HMDD v4.0: a database for experimentally supported human microRNA-disease associations.

MicroRNAs (miRNAs) are a class of important small non-coding RNAs with critical molecular functions in almost all biological processes, and thus, they play important roles in disease diagnosis and therapy. Human MicroRNA Disease Database (HMDD) represents an important and comprehensive resource for biomedical researchers in miRNA-related medicine. Here, we introduce HMDD v4.0, which curates 53530 miRNA-disease association entries from literatures. In comparison to HMDD v3.0 released five years ago, HMDD v4.0 contains 1.5 times more entries. In addition, some new categories have been curated, including exosomal miRNAs implicated in diseases, virus-encoded miRNAs involved in human diseases, and entries containing miRNA-circRNA interactions. We also curated sex-biased miRNAs in diseases. Furthermore, in a case study, disease similarity analysis successfully revealed that sex-biased miRNAs related to developmental anomalies are associated with a number of human diseases with sex bias. HMDD can be freely visited at http://www.cuilab.cn/hmdd.

LncRNADisease v3.0: an updated database of long non-coding RNA-associated diseases.

Systematic integration of lncRNA-disease associations is of great importance for further understanding their underlying molecular mechanisms and exploring lncRNA-based biomarkers and therapeutics. The database of long non-coding RNA-associated diseases (LncRNADisease) is designed for the above purpose. Here, an updated version (LncRNADisease v3.0) has curated comprehensive lncRNA (including circRNA) and disease associations from the burgeoning literatures. LncRNADisease v3.0 exhibits an over 2-fold increase in experimentally supported associations, with a total of 25440 entries, compared to the last version. Besides, each lncRNA-disease pair is assigned a confidence score based on experimental evidence. Moreover, all associations between lncRNAs/circRNAs and diseases are classified into general associations and causal associations, representing whether lncRNAs or circRNAs can directly lead to the development or progression of corresponding diseases, with 15721 and 9719 entries, respectively. In a case study, we used the data of LncRNADisease v3.0 to calculate the phenotypic similarity between human and mouse lncRNAs. This database will continue to serve as a valuable resource for potential clinical applications related to lncRNAs and circRNAs. LncRNADisease v3.0 is freely available at http://www.rnanut.net/lncrnadisease.

A BSL-2 compliant mouse model of SARS-CoV-2 infection for efficient and convenient antiviral evaluation.

Animal models of authentic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require operation in biosafety level 3 (BSL-3) containment. In the present study, we established a mouse model employing a single-cycle infectious virus replicon particle (VRP) system of SARS-CoV-2 that can be safely handled in BSL-2 laboratories. The VRP [ΔS-VRP(G)-Luc] contains a SARS-CoV-2 genome in which the spike gene was replaced by a firefly luciferase (Fluc) reporter gene (Rep-Luci), and incorporates the vesicular stomatitis virus glycoprotein on the surface. Intranasal inoculation of ΔS-VRP(G)-Luc can successfully transduce the Rep-Luci genome into mouse lungs, initiating self-replication of Rep-Luci and, accordingly, inducing acute lung injury mimicking the authentic SARS-CoV-2 pathology. In addition, the reporter Fluc expression can be monitored using a bioluminescence imaging approach, allowing a rapid and convenient determination of viral replication in ΔS-VRP(G)-Luc-infected mouse lungs. Upon treatment with an approved anti-SARS-CoV-2 drug, VV116, the viral replication in infected mouse lungs was significantly reduced, suggesting that the animal model is feasible for antiviral evaluation. In summary, we have developed a BSL-2-compliant mouse model of SARS-CoV-2 infection, providing an advanced approach to study aspects of the viral pathogenesis, viral-host interactions, as well as the efficacy of antiviral therapeutics in the future.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and pathogenic in humans; thus, research on authentic SARS-CoV-2 has been restricted to biosafety level 3 (BSL-3) laboratories. However, due to the scarcity of BSL-3 facilities and trained personnel, the participation of a broad scientific community in SARS-CoV-2 research had been greatly limited, hindering the advancement of our understanding on the basic virology as well as the urgently necessitated drug development. Previously, our colleagues Jin et al. had generated a SARS-CoV-2 replicon by replacing the essential spike gene in the viral genome with a Fluc reporter (Rep-Luci), which can be safely operated under BSL-2 conditions. By incorporating the Rep-Luci into viral replicon particles carrying vesicular stomatitis virus glycoprotein on their surface, and via intranasal inoculation, we successfully transduced the Rep-Luci into mouse lungs, developing a mouse model mimicking SARS-CoV-2 infection. Our model can serve as a useful platform for SARS-CoV-2 pathological studies and antiviral evaluation under BSL2 containment.

Deciphering gene contributions and etiologies of somatic mutational signatures of cancer.

Somatic mutational signatures (MSs) identified by genome sequencing play important roles in exploring the cause and development of cancer. Thus far, many such signatures have been identified, and some of them do imply causes of cancer. However, a major bottleneck is that we do not know the potential meanings (i.e. carcinogenesis or biological functions) and contributing genes for most of them. Here, we presented a computational framework, Gene Somatic Genome Pattern (GSGP), which can decipher the molecular mechanisms of the MSs. More importantly, it is the first time that the GSGP is able to process MSs from ribonucleic acid (RNA) sequencing, which greatly extended the applications of both MS analysis and RNA sequencing (RNAseq). As a result, GSGP analyses match consistently with previous reports and identify the etiologies for a number of novel signatures. Notably, we applied GSGP to RNAseq data and revealed an RNA-derived MS involved in deficient deoxyribonucleic acid mismatch repair and microsatellite instability in colorectal cancer. Researchers can perform customized GSGP analysis using the web tools or scripts we provide.

Defining the single base importance of human mRNAs and lncRNAs.

As the fundamental unit of a gene and its transcripts, nucleotides have enormous impacts on the gene function and evolution, and thus on phenotypes and diseases. In order to identify the key nucleotides of one specific gene, it is quite crucial to quantitatively measure the importance of each base on the gene. However, there are still no sequence-based methods of doing that. Here, we proposed Base Importance Calculator (BIC), an algorithm to calculate the importance score of each single base based on sequence information of human mRNAs and long noncoding RNAs (lncRNAs). We then confirmed its power by applying BIC to three different tasks. Firstly, we revealed that BIC can effectively evaluate the pathogenicity of both genes and single bases through single nucleotide variations. Moreover, the BIC score in The Cancer Genome Atlas somatic mutations is able to predict the prognosis of some cancers. Finally, we show that BIC can also precisely predict the transmissibility of SARS-CoV-2. The above results indicate that BIC is a useful tool for evaluating the single base importance of human mRNAs and lncRNAs.

NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells.

NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. However, its role in mammalian development remains unclear. Here, we found that NAT10 expression positively correlates with pluripotency in vivo and in vitro. High throughput ac4C-targeted RNA immunoprecipitation sequencing (ac4C-RIP-seq), NaCNBH3-based chemical ac4C sequencing (ac4C-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays revealed noticeable ac4C modifications in transcriptome of hESCs, among which transcripts encoding core pluripotency transcription factors are favorable targets of ac4C modification. Further validation assays demonstrate that genetic inactivation of NAT10, the ac4C writer enzyme, led to ac4C level decrease on target genes, promoted the core pluripotency regulator OCT4 (POU5F1) transcript decay, and finally impaired self-renewal and promoted early differentiation in hESCs. Together, our work presented here elucidates a previously unrecognized interconnectivity between the core pluripotent transcriptional network for the maintenance of human ESC self-renewal and NAT10-catalyzed ac4C RNA epigenetic modification.

Expanding the tolerance of segmented Influenza A Virus genome using a balance compensation strategy.

Reporter viruses provide powerful tools for both basic and applied virology studies, however, the creation and exploitation of reporter influenza A viruses (IAVs) have been hindered by the limited tolerance of the segmented genome to exogenous modifications. Interestingly, our previous study has demonstrated the underlying mechanism that foreign insertions reduce the replication/transcription capacity of the modified segment, impairing the delicate balance among the multiple segments during IAV infection. In the present study, we developed a "balance compensation" strategy by incorporating additional compensatory mutations during initial construction of recombinant IAVs to expand the tolerance of IAV genome. As a proof of concept, promoter-enhancing mutations were introduced within the modified segment to rectify the segments imbalance of a reporter influenza PR8-NS-Gluc virus, while directed optimization of the recombinant IAV was successfully achieved. Further, we generated recombinant IAVs expressing a much larger firefly luciferase (Fluc) by coupling with a much stronger compensatory enhancement, and established robust Fluc-based live-imaging mouse models of IAV infection. Our strategy feasibly expands the tolerance for foreign gene insertions in the segmented IAV genome, which opens up better opportunities to develop more versatile reporter IAVs as well as live attenuated influenza virus-based vaccines for other important human pathogens.

Paving new roads toward the advancement of broad-spectrum antiviral agents.

Broad-spectrum antivirals (BSAs) have the advantageous property of being effective against a wide range of viruses with a single drug, offering a promising therapeutic solution for the largely unmet need in treating both existing and emerging viral infections. In this review, we summarize the current strategies for the development of novel BSAs, focusing on either targeting the commonalities during the replication of multiple viruses or the systemic immunity of humans. In comparison to BSAs that target viral replication, these immuno-modulatory agents possess an expanded spectrum of antiviral activity. However, antiviral immunity is a double-edged sword, and maintaining immune homeostasis ultimately dictates the health status of hosts during viral infections. Therefore, establishing an ideal goal for immuno-modulation in antiviral interventions is crucial. Herein we propose a bionic approach for immuno-modulation inspired by mimicking bats, which possess a more robust immune system for combating viral invasions, compared to humans. In addition, we discuss an empirical approach to treat diverse viral infections using traditional Chinese medicines (TCMs), mainly through bidirectional immuno-modulation to restore the disrupted homeostasis. Advancing our understanding of both the immune system of bats and the mechanisms underlying antiviral TCMs will significantly contribute to the future development of novel BSAs.

Development of a virus-based affinity ultrafiltration method for screening virus-surface-protein-targeted compounds from complex matrixes: Herbal medicines as a case study.

Herbal medicines (HMs) are one of the main sources for the development of lead antiviral compounds. However, due to the complex composition of HMs, the screening of active compounds within these is inefficient and requires a significant time investment. We report a novel and efficient virus-based screening method for antiviral active compounds in HMs. This method involves the centrifugal ultrafiltration of viruses, known as the virus-based affinity ultrafiltration method (VAUM). This method is suitable to identify virus specific active compounds from complex matrices such as HMs. The effectiveness of the VAUM was evaluated using influenza A virus (IAV) H1N1. Using this method, four compounds that bind to the surface protein of H1N1 were identified from dried fruits of Terminalia chebula (TC). Through competitive inhibition assays, the influenza surface protein, neuraminidase (NA), was identified as the target protein of these four TC-derived compounds. Three compounds were identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS), and their anti-H1N1 activities were verified by examining the cytopathic effect (CPE) and by performing a virus yield reduction assay. Further mechanistic studies demonstrated that these three compounds directly bind to NA and inhibit its activity. In summary, we describe here a VAUM that we designed, one that can be used to accurately screen antiviral active compounds in HMs and also help improve the efficiency of screening antiviral drugs found in natural products.

Vitexin inhibits APEX1 to counteract the flow-induced endothelial inflammation.

Vascular endothelial cells are exposed to shear stresses with disturbed vs. laminar flow patterns, which lead to proinflammatory vs. antiinflammatory phenotypes, respectively. Effective treatment against endothelial inflammation and the consequent atherogenesis requires the identification of new therapeutic molecules and the development of drugs targeting these molecules. Using Connectivity Map, we have identified vitexin, a natural flavonoid, as a compound that evokes the gene-expression changes caused by pulsatile shear, which mimics laminar flow with a clear direction, vs. oscillatory shear (OS), which mimics disturbed flow without a clear direction. Treatment with vitexin suppressed the endothelial inflammation induced by OS or tumor necrosis factor-α. Administration of vitexin to mice subjected to carotid partial ligation blocked the disturbed flow-induced endothelial inflammation and neointimal formation. In hyperlipidemic mice, treatment with vitexin ameliorated atherosclerosis. Using SuperPred, we predicted that apurinic/apyrimidinic endonuclease1 (APEX1) may directly interact with vitexin, and we experimentally verified their physical interactions. OS induced APEX1 nuclear translocation, which was inhibited by vitexin. OS promoted the binding of acetyltransferase p300 to APEX1, leading to its acetylation and nuclear translocation. Functionally, knocking down APEX1 with siRNA reversed the OS-induced proinflammatory phenotype, suggesting that APEX1 promotes inflammation by orchestrating the NF-κB pathway. Animal experiments with the partial ligation model indicated that overexpression of APEX1 negated the action of vitexin against endothelial inflammation, and that endothelial-specific deletion of APEX1 ameliorated atherogenesis. We thus propose targeting APEX1 with vitexin as a potential therapeutic strategy to alleviate atherosclerosis.

Detection of diabetic patients in people with normal fasting glucose using machine learning.

BACKGROUND: Diabetes mellitus (DM) is a chronic metabolic disease that could produce severe complications threatening life. Its early detection is thus quite important for the timely prevention and treatment. Normally, fasting blood glucose (FBG) by physical examination is used for large-scale screening of DM; however, some people with normal fasting glucose (NFG) actually have suffered from diabetes but are missed by the examination. This study aimed to investigate whether common physical examination indexes for diabetes can be used to identify the diabetes individuals from the populations with NFG. METHODS: The physical examination data from over 60,000 individuals with NFG in three Chinese cohorts were used. The diabetes patients were defined by HbA1c ≥ 48 mmol/mol (6.5%). We constructed the models using multiple machine learning methods, including logistic regression, random forest, deep neural network, and support vector machine, and selected the optimal one on the validation set. A framework using permutation feature importance algorithm was devised to discover the personalized risk factors. RESULTS: The prediction model constructed by logistic regression achieved the best performance with an AUC, sensitivity, and specificity of 0.899, 85.0%, and 81.1% on the validation set and 0.872, 77.9%, and 81.0% on the test set, respectively. Following feature selection, the final classifier only requiring 13 features, named as DRING (diabetes risk of individuals with normal fasting glucose), exhibited reliable performance on two newly recruited independent datasets, with the AUC of 0.964 and 0.899, the balanced accuracy of 84.2% and 81.1%, the sensitivity of 100% and 76.2%, and the specificity of 68.3% and 86.0%, respectively. The feature importance ranking analysis revealed that BMI, age, sex, absolute lymphocyte count, and mean corpuscular volume are important factors for the risk stratification of diabetes. With a case, the framework for identifying personalized risk factors revealed FBG, age, and BMI as significant hazard factors that contribute to an increased incidence of diabetes. DRING webserver is available for ease of application ( http://www.cuilab.cn/dring ). CONCLUSIONS: DRING was demonstrated to perform well on identifying the diabetes individuals among populations with NFG, which could aid in early diagnosis and interventions for those individuals who are most likely missed.

Defining the functional divergence of orthologous genes between human and mouse in the context of miRNA regulation.

Animal models have a certain degree of similarity with human in genes and physiological processes, which leads them to be valuable tools for studying human diseases and for assisting drug development. However, translational researches adopting animal models are largely restricted by the species heterogeneity, which is also a major reason for the failure of drug research. Currently, computational method for exploring the functional differences between orthologous genes is still insufficient. For this purpose, here, we presented an algorithm, functional divergence score (FDS), by comprehensively evaluating the functional differences between the microRNAs regulating the paired orthologous genes. Given that mouse is one of the most popular model animals, currently, FDS was designed to dissect the functional divergence of orthologous genes between human and mouse. The results showed that gene FDS value is significantly associated with gene evolutionary characteristics and can discover expression divergence of human-mouse orthologous genes. Moreover, FDS performed well in distinguishing the targets of approved drugs and the failed ones. These results suggest that FDS is a valuable tool to evaluate the functional divergence of paired human and mouse orthologous genes. In addition, for each orthologous gene pair, FDS can provide detailed differences in functions and phenotypes. Our study provided a useful tool for quantifying the functional difference between human and mouse, and the presented framework is easily to be extended to the orthologous genes between human and other species. An online server of FDS is available at http://www.cuilab.cn/fds/.

Influenza virus cell entry and targeted antiviral development.

Influenza virus infection is currently one of the most prevalent and transmissible diseases in the world causing local outbreaks every year. It has the potential to cause devastating global pandemics as well. The development of anti-influenza drugs possessing novel mechanisms of action is urgently needed to control the spread of influenza infections; thus, drugs that inhibit influenza virus entry into target cells are emerging as a hot research topic. In addition to discussing the biological significance of hemagglutinin in viral replication, this article provides recent updates on the natural products, small molecules, proteins, peptides, and neutralizing antibody-like proteins that have anti-influenza potency.

Development of an HiBiT-tagged reporter H3N2 influenza A virus and its utility as an antiviral screening platform.

The balance of the segmented genome derived from naturally occurring influenza A viruses (IAVs) is delicate and vulnerable to foreign insertions, thus most reporter IAVs up to date are generated using the backbone of the laboratory-adapted strains. In this study, we constructed a reporter influenza A/H3N2 virus (A/NY-HiBiT) which was derived from a clinical isolate, by placing a minimized HiBiT tag to the N-terminus of the viral nuclear-export protein (NEP). Here, we show that this 11-amino acid HiBiT tag did not adversely impact the viral genome balance, and the recombinant A/NY-HiBiT virus maintains its relative stability. Moreover, the replication profile of the HiBiT-tagged virus can be measured by a simple Nano-Glo assay, providing a robust high-throughput screening (THS) platform. We used this platform to evaluate a collection of the pre-purified fractions which were derived from rare Chinese medicinal materials, and we identified three fractions, including wild Trametes robiniophila (50% methanol fraction), Ganoderma (water fraction), and wild Phellinus igniarius (ethyl acetate fraction), as potent anti-IAV actives. Our results demonstrate that this IAV reporter can be used as a powerful HTS platform for antiviral development.

Sulforaphane is a reversible covalent inhibitor of 3-chymotrypsin-like protease of SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a major public health threat worldwide and emphasizes an urgent need for effective therapeutics. Recently, Ordonez et al. identified sulforaphane (SFN) as a novel coronavirus inhibitor both in vitro and in mice, but the mechanism of action remains elusive. In this study, we independently discovered SFN for its inhibitory effect against SARS-CoV-2 using a target-based screening approach, identifying the viral 3-chymotrypsin-like protease (3CLpro ) as a target of SFN. Mechanistically, SFN inhibits 3CLpro in a reversible, mixed-type manner. Moreover, enzymatic kinetics studies reveal that SFN is a slow-binding inhibitor, following a two-step interaction. Initially, an encounter complex forms by specific binding of SFN to the active pocket of 3CLpro ; subsequently, the isothiocyanate group of SFN as "warhead" reacts covalently to the catalytic cysteine in a slower velocity, stabilizing the SFN-3CLpro complex. Our study has identified a new lead of the covalent 3CLpro inhibitors which has potential to be developed as a therapeutic agent to treat SARS-CoV-2 infection.

Licochalcone A plays dual antiviral roles by inhibiting RSV and protecting against host damage.

Respiratory syncytial virus (RSV) causes lower respiratory tract diseases and bronchiolitis in children and elderly individuals. There are no effective drugs currently available to treat RSV infection. In this study, we report that Licochalcone A (LCA) can inhibit RSV replication and mitigate RSV-induced cell damage in vitro, and that LCA exerts a protective effect by reducing the viral titer and inflammation in the lungs of infected mice in vivo. We suggest that the mechanism of action occurs through pathways of antioxidant stress and inflammation. Further mechanistic results demonstrate that LCA can induce nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus, activate heme oxygenase 1 (HO-1), and inhibit reactive oxygen species-induced oxidative stress. LCA also works to reverse the decrease in I-kappa-B-alpha (IкBα) levels caused by RSV, which in turn inhibits inflammation through the associated nuclear factor kappa B and tumor necrosis factor-α signaling pathways. The combined action of the two cross-talking pathways protects hosts from RSV-induced damage. To conclude, our study is the first of its kind to establish evidence of LCA as a viable treatment for RSV infection.

Discovery of ligustrazine and chalcone derivatives as novel viral nucleoprotein nuclear export inhibitors against influenza viruses.

Influenza viruses pose a significant threat to human health worldwide due to seasonal epidemics and occasional global pandemics. These viruses can cause severe upper respiratory tract infections that contribute to high morbidity and mortality rates. The emergence of drug-resistant influenza viruses has created the need for the development of novel broad-spectrum antivirals. Here, we present a novel anti-influenza agent with new targets and mechanisms of action to address this problem. Our findings led to the discovery of a novel influenza virus inhibitor, a ligustrazine derivative known as A9. We have found that it exhibits broad-spectrum antiviral properties against influenza A and B viruses (IAV and IBV, respectively), including oseltamivir-resistant strain. Through multiple bioassays such as time-of-addition assay, indirect immunofluorescence assay, and nuclear-cytoplasmic fractionation assay, we demonstrated that A9 inhibits the nuclear export of the viral ribonucleoprotein (vRNP). Furthermore, escape mutant analyses and affinity studies determined by surface plasmon resonance indicated that A9 specifically targets the nucleoprotein. In addition, four chalcone derivatives developed from A9 (B14, B29, B31, and B32), were found to effectively inhibit the replication of influenza virus through the same mechanism of action. In this manuscript we highlight A9 and its four derivatives as potential leads for the treatment of IAV and IBV infections, and their unique and novel mechanism of action probable benefit the field of anti-influenza drug discovery.

Hepatocellular cystathionine γ lyase/hydrogen sulfide attenuates nonalcoholic fatty liver disease by activating farnesoid X receptor.

BACKGROUND AND AIMS: Hydrogen sulfide (H2 S) plays a protective role in NAFLD. However, whether cystathionine γ lyase (CSE), a dominant H2 S generating enzyme in hepatocytes, has a role in the pathogenesis of NAFLD is currently unclear. APPROACH AND RESULTS: We showed that CSE protein expression is dramatically downregulated, especially in fibrotic areas, in livers from patients with NAFLD. In high-fat diet (HFD)-induced NAFLD mice or an oleic acid-induced hepatocyte model, the CSE/H2 S pathway is also downregulated. To illustrate a regulatory role for CSE in NAFLD, we generated a hepatocyte-specific CSE knockout mouse (CSELKO ). Feeding an HFD to CSELKO mice, they showed more hepatic lipid deposition with increased activity of the fatty acid de novo synthesis pathway, increased hepatic insulin resistance, and higher hepatic gluconeogenic ability compared to CSELoxp control mice. By contrast, H2 S donor treatment attenuated these phenotypes. Furthermore, the protection conferred by H2 S was blocked by farnesoid X receptor (FXR) knockdown. Consistently, serum deoxycholic acid and lithocholic acid (FXR antagonists) were increased, and tauro-ß-muricholic acid (FXR activation elevated) was reduced in CSELKO . CSE/H2 S promoted a post-translation modification (sulfhydration) of FXR at Cys138/141 sites, thereby enhancing its activity to modulate expression of target genes related to lipid and glucose metabolism, inflammation, and fibrosis. Sulfhydration proteomics in patients' livers supported the CSE/H2 S modulation noted in the CSELKO mice. CONCLUSIONS: FXR sulfhydration is a post-translational modification affected by hepatic endogenous CSE/H2 S that may promote FXR activity and attenuate NAFLD. Hepatic CSE deficiency promotes development of nonalcoholic steatohepatitis. The interaction between H2 S and FXR may be amenable to therapeutic drug treatment in NAFLD.

Parthenolide inhibits proliferation of cells infected with Kaposi's sarcoma-associated herpesvirus by suppression of the NF-κB signaling pathway.

The disease caused by Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the major causes of death of individuals with acquired immunodeficiency syndrome (AIDS). Development of anti-KSHV drugs is thus crucial. In this study, we investigated the effect of parthenolide (PTL) on the proliferation and NF-κB signaling pathway of KSHV-infected cells. iSLK.219 and KSHV-infected SH-SY5Y cells (SK-RG) were treated with PTL, TaqMan real-time quantitative PCR was used to determine the number of copies of the KSHV genome, and mRNA and protein expression of KSHV genes were analyzed by real-time PCR and immunocytochemistry. A cell viability test was used to measure cell proliferation, and flow cytometry was used to examine the effect of the drug on the cell cycle. Cyclin D1, CDK6, CDK4, and NF-κB-related proteins, including IKKß, P-p65, and P-IKB-α, were detected by Western blot. The results showed that PTL altered the morphology of the cells, reduced the KSHV copy number, and suppressed the production of ORF50, K8.1, and v-GPCR mRNA and the LANA, ORF50, and K8.1 proteins. It blocked the G1 phase in iSLK.219 cells and decreased the levels of cyclin D1, CDK6, and CDK4 as well as the levels of NF-κB signaling proteins, including IKKß, P-p65, and P-IKB-α. Together, these results suggest that PTL is a candidate drug that can decrease KSHV pathogenicity by suppressing cell proliferation and inhibiting the NF-κB signaling pathway in KSHV-infected cells.

Novel roles of an intragenic G-quadruplex in controlling microRNA expression and cardiac function.

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.