Single-exonuclease nanocircuits reveal the RNA degradation dynamics of PNPase and demonstrate potential for RNA sequencing.

The degradation process of RNA is decisive in guaranteeing high-fidelity translation of genetic information in living organisms. However, visualizing the single-base degradation process in real time and deciphering the degradation mechanism at the single-enzyme level remain formidable challenges. Here, we present a reliable in-situ single-PNPase-molecule dynamic electrical detector based on silicon nanowire field-effect transistors with ultra-high temporal resolution. These devices are capable of realizing real-time and label-free monitoring of RNA analog degradation with single-base resolution, including RNA analog binding, single-nucleotide hydrolysis, and single-base movement. We discover a binding event of the enzyme (near the active site) with the nucleoside, offering a further understanding of the RNA degradation mechanism. Relying on systematic analyses of independent reads, approximately 80% accuracy in RNA nucleoside sequencing is achieved in a single testing process. This proof-of-concept sets up a Complementary Metal Oxide Semiconductor (CMOS)-compatible playground for the development of high-throughput detection technologies toward mechanistic exploration and single-molecule sequencing.

Cisplatin resistance of NSCLC cells involves upregulation of visfatin through activation of its transcription and stabilization of mRNA.

Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Cisplatin (CDDP) has been used as a standard adjuvant therapy for advanced NSCLC patients, while chemoresistance is one of the most challenging problems to limit its clinical application. Our data showed that the expression of visfatin was significantly increased in CDDP resistant NSCLC cells as compared with that in their parental cells, while knockdown of visfatin or its neutralization antibody can restore the CDDP sensitivity of resistant NSCLC cells. The upregulation of visfatin in CDDP resistant NSCLC cells was due to the increased mRNA stability and promoter activity. Further, we found that signal transducer and activator of transcription 3 (STAT3), which was increased in chemoresistant cells, can increase the transcription of visfatin. While tristetraprolin (TTP), which can decease mRNA stability of visfatin, was decreased in chemoresistant cells. Inhibition of STAT3 or over expression of TTP can restore CDDP sensitivity of resistant NSCLC cells. Collectively, our data showed that STAT3 and TTP-regulated expression of visfatin was involved in CDDP resistance of NSCLC cells. It indicated that targeted inhibition of visfatin should be a potential approach to overcome CDDP resistance of NSCLC treatment.

Short-Chain Fatty Acids Promote Immunotherapy by Modulating Immune Regulatory Property in B Cells.

The dysfunction of regulatory B cells (Breg) may result in immune inflammation such as allergic rhinitis (AR); the underlying mechanism is not fully understood yet. Short-chain fatty acids, such as propionic acid (PA), have immune regulatory functions. This study is aimed at testing a hypothesis that modulates PA production alleviating airway allergy through maintaining Breg functions. B cells were isolated from the blood obtained from AR patients and healthy control (HC) subjects. The stabilization of IL-10 mRNA in B cells was tested with RT-qPCR. An AR mouse model was developed to test the role of PA in stabilizing the IL-10 expression in B cells. We found that the serum PA levels were negatively correlated with the serum Th2 cytokine levels in AR patients. Serum PA levels were positively associated with peripheral CD5+ B cell frequency in AR patients; the CD5+ B cells were also IL-10+. The spontaneous IL-10 mRNA decay was observed in B cells, which was prevented by the presence of PA through activating GPR43. PA counteracted the effects of Tristetraprolin (TTP) on inducing IL-10 mRNA decay in B cells through the AKT/T-bet/granzyme B pathway. Administration of Yupinfeng San, a Chinese traditional medical formula, or indole-3-PA, induced PA production by intestinal bacteria to stabilize the IL-10 expression in B cells, which promoted the allergen specific immunotherapy, and efficiently alleviated experimental AR. In summary, the data show that CD5+ B cells produce IL-10. The serum lower PA levels are associated with the lower frequency of CD5+ B cells in AR patients. Administration with Yupinfeng San or indole-3-PA can improve Breg functions and alleviate experimental AR.

Cinnamomum verum-derived O-methoxycinnamaldehyde prevents lipopolysaccharide-induced depressive-like behavior in mice via NFAT mRNA stability in T lymphocytes.

BACKGROUND: Depressive-like behaviors are related to inflammatory immune activation. Cinnamomum verum (CV) has anti-inflammatory effects, but the molecular mechanisms underlying the antidepressant effects after immunological activation still remain elusive. PURPOSE: The aim of the present study was to investigate the effect of CV in improving depressive-like behavior and explore its underlying mechanism in T lymphocytes. METHODS: Mice were randomly divided into Control, LPS, LPS plus fluoxetine, LPS plus CV, and LPS plus MCA groups. Behavior was evaluated using forced swimming test (FST) and tail suspension test (TST). The experimental group mice were exposed to LPS to induce depressive-like behavior. Cell viability was measured upon treating splenic T lymphocytes and Jurkat T cells with CV. Cytokine activity was measured using ELISA and RT-qPCR. The components of CV were analyzed by HPLC. NFAT expression was evaluated by western blotting, immunofluorescence, and luciferase assay. To verify the half-life of NFAT mRNA, Jurkat cells were treated with actinomycin D for 1.5, 3, and 4.5 h. RESULTS: CV effectively prevents inflammation-induced depressive-like behaviors. CV dose-dependently decreased protein and mRNA levels of TNFα and IL-2. Inhibition of TNFα and IL-2 production involves an MCA-mediated decrease in NFAT mRNA level, rather than inhibition of nuclear translocation. This mechanism was independent of NFAT transcription inducer p38 MAPK; it can be attributed to the promotion of NFAT mRNA decay. CONCLUSION: Overall, MCA might be an alternative or adjuvant to existing NFAT-targeting immunosuppressants for clinical prophylaxis or therapy in the context of inflammation-induced depressive disorder or other T-cell-associated inflammatory disorders.

Insights into how environment shapes post-mortem RNA transcription in mouse brain.

Most biological features that occur on the body after death were already deciphered by traditional medicine. However, the molecular mechanisms triggered in the cellular microenvironment are not fully comprehended yet. Previous studies reported gene expression alterations in the post-mortem condition, but little is known about how the environment could influence RNA degradation and transcriptional regulation. In this work, we analysed the transcriptome of mouse brain after death under three concealment simulations (air exposed, buried, and submerged). Our analyses identified 2,103 genes differentially expressed in all tested groups 48 h after death. Moreover, we identified 111 commonly upregulated and 497 commonly downregulated genes in mice from the concealment simulations. The gene functions shared by the individuals from the tested environments were associated with RNA homeostasis, inflammation, developmental processes, cell communication, cell proliferation, and lipid metabolism. Regarding the altered biological processes, we identified that the macroautophagy process was enriched in the upregulated genes and lipid metabolism was enriched in the downregulated genes. On the other hand, we also described a list of biomarkers associated with the submerged and buried groups, indicating that these environments can influence the post-mortem RNA abundance in its particular way.

Snord116 Post-transcriptionally Increases Nhlh2 mRNA Stability: Implications for Human Prader-Willi Syndrome.

The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work in the field revealed the tantalizing possibility that expression of NHLH2, a gene previously implicated in both obesity and hypogonadism, was downregulated in PWS patients and differentiated stem cells. In silico RNA: RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3'-UTR, linked to a c-myc tag was transfected into a hypothalamic neuron cell line in the presence and absence of exogenously-expressed Snord116. Nhlh2 mRNA expression was upregulated in the presence of Snord116 dependent on the length and type of 3'UTR used on the construct. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA in the 45 minutes immediately following transcription. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could reduce the predicted interaction strength of the NHLH2:SNORD116 diad. Indeed, use of an Nhlh2 mRNA construct containing this SNV significantly reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes.

Deficiency of Tristetraprolin Triggers Hyperthermia through Enhancing Hypothalamic Inflammation.

Tristetraprolin (TTP), an RNA-binding protein, controls the stability of RNA by capturing AU-rich elements on their target genes. It has recently been identified that TTP serves as an anti-inflammatory protein by guiding the unstable mRNAs of pro-inflammatory proteins in multiple cells. However, it has not yet been investigated whether TTP affects the inflammatory responses in the hypothalamus. Since hypothalamic inflammation is tightly coupled to the disturbance of energy homeostasis, we designed the current study to investigate whether TTP regulates hypothalamic inflammation and thereby affects energy metabolism by utilizing TTP-deficient mice. We observed that deficiency of TTP led to enhanced hypothalamic inflammation via stimulation of a variety of pro-inflammatory genes. In addition, microglial activation occurred in the hypothalamus, which was accompanied by an enhanced inflammatory response. In line with these molecular and cellular observations, we finally confirmed that deficiency of TTP results in elevated core body temperature and energy expenditure. Taken together, our findings unmask novel roles of hypothalamic TTP on energy metabolism, which is linked to inflammatory responses in hypothalamic microglial cells.

De novo 3D models of SARS-CoV-2 RNA elements from consensus experimental secondary structures.

The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta's FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5' UTR; the reverse complement of the 5' UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3' UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m and 3' UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets ('FARFAR2-SARS-CoV-2', https://github.com/DasLab/FARFAR2-SARS-CoV-2; and 'FARFAR2-Apo-Riboswitch', at https://github.com/DasLab/FARFAR2-Apo-Riboswitch') include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.

Berberine inhibits colorectal tumor growth by suppressing SHH secretion.

Hedgehog plays an important role in a wide range of physiological and pathological conditions. Paracrine activation of Hedgehog pathway in stromal cells increases the expression of VEGF, which promotes neovascularization in colorectal cancer and ultimately the growth of colorectal cancer. Berberine (BBR) has anticancer activity. In this study we investigated whether BBR inhibited the growth of colon cancer through suppressing the paracrine sonic hedgehog (SHH) signaling in vitro and in vivo. We showed that BBR (1-10 µM) dose-dependently inhibited the secretion and expression of SHH protein in HT-29 and SW480 cells. BBR did not influence the transcription of SHH, but promoted the degradation of SHH mRNA, thus decreased the SHH mRNA expression in the colorectal cancer cells. In nude mice bearing HT-29 xenograft, oral administration of BBR (100 mg · kg-1 · d-1) or a positive control drug GDC-0449 (100 mg · kg-1 · d-1) for 4 weeks markedly suppressed the growth of HT-29 tumor with BBR exhibiting a better antitumor efficacy. The tumor growth inhibition caused by BBR or GDC-0449 was comparable to their respective inhibitory effect on the mouse-specific Gli mRNA expression in the tumor. However, BBR (20 µM) did not affect the expression of human transcription factor Gli1 mRNA in HT-29 and SW480 cells. In conclusion, BBR promotes the degradation of SHH mRNA in colorectal cancer cells, interrupting the paracrine Hedgehog signaling pathway activity thus suppresses the colorectal cancer growth. This study reveals a novel molecular mechanism underlying the anticancer action of BBR.

LncRNA PCAT6 Regulated by YY1 Accelerates the Progression of Glioblastoma via miR-513/IGF2BP1.

Glioblastoma is one of the most frequent and aggressive primary tumor of glial brain tumors. Long non-coding RNA Prostate cancer-associated ncRNA transcript 6 (PCAT6) has been identified to influence the progression of many cancers, but its expression and functions in glioblastoma remain unclear. In this study, we intended to investigate the expression, functions and the corresponding mechanisms of PCAT6 in glioblastoma. We observed that PCAT6 expression was upregulated in glioblastoma tissues and cell lines and its high expression was due to the transcriptional activation by Yin Yang 1. miR-513 was a target of PCAT6 and Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) was a target of miR-513. Hence, PCAT6 upregulated IGF2BP1 expression via miR-513 in a competing endogenous RNAs manner. PCAT6 and IGF2BP1 functioned as oncogenes while miR-513 acted as a tumor suppressor gene in glioblastoma. PCAT6 and miR-513 modulated the proliferation and survival of glioblastoma cells via AKT signaling by mediating IGF2BP1. IGF2BP1 raised the expression of PCAT6 by increasing its stability. In conclusion, our results indicate that PCAT6/miR-513/IGF2BP1 positive feedback loop plays a crucial role in facilitating glioblastoma progression.

smi-miR396b targeted SmGRFs, SmHDT1, and SmMYB37/4 synergistically regulates cell growth and active ingredient accumulation in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: MIR396b had been cloned and overexpressed in Salvia miltiorrhiza hairy roots. MiR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to regulate cell growth and secondary metabolism in S. miltiorrhiza hairy roots. Danshen (Salvia miltiorrhiza Bunge) is a valuable medicinal herb with two kinds of clinically used natural products, salvianolic acids and tanshinones. miR396 is a conserved microRNA and plays extensive roles in plants. However, it is still unclear how miR396 works in S. miltiorrhiza. In this study, an smi-MIR396b has been cloned from S. miltiorrhiza. Overexpression of miR396b in danshen hairy roots inhibited hairy root growth, reduced salvianolic acid concentration, but enhanced tanshinone accumulation, resulting in the biomass and total salvianolic acids respectively reduced to 55.5 and 72.1% of the control and total tanshinones increased up to 1.91-fold of the control. Applied degradome sequencing, 5'RLM-RACE, and qRT-PCR, 13 targets for miR396b were identified including seven conserved SmGRF1-7 and six novel ones. Comparative transcriptomics and microRNomics analysis together with qRT-PCR results confirmed that miR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to mediate the phytohormone, especially gibberellin signaling pathways and consequentially resulted in the phenotype variation of miR396b-OE hairy roots. Furthermore, miR396b could be activated by methyl jasmonate, abscisic acid, gibberellin, salt, and drought stresses. The findings in this study indicated that smi-miR396b acts as an upstream and central regulator in cell growth and the biosynthesis of tanshinones and salvianolic acids, shedding light on the coordinated regulation of plant growth and biosynthesis of active ingredients in S. miltiorrhiza.

IL-10 regulates the malignancy of hemangioma-derived endothelial cells via regulation of PCNA.

Hemangioma (HA) is the most common benign tumor and formed by the proliferating endothelial cells of blood vessels. Interleukins (ILs) have been reported to be critical for HA progression. Our present study found that the expression of IL-10 was decreased in HA cells and tissues as compared to their corresponding controls. Treatment with recombinant IL-10 (rIL-10) can suppress the proliferation of HA cells via suppression of proliferating cell nuclear antigen (PCNA), while over expression of PCNA can attenuate rIL-10-inhibited cell proliferation. Further, rIL-10 can decrease the promoter activity and mRNA stability of PCNA in HA cells. Mechanistically, rIL-10 can increase expression of miR-27b-3p to decrease mRNA stability of PCNA, while down regulation of YY1 is involved in rIL-10 suppressed transcription of PCNA. Collectively, IL-10 can suppress the expression of PCNA via miR-27b-3p mediated suppression of mRNA stability and YY1 mediated down regulation of transcription. It suggested that rIL-10 might be a potential therapeutic approach for HA development and progression.

Progress toward the development of the small molecule equivalent of small interfering RNA.

Given that many small molecules could bind to structured regions at sites that will not affect function, approaches that trigger degradation of RNA could provide a general way to affect biology. Indeed, targeted RNA degradation is an effective strategy to selectively and potently modulate biology. We describe several approaches to endow small molecules with the power to cleave RNAs. Central to these strategies is Inforna, which designs small molecules targeting RNA from human genome sequence. Inforna deduces the uniqueness of a druggable pocket, enables generation of hypotheses about functionality of the pocket, and defines on- and off-targets to drive compound optimization. RNA-binding compounds are then converted into cleavers that degrade the target directly or recruit an endogenous nuclease to do so. Cleaving compounds have significantly contributed to understanding and manipulating biological functions. Yet, there is much to be learned about how to affect human RNA biology with small molecules.

Cephalotaxine inhibits Zika infection by impeding viral replication and stability.

The Zika virus (ZIKV) is a mosquito-borne flavivirus that has reemerged as a serious public health problem around the world. Syndromes of infected people range from asymptomatic infections to severe neurological disorders, such as Guillain-Barré syndrome and microcephaly. Screening anti-ZIKV drugs derived from Chinese medicinal herbs is one method of identifying antiviral agents. In this paper, we report that (1) Cephalotaxine (CET), an alkaloid isolated from Cephalotaxus drupacea, was effective in inhibiting ZIKV activity in vitro (i.e., in Vero and A549 cell lines) and (2) the mechanisms which underlie these effects involve virucidal activity and a decrease in viral replication. Specifically, CET was found to decrease ZIKV RNA and viral protein expression, inhibit ZIKV replication, and inhibit ZIKV mRNA/protein production. We also determined that CET is effective in inhibiting dengue virus 1-4 (DENV1-4). Taken together, our findings indicate that CET could be an effective lead compound in the treatment of ZIKV and also suggest that further investigation and development of CET-derived drugs may lead to a new class of anti-Flavivirus medications.

Unfolded protein-independent IRE1 activation contributes to multifaceted developmental processes in Arabidopsis.

In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay under ER stress, whereas the mutant does not exhibit severe developmental defects under normal conditions. In this study, we focused on the Arabidopsis IRE1C gene, whose product lacks a sensor domain. We found that the ire1a/b/c triple mutant is lethal, and heterozygous IRE1C (ire1c/+) mutation in the ire1a/b mutants resulted in growth defects and reduction of the number of pollen grains. Genetic analysis revealed that IRE1C is required for male gametophyte development in the ire1a/b mutant background. Expression of a mutant form of IRE1B that lacks the luminal sensor domain (ΔLD) complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the ΔLD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate regulated IRE1-dependent decay but not bZIP60 splicing. These observations suggest that IRE1 contributes to plant development, especially male gametogenesis, using an alternative activation mechanism that bypasses the unfolded protein-sensing luminal domain.

Molecular mechanism of tumour necrosis factor alpha regulates hypocretin (orexin) expression, sleep and behaviour.

Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF-α) is an important neuroinflammation mediator. Here, we examined the effects of TNF-α treatment on hypocretin expression in vivo and behaviour in mice. TNF-α decreased hypocretin 1 and hypocretin 2 expression in a dose-dependent manner in cultured hypothalamic neurons. TNF-α decreased mRNA stability of prepro-hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF-α demonstrated decreased expression of prepro-hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF-α, prepro-hypocretin mRNA decay was increased in hypothalamus. TNF-α neutralizing antibody restored the expression of prepro-hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF-α challenged mice, supporting hypocretin system can be impaired by increased TNF-α through decreasing hypocretin expression. Repeated TNF-α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF-α neutralizing antibody blocked the effects of TNF-α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF-α. The data support that TNF-α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

Thymoquinone-Induced Tristetraprolin Inhibits Tumor Growth and Metastasis through Destabilization of MUC4 mRNA.

Tristetraprolin (TTP), a well-characterized AU-rich element (ARE) binding protein, functions as a tumor suppressor gene. The purpose of this study was to investigate whether a bioactive substance derived from a natural medicinal plant affects the induction of TTP and to elucidate its mechanism. We examined the effects of natural bioactive materials including Resveratrol (RSV), thymoquinone (TQ) and curcumin on the expression of TTP in cancer cell. TQ derived from a natural plant Nigella sativa increased the expression levels of TTP mRNA and proteins in a dose-dependent manner in gastric and breast cancer cells. TQ-induced TTP increased the instability of MUC4 mRNA by direct binding of TTP to ARE in the 3'UTR of MUC4 mRNA. The induction of TTP by TQ also reduced the proliferation, migration and invasion of cancer cells. The expression of the epithelial-mesenchymal (EMT)-related genes, which were target genes of TTP, was also decreased by the TQ treatment. In the in vivo experiments using mouse melanoma cells, TQ-induced TTP inhibited metastasis of tumor cells. We have found that TQ-induced TTP might inhibit metastasis by reducing tumor cell migration and invasion through destabilization of MUC4 mRNA, which suggest the MUC4 as a novel target to TTP.

Selection of reference genes for qPCR normalization in buffalobur (Solanum rostratum Dunal).

Buffalobur (Solanum rostratum Dunal), which belongs to the Solanaceae family, is a worldwide noxious invasive weed and is listed as one of the top 10 alien invasive species in China. It is harmful to humans and livestock because the entire plant is covered with spines containing toxins. Many studies have analysed the gene expression in this weed species under different stress conditions using quantitative real-time PCR (qPCR). However, until now, there has been no report on suitable reference genes in buffalobur. Herein, 14 candidate reference genes were selected and evaluated for their expression stability in buffalobur in different tissues, at different developmental stages, and in response to several stress conditions using the geNorm, NormFinder, BestKeeper and RefFinder statistical algorithms. The results showed that EF1α, ACT and SAND are suitable reference genes across all samples tested. We recommend the normalization of target gene expression under different experimental conditions using these three genes together. Validation of selected reference genes was achieved by assessing the relative expression levels of P5CS and GI. This work identified the appropriate reference genes for transcript normalization in buffalobur, which will be helpful in future genetic studies of this invasive weed.

The anti-angiogenic effect of tryptanthrin is mediated by the inhibition of apelin promoter activity and shortened mRNA half-life in human vascular endothelial cells.

BACKGROUND: Anti-angiogenesis is an important strategy of psoriasis treatment, but the side effects of systemic agents remain difficult to overcome. Topical use of indigo naturalis ointment has been proved to improve the skin lesion of psoriasis effectively and safely and one of its major components, tryptanthrin, has been demonstrated to have anti-angiogenic effect. Apelin, which has been reported to act as an angiogenic factor that could stimulate the proliferation and migration of vascular endothelial cells and proved to be elevated in psoriasis patients, is a potential target of anti-angiogenic therapy. PURPOSE: We aim to find out if tryptanthrin works on the apelin pathway and study its anti-angiogenic mechanism. STUDY DESIGN: Human umbilical vein endothelial cells (HUVECs) were used as the in vitro model. METHODS: The effect of tryptanthrin on the expression of apelin and its receptor, APJ, was examined. The mRNA stability, promoter activity, and bioactivity of apelin, were also investigated. Migration and tube formation assay were used to evaluate the relationship between tryptanthrin and apelin. PD98059 and wortmannin were used to study the role of ERK1/2 MAPK and PI3K in apelin signaling pathway. RESULTS: We demonstrated that tryptanthrin could inhibit the expression of apelin, attenuated the stability of apelin mRNA, and significantly inhibited the apelin promoter activity. The addition of apelin-13 restored the suppression of tube formation and migration by tryptanthrin. Both PD98059 and wortmannin could down-regulate the apelin mRNA expression suggesting the important signaling role of ERK1/2 MAPK and PI3K in the gene expression of apelin. CONCLUSION: The anti-angiogenic effect of tryptanthrin was mediated by down-regulating apelin gene expression through suppression of promoter activity and decrease of mRNA stability in human vascular endothelial cells.

Targeting RNA: A Transformative Therapeutic Strategy.

The therapeutic pathways that modulate transcription mechanisms currently include gene knockdown and splicing modulation. However, additional mechanisms may come into play as more understanding of molecular biology and disease etiology emerge. Building on advances in chemistry and delivery technology, oligonucleotide therapeutics is emerging as an established, validated class of drugs that can modulate a multitude of genetic targets. These targets include over 10,000 proteins in the human genome that have hitherto been considered undruggable by small molecules and protein therapeutics. The approval of five oligonucleotides within the last 2 years elicited unprecedented excitement in the field. However, there are remaining challenges to overcome and significant room for future innovation to fully realize the potential of oligonucleotide therapeutics. In this review, we focus on the translational strategies encompassing preclinical evaluation and clinical development in the context of approved oligonucleotide therapeutics. Translational approaches with respect to pharmacology, pharmacokinetics, cardiac safety evaluation, and dose selection that are specific to this class of drugs are reviewed with examples. The mechanism of action, chemical evolution, and intracellular delivery of oligonucleotide therapies are only briefly reviewed to provide a general background for this class of drugs.