Bacterial translation machinery for deliberate mistranslation of the genetic code.

Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.

Mapping the regulatory effects of herbal organic compounds on gut bacteria.

Herbal organic compounds (HOCs) are bioactive natural products from medicinal plants and some traditional Chinese medicines (TCMs). Recently, ingestion of a few HOCs with low bioavailability has been associated with alterations in gut microbiota, but the extent of this phenomenon remains unclear. Here, we systematically screened 481 HOCs against 47 representative gut bacterial strains in vitro and found that almost one-third of the HOCs exhibited unique anticommensal activity. Quinones showed a potent anticommensal activity, while saturated fatty acids exhibited stronger inhibition of the Lactobacillus genus. Flavonoids, phenylpropanoids, terpenoids, triterpenoids, alkaloids and phenols displayed weaker anticommensal activity, but steroids, saccharides and glycosides had hardly any effect on strain growth. Notably, S-configuration HOCs demonstrated stronger anticommensal activity than R-configuration HOCs. The strict screening conditions ensured high accuracy (95%) through benchmarking validation. Additionally, the effects of HOCs on human fecal microbiota profiling were positively correlated with their anticommensal activity against bacterial strains. Molecular and chemical features such as AATS3i and XLogP3 were correlated with the anticommensal activity of the HOCs in the random forest classifier. Finally, we validated that curcumin, a polyhydric phenol with anticommensal activity, improved insulin resistance in HFD mice by modulating the composition and metabolic function of gut microbiota. Our results systematically mapped the profile of HOCs directly affecting human gut bacterial strains, offering a resource for future research on HOC-microbiota interaction, and broadening our understanding of natural product utilization through gut microbiota modulation.

Recent progress in unraveling the biosynthesis of natural sunscreens mycosporine-like amino acids.

Exposure to ultraviolet (UV) rays is a known risk factor for skin cancer, which can be notably mitigated through the application of sun care products. However, escalating concerns regarding the adverse health and environmental impacts of synthetic anti-UV chemicals underscore a pressing need for the development of biodegradable and eco-friendly sunscreen ingredients. Mycosporine-like amino acids (MAAs) represent a family of water-soluble anti-UV natural products synthesized by various organisms. These compounds can provide a two-pronged strategy for sun protection as they not only exhibit a superior UV absorption profile but also possess the potential to alleviate UV-induced oxidative stresses. Nevertheless, the widespread incorporation of MAAs in sun protection products is hindered by supply constraints. Delving into the biosynthetic pathways of MAAs can offer innovative strategies to overcome this limitation. Here, we review recent progress in MAA biosynthesis, with an emphasis on key biosynthetic enzymes, including the dehydroquinate synthase homolog MysA, the adenosine triphosphate (ATP)-grasp ligases MysC and MysD, and the nonribosomal peptide synthetase (NRPS)-like enzyme MysE. Additionally, we discuss recently discovered MAA tailoring enzymes. The enhanced understanding of the MAA biosynthesis paves the way for not only facilitating the supply of MAA analogs but also for exploring the evolution of this unique family of natural sunscreens. ONE-SENTENCE SUMMARY: This review discusses the role of mycosporine-like amino acids (MAAs) as potent natural sunscreens and delves into recent progress in their biosynthesis.

Tongmai Yangxin pill alleviates myocardial no-reflow by activating GPER to regulate HIF-1α signaling and downstream potassium channels.

CONTEXT: The Tongmai Yangxin pill (TMYX) has potential clinical effects on no-reflow (NR); however, the effective substances and mechanisms remain unclear. OBJECTIVE: This study evaluates the cardioprotective effects and molecular mechanisms of TMYX against NR. MATERIALS AND METHODS: We used a myocardial NR rat model to confirm the effect and mechanism of action of TMYX in alleviating NR. Sprague-Dawley (SD) rats were divided into Control (Con), sham, NR, TMYX (4.0 g/kg), and sodium nitroprusside (SNP, 5.0 mg/kg), and received their treatments once a day for one week. In vitro studies in isolated coronary microvasculature of NR rats and in silico network pharmacology analyses were performed to reveal the underlying mechanisms of TMYX and determine the main components, targets, and pathways of TMYX, respectively. RESULTS: TMYX (4.0 g/kg) showed therapeutic effects on NR by improving the cardiac structure and function, reducing NR, ischemic areas, and cardiomyocyte injury, and decreasing the expression of cardiac troponin I (cTnI). Moreover, the mechanism of TMYX predicted by network pharmacology is related to the HIF-1, NF-κB, and TNF signaling pathways. In vivo, TMYX decreased the expression of MPO, NF-κB, and TNF-α and increased the expression of GPER, p-ERK, and HIF-1α. In vitro, TMYX enhanced the diastolic function of coronary microvascular cells; however, this effect was inhibited by G-15, H-89, L-NAME, ODQ and four K+ channel inhibitors. CONCLUSIONS: TMYX exerts its pharmacological effects in the treatment of NR via multiple targets. However, the contribution of each pathway was not detected, and the mechanisms should be further investigated.

The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide.

TxtE is a cytochrome P450 (CYP) homologue that mediates the nitric oxide (NO)-dependent direct nitration of l-tryptophan (Trp) to form 4-nitro-l-tryptophan (4-NO2-Trp). A recent report showed evidence that TxtE activity requires NO to react with a ferric-superoxo intermediate. Given this minimal mechanism, it is not clear how TxtE avoids Trp hydroxylation, a mechanism that also traverses the ferric-superoxo intermediate. To provide insight into canonical CYP intermediates that TxtE can access, electron coupling efficiencies to form 4-NO2-Trp under single- or limited-turnover conditions were measured and compared to steady-state efficiencies. As previously reported, Trp nitration by TxtE is supported by the engineered self-sufficient variant, TB14, as well as by reduced putidaredoxin. Ferrous (FeII) TxtE exhibits excellent electron coupling (70%), which is 50-fold higher than that observed under turnover conditions. In addition, two- or four-electron reduced TB14 exhibits electron coupling (∼6%) that is 2-fold higher than that of one-electron reduced TB14 (3%). The combined results suggest (1) autoxidation is the sole TxtE uncoupling pathway and (2) the TxtE ferric-superoxo intermediate cannot be reduced by these electron transfer partners. The latter conclusion is further supported by ultraviolet-visible absorption spectral time courses showing neither spectral nor kinetic evidence for reduction of the ferric-superoxo intermediate. We conclude that resistance of the ferric-superoxo intermediate to reduction is a key feature of TxtE that increases the lifetime of the intermediate and enables its reaction with NO and efficient nitration activity.

Biosynthesis and Heterologous Production of Mycosporine-Like Amino Acid Palythines.

Mycosporine-like amino acids (MAAs) are a family of natural products that are produced by a variety of organisms for protection from ultraviolet damage. In this work, we combined different bioinformatic approaches to assess the distribution of the MAA biosynthesis and identified a putative gene cluster from Nostoc linckia NIES-25 that encodes a short-chain dehydrogenase/reductase and a nonheme iron(II)- and 2-oxoglutarate-dependent oxygenase (MysH) as potential new biosynthetic enzymes. Heterologous expression of refactored gene clusters in E. coli produced two known biosynthetic intermediates, 4-deoxygadusol and mycosporine-glycine, and three disubstituted MAA analogues, porphyra-334, shinorine, and mycosporine-glycine-alanine. Importantly, the disubstituted MAAs were converted into palythines by MysH. Furthermore, biochemical characterization revealed the substrate preference of recombinant MysD, a d-Ala-d-Ala ligase-like enzyme for the formation of disubstituted MAAs. Our study advances the biosynthetic understanding of an important family of natural UV photoprotectants and opens new opportunities to the development of next-generation sunscreens.

Heterologous production of cyanobacterial compounds.

Cyanobacteria produce a plethora of compounds with unique chemical structures and diverse biological activities. Importantly, the increasing availability of cyanobacterial genome sequences and the rapid development of bioinformatics tools have unraveled the tremendous potential of cyanobacteria in producing new natural products. However, the discovery of these compounds based on cyanobacterial genomes has progressed slowly as the majority of their corresponding biosynthetic gene clusters (BGCs) are silent. In addition, cyanobacterial strains are often slow-growing, difficult for genetic engineering, or cannot be cultivated yet, limiting the use of host genetic engineering approaches for discovery. On the other hand, genetically tractable hosts such as Escherichia coli, Actinobacteria, and yeast have been developed for the heterologous expression of cyanobacterial BGCs. More recently, there have been increased interests in developing model cyanobacterial strains as heterologous production platforms. Herein, we present recent advances in the heterologous production of cyanobacterial compounds in both cyanobacterial and noncyanobacterial hosts. Emerging strategies for BGC assembly, host engineering, and optimization of BGC expression are included for fostering the broader applications of synthetic biology tools in the discovery of new cyanobacterial natural products.

Screening and identifying antioxidants from Oplopanax elatus using 2,2'-diphenyl-1-picrylhydrazyl with off-line two-dimensional HPLC coupled with diode array detection and tandem time-of-flight mass spectrometry.

The root of Oplopanax elatus (Nakai) Nakai has a well-known history of use for the treatment of diseases such as neurasthenia, cardiovascular disorders, and cancer by the native people in northeast China. It is important to screen and identify the bioactive molecules from its root rapidly. Hereby, an off-line two-dimensional high performance liquid chromatography coupled with diode array detection and tandem time-of-flight mass spectrometry together with 2,2'-diphenyl-1-picrylhydrazyl was established to screen antioxidants from the root of O. elatus. A Waters cyanogen column (150 × 3.9 mm, id, 4 µm) was used for the first dimensional liquid chromatography, while a Hypersil BDS-C18 column (250 × 4.6 mm, id, 5 µm) was installed for the second dimension liquid chromatographic analysis. Twenty-eight compounds had been tentatively identified from the methanol extract of the air-dried root of O. elatus including six polyynes and eight phenolic derivatives were screened with antioxidant activity. The developed method could be expedient for screening and identifying antioxidants from O. elatus.

Biosynthesis of Dolastatin 10 in Marine Cyanobacteria, a Prototype for Multiple Approved Cancer Drugs.

Dolastatin 10, a potent tubulin-targeting marine anticancer natural product, provided the basis for the development of six FDA-approved antibody-drug conjugates. Through the screening of cyanobacterial Caldora penicillata environmental DNA libraries and metagenome sequencing, we identified its biosynthetic gene cluster. Functional prediction of 10 enzymes encoded in the 39 kb cluster supports the dolastatin 10 biosynthesis. The nonheme diiron monooxygenase DolJ was biochemically characterized to mediate the terminal thiazole formation in dolastatin 10.

Direct aromatic nitration by bacterial P450 enzymes.

Nitro aromatics have broad applications in industry, agriculture, and pharmaceutics. However, their industrial production is faced with many challenges including poor selectivity, heavy pollution and safety concerns. Nature provides multiple strategies for aromatic nitration, which opens the door for the development of green and efficient biocatalysts. Our group's efforts focused on a unique bacterial cytochrome P450 TxtE that originates from the biosynthetic pathway of phytotoxin thaxtomins, which can install a nitro group at C4 of l-Trp indole ring. TxtE is a Class I P450 and its reaction relies on a pair of redox partners ferredoxin and ferredoxin reductase for essential electron transfer. To develop TxtE as an efficient nitration biocatalyst, we created artificial self-sufficient P450 chimeras by fusing TxtE with the reductase domain of the bacterial P450BM3 (BM3R). We evaluated the catalytic performance of the chimeras with different lengths of the linker connecting TxtE and BM3R domains and identified one with a 14-amino-acid linker (TB14) to give the best activity. In addition, we demonstrated the broad substrate scope of the engineered biocatalyst by screening diverse l-Trp analogs. In this chapter, we provide a detailed procedure for the development of aromatic nitration biocatalysts, including the construction of P450 fusion chimeras, biochemical characterization, determination of catalytic parameters, and testing of enzyme-substrate scope. These protocols can be followed to engineer other P450 enzymes and illustrate the processes of biocatalytic development for the synthesis of nitro chemicals.

Biotransformation differences of ginsenoside compound K mediated by the gut microbiota from diabetic patients and healthy subjects.

Many natural products can be bio-converted by the gut microbiota to influence pertinent efficiency. Ginsenoside compound K (GCK) is a potential anti-type 2 diabetes (T2D) saponin, which is mainly bio-transformed into protopanaxadiol (PPD) by the gut microbiota. Studies have shown that the gut microbiota between diabetic patients and healthy subjects are significantly different. Herein, we aimed to characterize the biotransformation of GCK mediated by the gut microbiota from diabetic patients and healthy subjects. Based on 16S rRNA gene sequencing, the results indicated the bacterial profiles were considerably different between the two groups, especially Alistipes and Parabacteroides that increased in healthy subjects. The quantitative analysis of GCK and PPD showed that gut microbiota from the diabetic patients metabolized GCK slower than healthy subjects through liquid chromatography tandem mass spectrometry (LC-MS/MS). The selected strain A. finegoldii and P. merdae exhibited a different metabolic capability of GCK. In conclusion, the different biotransformation capacity for GCK may impact its anti-diabetic potency.

Bacteroides ovatus accelerates metformin-induced vitamin B12 deficiency in type 2 diabetes patients by accumulating cobalamin.

Vitamin B12 (VB12) deficiency, which may lead to hematologic and neurologic symptoms, has been associated with metformin use, but the underlying mechanism is unclear. Here we report the B. ovatus as an effective VB12 catcher which was enriched in the type 2 diabetes patients suffered from VB12 deficiency after 3 to 6 months of metformin treatment. Colonization of B. ovatus increased the plasma levels of methylmalonic acid and homocysteine in high-fat diet (HFD)-fed mice treated with metformin, and compromised the efficacy of metformin against the HFD-induced metabolic disorders. Mechanistically, metformin increased the intracellular accumulation of VB12 in B. ovatus via btuB upregulation and promoted ATP production for energy-dependent translocation of VB12 transporters at the inner membrane, leading to an enhanced colonization of B. ovatus to compete for VB12 with hosts and subsequently an aggravated VB12 deficiency in the host. Our findings illustrate a previously unappreciated mechanism of metformin leads to host VB12 deficiency by acting directly on gut bacteria to increase their VB12 uptake and consumption, and suggest that inter-host-microbe competition for nutrients may broadly impact human health and drug safety.

Design, Synthesis, and Evaluation of Carbonate-Linked Halogenated Phenazine-Quinone Prodrugs with Improved Water-Solubility and Potent Antibacterial Profiles.

Pathogenic bacteria have devastating impacts on human health as a result of acquired antibiotic resistance and innate tolerance. Every class of our current antibiotic arsenal was initially discovered as growth-inhibiting agents that target actively replicating (individual, free-floating) planktonic bacteria. Bacteria are notorious for utilizing a diversity of resistance mechanisms to overcome the action of conventional antibiotic therapies and forming surface-attached biofilm communities enriched in (non-replicating) persister cells. To address problems associated with pathogenic bacteria, our group is developing halogenated phenazine (HP) molecules that demonstrate potent antibacterial and biofilm-eradicating activities through a unique iron starvation mode of action. In this study, we designed, synthesized, and investigated a focused collection of carbonate-linked HP prodrugs bearing a quinone trigger to target the reductive cytoplasm of bacteria for bioactivation and subsequent HP release. The quinone moiety also contains a polyethylene glycol group, which dramatically enhances the water-solubility properties of the HP-quinone prodrugs reported herein. We found carbonate-linked HP-quinone prodrugs 11, 21-23 to demonstrate good linker stability, rapid release of the active HP warhead following dithiothreitol (reductive) treatment, and potent antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, and Enterococcus faecalis. In addition, HP-quinone prodrug 21 induced rapid iron starvation in MRSA and S. epidermidis biofilms, illustrating prodrug action within these surface-attached communities. Overall, we are highly encouraged by these findings and believe that HP prodrugs have the potential to address antibiotic resistant and tolerant bacterial infections.

Design, synthesis and evaluation of halogenated phenazine antibacterial prodrugs targeting nitroreductase enzymes for activation.

It is of great importance to develop new strategies to combat antibiotic resistance. Our lab has discovered halogenated phenazine (HP) analogues that are highly active against multidrug-resistant bacterial pathogens. Here, we report the design, synthesis, and study of a new series of nitroarene-based HP prodrugs that leverage intracellular nitroreductase (NTR) enzymes for activation and subsequent release of active HP agents. Our goals of developing HP prodrugs are to (1) mitigate off-target metal chelation (potential toxicity), (2) possess motifs to facilitate intracellular, bacterial-specific HP release, (3) improve water solubility, and (4) prevent undesirable metabolism (e.g., glucuronidation of HP's phenol). Following the synthesis of HP-nitroarene prodrugs bearing a sulfonate ester linker, NTR-promoted release experiments demonstrated prodrug HP-1-N released 70.1% of parent HP-1 after 16 hours (with only 6.8% HP-1 release without NTR). In analogous in vitro experiments, no HP release was observed for control sulfonate ester compounds lacking the critical nitro group. When compared to parent HP compounds, nitroarene prodrugs evaluated during these studies demonstrate similar antibacterial activities in MIC and zone of inhibition assays (against lab strains and clinical isolates). In conclusion, HP-nitroarene prodrugs could provide a future avenue to develop potent agents that target antibiotic resistant bacteria.

Panax notoginseng Alleviates Colitis via the Regulation of Gut Microbiota.

Gut microbiota are significantly associated with the occurrence and development of inflammatory bowel disease (IBD). Panax notoginseng saponins (PNS) could be used for colitis and to modulate gut microbiota. However, the mechanism behind the effects of PNS on anti-colitis that are pertinent to gut microbiota is largely unknown. This study aimed to evaluate the anti-colitis effects of PNS and explore the involved mechanism as it is related to gut microbiota. Results showed that PNS significantly alleviated dextran sulfate sodium (DSS)-induced colitis. Meanwhile, after PNS treatment, the tight junction proteins were enhanced and proinflammatory cytokines, such as TNF-[Formula: see text], IL-6, IL-1[Formula: see text], and IL-17, were decreased. Furthermore, Bacteroides spp. were significantly increased after modeling, while PNS reduced their abundance and significantly increased the amount of Akkermansia spp. in vivo. Importantly, Akkermansia spp. and Bacteroides spp. were correlated with the IBD disease indicators. Moreover, fecal microbiota transplantation (FMT) experiments confirmed that PNS-reshaped gut microbiota significantly alleviated DSS-induced colitis, while A. muciniphila significantly reduced the levels of the LPS-induced cellular inflammatory factors IL-1[Formula: see text] and TNF-[Formula: see text]. In conclusion, PNS alleviated colitis pertinent to the upregulation of Akkermania spp. and downregulation of Bacteroides spp. in the gut.

Heterologous Production of the C33-C45 Polyketide Fragment of Anticancer Apratoxins in a Cyanobacterial Host.

A polyketide synthase subcluster of cytotoxic apratoxin A was isolated from a Moorena bouillonii environmental DNA library and engineered with a thioesterase II domain for heterologous expression in the filamentous cyanobacterium Anabaena sp. PCC7120. Further engineering with a rhamnose-inducible promoter led to the production of (2R,3R,5R,7R)-3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid, a stereogenically rich chiral building block that is important to the efficient synthesis of apratoxin analogues, representing the first synthetic biology attempt for this type of polyketide fragment.

Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization.

Hydrochlorothiazide (HCTZ) is reported to impair glucose tolerance and may induce new onset of diabetes, but the pharmacomicrobiomics of the adverse effect for HCTZ remains unknown. Mice-fed HCTZ exhibited insulin resistance and impaired glucose tolerance. By using FMT and antibiotic cocktail models, we found that HCTZ-induced metabolic disorder was mediated by commensal microbiota. HCTZ consumption disturbed the structure of the intestinal microbiota, causing abnormal elevation of Gram-negative Enterobacteriaceae and lipopolysaccharide (LPS) then leading to intestinal barrier dysfunction. Additionally, HCTZ activated TLR4 signaling and induced macrophage polarization and inflammation in the liver. Furthermore, HCTZ-induced macrophage polarization and metabolic disorder were abrogated by blocking TLR4 signaling. HCTZ consumption caused a significant increase in Gram-negative Enterobacteriaceae, which elevated the levels of LPS, thereby activating LPS/TLR4 pathway, promoting inflammation and macrophage polarization, and resulting in metabolic disorders. These findings revealed that the gut microbiome is the key medium underlying HCTZ-induced metabolic disorder.

Chemical and genomic characterization of a potential probiotic treatment for stony coral tissue loss disease.

Considered one of the most devastating coral disease outbreaks in history, stony coral tissue loss disease (SCTLD) is currently spreading throughout Florida's coral reefs and the greater Caribbean. SCTLD affects at least two dozen different coral species and has been implicated in extensive losses of coral cover. Here we show Pseudoalteromonas sp. strain McH1-7 has broad-spectrum antibacterial activity against SCTLD-associated bacterial isolates. Chemical analyses indicated McH1-7 produces at least two potential antibacterials, korormicin and tetrabromopyrrole, while genomic analysis identified the genes potentially encoding an L-amino acid oxidase and multiple antibacterial metalloproteases (pseudoalterins). During laboratory trials, McH1-7 arrested or slowed disease progression on 68.2% of diseased Montastraea cavernosa fragments treated (n = 22), and it prevented disease transmission by 100% (n = 12). McH1-7 is the most chemically characterized coral probiotic that is an effective prophylactic and direct treatment for the destructive SCTLD as well as a potential alternative to antibiotic use.

Effects of ginsenoside compound K on colitis-associated colorectal cancer and gut microbiota profiles in mice.

Background: Ginsenoside compound K (GC-K), generated from ginseng saponins bioconverted by gut microbiota, has potential anti-colorectal cancer (CRC) effects. Meanwhile, GC-K may interact with gut microbiota, playing important roles in the occurrence and development of CRC. However, the effects of gut microbiota on the preventive and therapeutic effects of GC-K in CRC remain to be elucidated. Methods: The anti-CRC effects of GC-K were evaluated in an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-associated CRC Balb/c mice model under the dosage of 30 and 60 mg/kg. Stool samples were collected during the experiments for profiling gut microbiota by 16S rRNA sequencing. Correlative analysis between gut microbiota and anti-CRC effect of GC-K was also assessed. Finally, the anti-CRC effect of Akkermansia muciniphila (A. muciniphila) was validated in CRC cell lines. Results: GC-K could significantly suppress tumor growth in vivo at the dosage of 60 mg/kg without exogenous interference of gut microbiota. Moreover, dysbiosis of gut microbiota was observed in the CRC model group, which could be recovered by GC-K treatment. In particular, A. muciniphila, which could inhibit the proliferation of HCT-116, HT-29, and LOVO cells, was significantly up-regulated by GC-K. Conclusions: GC-K was verified to suppress the tumor growth of AOM/DSS-induced colitis-associated CRC through the modulation of gut microbiota, partially by up-regulation of A. muciniphila.

LncRNA weighted gene co-expression network analysis reveals novel biomarkers related to prostate cancer metastasis.

BACKGROUND: Most prostate cancer patients die from metastasis and lack accurate efficacious biomarkers to monitor the disease behavior, optimize treatment and assess prognosis. Herein, we aimed to identify meaningful lncRNA biomarkers associated with prostate cancer metastatic progression. METHODS: By repurposing microarray probes, 11,624 lncRNAs in prostate cancer were obtained from Gene Expression Omnibus  database (GSE46691, N = 545; GSE29079, N = 235; GSE94767, N = 130). Weighted gene co-expression network analysis was applied to determine the co-expression lncRNA network pertinent to metastasis. Hub lncRNAs were screened. RNA-seq and clinical data from the Cancer Genome Atlas prostate cancer (TCGA-PRAD) cohort (N = 531) were analyzed. Transwell assay and bioinformatic analysis were performed for mechanism research. RESULTS: The high expression levels of nine hub lncRNAs (FTX, AC005261.1, NORAD, LINC01578, AC004542.2, ZFAS1, EBLN3P, THUMPD3-AS1, GAS5) were significantly associated with Gleason score and increased probability of metastatic progression. Among these lncRNAs, ZFAS1 had the consistent trends of expression in all of the analysis from different cohorts, and the Kaplan-Meier survival analyses showed higher expression of ZFAS1 was associated with shorter relapse free survival. In-vitro studies confirmed that downregulation of ZFAS1 decreased prostate cancer cell migration. CONCLUSION: We offered some new insights into discovering lncRNA markers correlated with metastatic progression of prostate cancer using the WGCNA. Some may serve as potential prognostic biomarkers and therapeutic targets for advanced metastatic prostate cancer.