Aptamer‐conjugated mesoporous polydopamine for docetaxel targeted delivery and synergistic photothermal therapy of prostate cancer

ObjectivesIt is imperative to develop efficient strategies on the treatment of prostate cancer. Here, we constructed multifunctional nanoparticles, namely AS1411@MPDA‐DTX (AMD) for targeted and synergistic chemotherapy/photothermal therapy of prostate cancer.Materials and MethodsMesoporous polydopamine (MPDA) nanoparticles were prepared by a one‐pot synthesis method, DTX was loaded through incubation, and AS1411 aptamer was modified onto MPDA by the covalent reaction. The prepared nanoparticles were characterized by ultra‐micro spectrophotometer, Fourier transform infrared spectra, transmission electron microscope, and so on. The targeting ability was detected by selective uptake and cell killing. The mechanism of AMD‐mediated synergistic therapy was detected by Western blot and immunofluorescence.ResultsThe prepared nanoparticles can be easily synthesized and possessed excellent water solubility, stability, and controlled drug release ability, preferentially in acidic context. Based on in vitro and in vivo results, the nanoparticles can efficiently target prostate cancer cells, promote DTX internalization, and enhance the antitumor effects of chemo‐photothermal therapy strategies under the NIR laser irradiation.ConclusionsAs a multifunctional nanoplatform, AS1411@MPDA‐DTX could efficiently target prostate cancer cells, promote DTX internalization, and synergistically enhance the antiprostate cancer efficiency by combining with NIR irradiation.     

SQLE facilitates the pancreatic cancer progression via the lncRNA‐TTN‐AS1/miR‐133b/SQLE axis

Studies have shown that SQLE is highly expressed in a variety of tumours and promotes tumour progression. However, the role of SQLE in pancreatic cancer (PC) has not been reported. Here, we aim to study the role and molecular mechanism of SQLE in PC. Immunohistochemistry and functional experiments showed that SQLE was highly expressed in PC tissues and promoted the proliferation and invasion of PC cells. Terbinafine, an inhibitor of SQLE, inhibited this effect. In order to further study the upstream mechanism that regulates SQLE, we used bioinformatics technology to lock miR‐133b and lncRNA‐TTN‐AS. In situ hybridization was used to detect the expression of miR‐133b and lncRNA‐TTN‐AS1 in PC tissues. The luciferase reporter gene experiment was used to confirm the binding of miR‐133b and lncRNA‐TTN‐AS1. The results showed that miR‐133b was down‐regulated in PC tissues and negatively correlated with the expression of SQLE. LncRNA‐TTN‐AS1 was upregulated in pancreatic cancer tissues and positively correlated with the expression of SQLE. Luciferase gene reporter gene analysis confirmed lncRNA‐TTN‐AS1 directly binded to miR‐133b. Therefore, we propose that targeting the lncRNA‐TTN‐AS1/miR‐133b/SQLE axis is expected to provide new ideas for the clinical treatment of PC patients.     

Neutralization of SARS‐CoV‐2 by highly potent,hyperthermostable, and mutation‐tolerant nanobodies

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.     

m6A‐mediated alternative splicing coupled with nonsense‐mediated mRNA decay regulates SAM synthetase homeostasis

Alternative splicing of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS‐NMD) in an organism, we performed long‐read RNA sequencing of poly(A)⁺ RNAs from an NMD‐deficient mutant strain of Caenorhabditis elegans, and obtained full‐length sequences for mRNA isoforms from 259 high‐confidence AS‐NMD genes. Among them are the S‐adenosyl‐L‐methionine (SAM) synthetase (sams) genes sams‐3 and sams‐4. SAM synthetase activity autoregulates sams gene expression through AS‐NMD in a negative feedback loop. We furthermore find that METT‐10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3′ splice site (3′SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m⁶A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m⁶A modification at the 3′SS of the sams genes.     

Characterization and prognostic significance of alternative splicing events in lower‐grade diffuse gliomas

Alternative splicing (AS) is assumed to play important roles in the progression and prognosis of cancer. Currently, the comprehensive analysis and clinical relevance of AS in lower‐grade diffuse gliomas have not been systematically addressed. Here, we gathered alternative splicing data of lower‐grade diffuse gliomas from SpliceSeq. Based on the Percent Spliced In (PSI) values of 515 lower‐grade diffuse glioma patients from the Cancer Genome Atlas (TCGA), we performed subtype‐differential AS analysis and consensus clustering to determine robust clusters of patients. A total of 48 050 AS events in 10 787 genes in lower‐grade diffuse gliomas were profiled. Subtype‐differential splicing analysis and functional annotation revealed that spliced genes were significantly enriched in numerous cancer‐related biological phenotypes and signalling pathways. Consensus clustering using AS events identified three robust clusters of patients with distinguished pathological and prognostic features. Moreover, each cluster was also associated with distinct genomic alterations. Finally, we developed and validated an AS‐related signature with Cox proportional hazards model. The signature, significantly associated with clinical and molecular features, could serve as an independent prognostic factor for lower‐grade diffuse gliomas. Thus, our results indicated that AS events could discriminate molecular subtypes and have prognostic impact in lower‐grade diffuse gliomas.     

Insulin binding to the analytical antibody sandwich pair OXI‐005 and HUI‐018: Epitope mapping and binding properties

The insulin epitopes for two monoclonal antibodies (mAbs), OXI‐005 and HUI‐018, commonly used in combination for insulin concentration determination in sandwich assays, were determined using X‐ray crystallography. The crystal structure of the HUI‐018 Fab in complex with human insulin (HI) was determined and OXI‐005 Fab crystal structures were determined in complex with HI and porcine insulin (PI) as well as on its own. The OXI‐005 epitope comprises insulin residues 1,3,4,19–21 (A‐chain) and 25–30 (B‐chain) and for HUI‐018 residues 7,8,10–14,17 (A‐chain) and 5–7, 10, 14 (B‐chain). The areas of insulin involved in interactions with the mAb are 20% (OXI‐005) and 24% (HUI‐018) of the total insulin surface. Based on the Fab complex crystal structures with the insulins a molecular model for simultaneous binding of the Fabs to PI was built and this model was validated by small angle X‐ray scattering measurements for the ternary complex. The epitopes for the mAbs on insulin were found well separated from each other as expected from luminiscent oxygen channeling immunoassay results for different insulins (HI, PI, bovine insulin, DesB30 HI, insulin glargine, insulin lispro). The affinities of the OXI‐005 and HUI‐018 Fabs for HI, PI, and DesB30 HI were determined using surface plasmon resonance. The K _Ds were found to be in the range of 1–4 nM for the HUI‐018 Fab, while more different for the OXI‐005 Fab (50 nM for HI, 20 nM for PI and 400 nM for DesB30 HI) supporting the importance of residue B30 for binding to OXI‐005.     

Dual impacts of lncRNA XIST and lncRNA SNHG5 on inflammatory reaction and apoptosis of endothelial cells via regulating miR‐155/CARHSP1 axis

Considering the significance of lncRNA/miRNA axis in explaining atherosclerosis (AS) progression, this investigation was intended to clarify whether lncRNAs XIST/SNHG5 would regulate AS aetiology by sponging miR‐155, an AS‐promoting molecule. We altogether recruited 367 patients who were examined by coronary angiography, and meanwhile, human coronary artery endothelial cells (HCAECs) were purchased to establish cells models via ox‐LDL treatment. The study results indicated that lowly expressed XIST/SNHG5 and highly expressed miR‐155 were frequently detectable among AS patients who showed severe stenosis and possessed high triglyceride (TG), low‐density lipoprotein cholesterol (LDL‐C) and high‐sensitivity C‐reactive protein (hs‐CRP) levels. Besides, HCAECs treated by ox‐LDL released large amounts of inflammatory cytokines, and their apoptosis rate was also raised. Moreover, expressions of XIST and SNHG5 declined markedly within ox‐LDL‐treated HCAECs, whereas miR‐155 expression significantly ascended. Transfection of pcDNA‐XIST and pcDNA‐SNHG5 both reduced the expression of TNF‐α, IL‐6, IL‐8 and IL‐1β within HCAECs and also dampened the apoptotic tendency of HCAECs. Co‐treatment of pcDNA‐XIST and pcDNA‐SNHG5 produced a larger effect on HCAEC activity than pcDNA‐XIST or pcDNA‐SNHG5 alone. Furthermore, miR‐155, modified by XIST and SNHG5, was capable of reversing the impacts of XIST and SNHG5 on HCAEC activity. Eventually, CARHSP1 was activated by XIST and SNHG5, and its overexpression dwindled impacts of miR‐155 mimic on proliferation and inflammation response of HCAECs. In conclusion, targeting XIST and SNHG5 might be an ideal alternative in delaying AS progression, allowing for their repression of downstream miR‐155.     

Therapeutic effects of CXCR4+ subpopulation of transgene‐free induced cardiosphere‐derived cells on experimental myocardial infarction

ObjectivesMyocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor‐like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus‐based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene‐free approaches.Materials and MethodsFour transgene‐free approaches for somatic reprogramming, including episome, minicircle, self‐replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations.ResultsThe self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C‐X‐C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach.ConclusionThus, the optimized strategy for iCS generation is safer and has more therapeutic potentials.     

Cryo‐EM reveals mechanisms of angiotensin I‐converting enzyme allostery and dimerization

Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I‐converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X‐ray crystallography and molecular dynamics simulations. Here, we report the first cryo‐EM structures of full‐length, glycosylated, soluble sACE (sACE^S1211). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N‐ and C‐terminal domains of monomeric sACE^S1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N‐terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.     

miR‐126‐5p enhances radiosensitivity of lung adenocarcinoma cells by inhibiting EZH2 via the KLF2/BIRC axis

Radiotherapy is a common method for the treatment of lung adenocarcinoma, but it often fails due to the relative non‐susceptibility of lung adenocarcinoma cells to radiation. We aimed to discuss the related mechanisms by which miR‐126‐5p might mediate radiosensitivity of lung adenocarcinoma cells. The binding affinity between miR‐126‐5p and EZH2 and between KLF2 and BIRC5 was identified using multiple assays. A549 and H1650 cells treated with X‐ray were transfected with miR‐126‐5p mimic/inhibitor, oe‐EZH2, or si‐KLF2 to detect cell biological functions and radiosensitivity. Finally, lung adenocarcinoma nude mouse models were established. miR‐126‐5p and KLF2 were poorly expressed, while EZH2 and BIRC5 were upregulated in lung adenocarcinoma tissues and cells. miR‐126‐5p targeted EZH2 to promote the KLF2 expression so as to inhibit BIRC5 activation. Both in vitro and in vivo experiments verified that elevated miR‐126‐5p inhibited cell migration and promoted apoptosis to enhance the sensitivity of lung adenocarcinoma cells to radiotherapy via the EZH2/KLF2/BIRC5 axis. Collectively, miR‐126‐5p downregulated EZH2 to facilitate the sensitivity of lung adenocarcinoma cells to radiotherapy via KLF2/BIRC5.     

A new l‐arginine oxidase engineered from l‐glutamate oxidase

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l‐Glutamate oxidase (LGOX) from Streptomyces sp. X‐119‐6 catalyzes the oxidative deamination of l‐glutamate to produce 2‐ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l‐glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l‐glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l‐arginine. The oxidative deamination activity of LGOX to l‐arginine is higher than that of l‐arginine oxidase form from Pseudomonas sp. TPU 7192. X‐ray crystal structure analysis revealed that the guanidino group of l‐arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild‐type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l‐arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l‐arginine by LGOX R305E, is proportional to the concentration of l‐arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l‐arginine. Thus, LGOX R305E is suitable for the determination of l‐arginine.     

Toll‐7 promotes tumour growth and invasion in Drosophila

Objectives Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. Here, by using well‐established Drosophila tumour models, we identified Toll‐7 as a novel regulator of tumour growth and invasion.Materials and methodsTransgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Immunostaining and RT‐qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0.ResultsLoss of Toll‐7 suppresses Ras^V12/lgl ^−/−‐induced tumour growth and invasion, as well as cell polarity disruption‐induced invasive cell migration, whereas expression of a constitutively active allele of Toll‐7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr‐JNK signalling is necessary and sufficient for Toll‐7‐induced invasive cell migration. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll‐7 activates the EGFR‐Ras signalling, which cooperates with the Egr‐JNK signalling to promote Yki‐mediated cell proliferation and tissue overgrowth. Finally, Toll‐7 is necessary and sufficient for the proper maintenance of EGFR protein level.ConclusionsOur findings characterized Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion in Drosophila, which shed light on the pro‐tumour function of mammalian Toll‐like receptors (TLRs).     

LOXL1‐AS1 communicating with TIAR modulates vasculogenic mimicry in glioma via regulation of the miR‐374b‐5p/MMP14 axis

At present, growing evidence indicates that long non‐coding RNAs (lncRNAs) participate in the progression of glioma. The function of LOXL1‐AS1 in vasculogenic mimicry (VM) in glioma remains unclear. First, the expressions of TIAR, the lncRNA LOXL1‐AS1, miR‐374b‐5p and MMP14 were examined by qRT‐PCR and Western blot in both, glioma tissues and glioma cell lines. Proliferation, migration, invasion and tube formation assays were conducted to evaluate the roles of TIAR, LOXL1‐AS1, miR‐374b‐5p and MMP14 in malignant cellular behaviours in glioma cells. A nude mouse xenograft model and dual staining for CD34 and PAS were used to assess whether VM was affected by TIAR, LOXL1‐AS1 or miR‐374b‐5p in vivo. In this study, low levels of TIAR and high levels of LOXL1‐AS1 were found in glioma cells and tissues. TIAR downregulated the expression of LOXL1‐AS1 by destabilizing it. LOXL1‐AS1 acted like a miRNA sponge towards miR‐374b‐5p so that downregulation of the former greatly inhibited cell proliferation, migration, invasion and VM. Additionally, miR‐374b‐5p overexpression repressed malignant biological behaviours and VM in glioma by modifying MMP14. In summary, we demonstrated that TIAR combined with LOXL1‐AS1 modulates VM in glioma via the miR‐374b‐5p/MMP14 axis, revealing novel targets for glioma therapy.     

MicroRNA‐148a‐3p suppresses epithelial‐to‐mesenchymal transition and stemness properties via Wnt1‐mediated Wnt/β‐catenin pathway in pancreatic cancer

Although miR‐148a‐3p has been reported to function as a tumour suppressor in various cancers, the molecular mechanism of miR‐148a‐3p in regulating epithelial‐to‐mesenchymal transition (EMT) and stemness properties of pancreatic cancer (PC) cells remains to be elucidated. In the present study, we demonstrated that miR‐148a‐3p expression was remarkably down‐regulated in PC tissues and cell lines. Moreover, low expression of miR‐148a‐3p was associated with poorer overall survival (OS) in patients with PC. In vitro, gain‐of‐function and loss‐of‐function experiments showed that miR‐148a‐3p suppressed EMT and stemness properties as well as the proliferation, migration and invasion of PC cells. A dual‐luciferase reporter assay demonstrated that Wnt1 was a direct target of miR‐148a‐3p, and its expression was inversely associated with miR‐148a‐3p in PC tissues. Furthermore, miR‐148a‐3p suppressed the Wnt/β‐catenin pathway via down‐regulation of Wnt1. The effects of ectopic miR‐148a‐3p were rescued by Wnt1 overexpression. These biological functions of miR‐148a‐3p in PC were also confirmed in a nude mouse xenograft model. Taken together, these findings suggest that miR‐148a‐3p suppresses PC cell proliferation, invasion, EMT and stemness properties via inhibiting Wnt1‐mediated Wnt/β‐catenin pathway and could be a potential prognostic biomarker as well as a therapeutic target in PC.     

Integrating systematic biological and proteomics strategies to explore the pharmacological mechanism of danshen yin modified on atherosclerosis

This research utilized the systematic biological and proteomics strategies to explore the regulatory mechanism of Danshen Yin Modified (DSYM) on atherosclerosis (AS) biological network. The traditional Chinese medicine database and HPLC was used to find the active compounds of DSYM, Pharmmapper database was used to predict potential targets, and OMIM database and GeneCards database were used to collect AS targets. String database was utilized to obtain the other protein of proteomics proteins and the protein‐protein interaction (PPI) data of DSYM targets, AS genes, proteomics proteins and other proteins. The Cytoscape 3.7.1 software was utilized to construct and analyse the network. The DAVID database is used to discover the biological processes and signalling pathways that these proteins aggregate. Finally, animal experiments and proteomics analysis were used to further verify the prediction results. The results showed that 140 active compounds, 405 DSYM targets and 590 AS genes were obtained, and 51 differentially expressed proteins were identified in the DSYM‐treated ApoE‐/‐ mouse AS model. A total of 4 major networks and a number of their derivative networks were constructed and analysed. The prediction results showed that DSYM can regulate AS‐related biological processes and signalling pathways. Animal experiments have also shown that DSYM has a therapeutic effect on ApoE‐/‐mouse AS model (P < .05). Therefore, this study proposed a new method based on systems biology, proteomics, and experimental pharmacology, and analysed the pharmacological mechanism of DSYM. DSYM may achieve therapeutic effects by regulating AS‐related signalling pathways and biological processes found in this research.     

METTL3‐mediated maturation of miR‐589‐5p promotes the malignant development of liver cancer

MiR‐589‐5p could promote liver cancer, but the specific mechanisms are largely unknown. This study examined the role and mechanisms of miR‐589‐5p in liver cancer. The expressions of miR‐589‐5p, METTL3 and m6A in liver cancers were determined by RT‐qPCR. The relationship between miR‐589‐5p and METTL3‐mediated m6A methylation was examined by m6A RNA immunoprecipitation. After transfection, the viability, migration, invasion and expressions of METTL3 and miR‐589‐5p in liver cancer cells were detected by CCK‐8, wound‐healing, transwell and RT‐qPCR. After the xenograft tumour was established in mice, the tumour volume was determined and the expressions of METTL3, miR‐589‐5p, MMP‐2, TIMP‐2, E‐cadherin, N‐cadherin and Vimentin in tumour tissue were detected by RT‐qPCR and Western blotting. In vitro study showed that miR‐589‐5p and METTL3 were highly expressed in liver cancer. METTL3 was positively correlated with miR‐589‐5p. METTL3 up‐regulated the expression of miR‐589‐5p and promoted the maturation of miR‐589‐5p. Overexpressed miR‐589‐5p and METTL3 promoted the viability, migration and invasion of liver cancer cells, while the effects of silencing miR‐589‐5p and METTL3 on the cells were the opposite. The effects of METTL3 overexpression and silencing were reversed by miR‐589‐5p inhibitor and mimic, respectively. In vivo study showed that METLL3 silencing inhibited the growth of xenograft tumour and the expressions of METTL3, MMP‐2, N‐cadherin and Vimentin, promoted the expressions of TIMP‐2 and E‐cadherin, while miR‐589‐5p mimic caused the opposite results and further reversed the effects of METLL3 silencing. In summary, this study found that METTL3‐mediated maturation of miR‐589‐5p promoted the malignant development of liver cancer.     

Cell‐extracellular matrix interactions in the fluidic phase direct the topology and polarity of self‐organized epithelial structures

IntroductionIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell‐ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood.MethodsHere, using in vitro assay with engineered MDCK cells expressing H2B‐mCherry (nucleus) and gp135/Podocalyxin‐GFP (apical marker), we show in multi‐dimensions that such coordination for epithelial morphogenesis can be determined by cell‐soluble ECM interaction in the fluidic phase.ResultsThe coordination depends on the native topology of ECM components such as sheet‐like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close‐ended (open‐ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time‐lapse two‐photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135‐GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi‐lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment.ConclusionOur results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell‐soluble ECM interaction and self‐assembly.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.