biomarker and a target for new therapies in CRC patients.

BackgroundAccumulated studies indicate that aberrant expression of long noncoding RNAs (lncRNAs) is associated with tumorigenesis and progression of colon cancer. In the present study, long intergenic non-protein coding RNA 1287 (LINC01287) was identified to up-regulate in colon cancer by transcriptome RNA-sequencing, but the exact function remained unclear.MethodsTranscriptome RNA-sequencing was conducted to identify dysregulated lncRNAs. Expression of LINC01287 was evaluated by real-time quantitative PCR. The downstream targets of LINC01287 and miR-4500 were verified by luciferase reporter assay, pull down assay and western blot. The potential functions of LINC01287 were evaluated by cell viability assay, colony formation assay, soft agar assay, flow cytometry, transwell migration and invasion assay, and tumor xenograft growth in colon cancer cells.ResultsOur results indicated that LINC01287 was up-regulated in colon cancer patients. High LINC01287 expression was associated with advanced TNM stage, lymph node metastasis, distant metastasis and shorter overall survival. Knockdown of LINC01287 inhibited cell growth, colony formation in plates and soft agar, transwell cell migration and invasion, and epithelial-mesenchymal transition (EMT) of colon cancer cells, while LINC01287 overexpression had contrary effects. In addition, LINC01287 mediated MAP3K13 expression by sponging miR-4500, thus promoted NF-κB p65 phosphorylation. Restored MAP3K13 expression or miR-4500 knockdown partially abrogated the effects of silencing LINC01287 in colon cancer cells.ConclusionOur findings demonstrated that the LINC01287/miR-4500/MAP3K13 axis promoted progression of colon cancer. Therefore, LINC01287 might be a potential therapeutic target and prognostic marker for colon cancer patients.

Respiratory syncytial virus (RSV) is an enveloped RNA virus which is responsible for approximately 80% of lower respiratory tract infections in children. Current lines of evidence have supported the functional involvement of long noncoding RNA (lncRNA) in many viral infectious diseases. However, the overall biological effect and clinical role of lncRNAs in RSV infection remain unclear. In this study, lncRNAs related to respiratory virus infection were obtained from the lncRNA database, and we collected 144 clinical sputum specimens to identify lncRNAs related to RSV infection. Quantitative PCR (qPCR) detection indicated that the expression of lncRNA negative regulator of antiviral response (NRAV) in RSV-positive patients was significantly lower than that in uninfected patients, but lncRNA psoriasis-associated non-protein coding RNA induced by stress (PRINS), nuclear paraspeckle assembly transcript 1 (NEAT1), and Nettoie Salmonella pas Theiler's (NeST) showed no difference in vivo and in vitro Meanwhile, overexpression of NRAV promoted RSV proliferation in A549 and BEAS-2B cells, and vice versa, indicating that the downregulation of NRAV was part of the host antiviral defense. RNA fluorescent in situ hybridization (FISH) confirmed that NRAV was mainly located in the cytoplasm. Through RNA sequencing, we found that Rab5c, which is a vesicle transporting protein, showed the same change trend as NRAV. Subsequent investigation revealed that NRAV was able to favor RSV production indirectly by sponging microRNA miR-509-3p so as to release Rab5c and facilitate vesicle transportation. The study provides a new insight into virus-host interaction through noncoding RNA, which may contribute to exploring potential antivirus targets for respiratory virus.IMPORTANCE The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection.

Long non-coding RNA LINC01004 promotes malignant behaviors of pituitary adenoma via miR-323a-3p/136–5p/RCN2 axis

Background Acute inflammation leads to increased myelopoiesis in the bone marrow and recruitment of immune cells to respective sites of injury. Yet, in certain cases of high demand such as acute tissue damage as in acute myocardial infarction (AMI), innate immune cells can be additionally recruited from lymphoid organs such as the spleen. Monocytes are important in both onset and resolution of inflammation comprising a huge repertoire of coding and non-coding RNA molecules regulating a fast-acting immune response. Long non-coding RNAs (lncRNA) emerge as novel regulatory cell-specific molecules, which are able to influence cell physiology in numerous ways. Purpose Delineate monocyte subpopulations from patients with AMI compared to healthy controls based on their non-coding transcriptome, different isoform usage and associated source of origin. Methods and results Next generation bulk RNA sequencing has been performed from human monocytes after pre-sorting for CD14 expression and subsequent separation into three major subpopulations based on their surface markers CD14 and CD16 using a FACS Aria II cell sorter. Non-coding RNA expression shows clear differences between cells from healthy controls versus patients with AMI and among subpopulations. Intensive bioinformatics analysis revealed numerous annotated lncRNA, antisense, pseudogene and circular RNAs displaying significant differences in their expression profiles. Some lncRNA show positive or negative correlations with their respective coding antisense gene or their convergent or divergent sense neighbouring gene, such as linc02207 vs MCTP2, SAP30-AS vs SAP30, BID-AS vs BID, HLX-AS vs HLX or TNFAIP3-AS vs TNFAIP3. We also identified the existence of different isoforms of certain lncRNA and a distinct preference thereof, such as linc02207- 217 isoform being most abundant. Real-time PCR was applied for validation of lncRNA expression profile and their nucleocytoplasmic distribution observing preferential nuclear expression for most lncRNA. In vitro assays for silencing and overexpression approaches as well as treatment with inflammatory stimuli and in silico bioinformatics analysis will help to unravel their functionality. Additionally mapping our bulk RNA data to deconvoluted single cell RNAseq data from publicly available human cell atlas (HCA), is applied to additionally unravel the origin of particular subpopulations within the AMI monocytes, associating their origin to either spleen or bone marrow. Conclusions High read depth bulk RNA sequencing revealed a human monocyte-specific long non-coding RNA transcriptome differentially expressed in three predefined subpopulations and disease stages. Distinct expression of lncRNA and associated coding genes as well as distinct isoform preference implies specific functionalities. Studying these molecules and their interaction partners will unravel functional participants in the development of therapeutic approaches for cardiovascular diseases. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DZHK, SNF