Comprehensive analysis of non-synonymous missense SNPs of human galactose mutarotase (GALM) gene: an integrated computational approach.

Missense Non-synonymous single nucleotide polymorphisms (nsSNPs) of Galactose Mutarotase (GALM) are associated with the Novel type of Galactosemia (Galactosemia type 4) together with symptoms such as high blood galactose levels and eye cataracts. The objective of the present study was to identify deleterious nsSNPs of GALM recorded on the dbSNP database through comprehensive insilico analysis. Among the 319 missense nsSNPs reported, various insilco tools predicted R78S, R82G, A163E, P210S, Y281C, E307G and F339C as the most deleterious mutations. Structural analysis, PTM analysis and molecular dynamics simulations (MDS) were carried out to understand the effect of these mutations on the structural and physicochemical properties of the GALM protein. The residues R82G and E307G were found to be part of the binding site that resulted in decreased surface accessibility. Replacing the charged wild-type residue with a neutral mutant type affected its substrate binding. All 7 mutations were found to increase the rigidity of the protein structure, which is unfavorable during ligand binding. The mutation F339E made the protein structure more rigid than all the other mutations. Y281 is a phosphorylated site, and therefore, less significant structural changes were observed when compared to other mutations; however, it may have significant differences in the usual functioning of the protein. In summary, the structural and functional analysis of missense SNPs of GALM is important to reduce the number of potential mutations to be evaluated in vitro to understand the association with some genetic diseases.Communicated by Ramaswamy H. Sarma.

FUCA2 and TSTA3 expression in gastric cancer: candidate biomarkers of malignant transformation.

INTRODUCTION: Aberrant fucosylation is closely related to malignant transformation, cancer detection, and evaluation of treatment efficacy. The fucosylation process requires GDP-L-fucose, fucosyltransferases, and fucosidases. In gastric cancer (GC), fucosylation alterations were associated with tumor formation, metastasis inhibition, and multi-drug resistance. It is not clear whether tissue-specific transplantation antigen P35B (TSTA3) and alpha-L-fucosidase 2 (FUCA2) have any effect on the development of GC. MATERIALS AND METHODS: We used immunohistochemistry to assess the expression of TSTA3 and FUCA2 in 71 gastric adenocarcinoma samples and their relationship with clinicopathological parameters. RESULTS: TSTA3 expression was associated with lower histological grade I and II (P = 0.0120) and intestinal type Lauren classification (P = 0.0120). TSTA3 immunopositivity could predict Lauren's classification. Analysis of mRNA expression in GC validation cohorts corroborates the significant TSTA3 association with histological grade observed in our study. However, no associations were found between TSTA3 staining and overall survival. FUCA2 expression was markedly increased in GC tissues compared with non-tumoral tissues (P < 0.0001) and was associated with surgical staging III and IV (P = 0.0417) and advanced histological grade tumor states (P = 0.0125). CONCLUSIONS: Alterations of FUCA2 and TSAT3 immunoexpression could lay the basis for future studies using cell glycosylation as a biomarker for the planning of therapeutic strategy in primary gastric cancer.

The Effect of Mutation in Lipopolysaccharide Biosynthesis on Bacterial Fitness.

This paper presents the genome sequence of a Shigella sonnei mutant strain (S. sonnei 4351) and the effect of mutation in lipopolysaccharide biosynthesis on bacterial fitness. Lipopolysaccharides are the major component of the outer leaflet of the Gram-negative outer membrane. We report here a frameshift mutation of the gene gmhD in the genome of S. sonnei 4351. The mutation results in a lack of epimerization of the core heptose while we also found increased thermosensitivity, abnormal cell division, and increased susceptibility to erythromycin and cefalexin compared to the S. sonnei 4303. Comparative genomic analysis supplemented with structural data helps us to understand the effect of specific mutations on the virulence of the bacteria and may provide an opportunity to study the effect of short lipopolysaccharides.

Spinning sugars in antigen biosynthesis: characterization of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases.

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3'',5'' double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3''-position than the 5''-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.

Functional characterization of GNE mutations prevalent in Asian subjects with GNE myopathy, an ultra-rare neuromuscular disorder.

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is a bifunctional enzyme (N-terminal epimerase and C-terminal Kinase domain) that catalyses the rate limiting step in sialic acid biosynthesis. More than 200 homozygous missense or compound heterozygous mutations in GNE have been reported worldwide to cause a rare neuromuscular disorder, GNE myopathy. It is characterized by a slowly progressive defect in proximal and distal skeletal muscles with patients becoming wheel-chair-bound. There are no current approved therapies available for GNE myopathy. ManNAc therapy is currently in advanced clinical trials and has shown signs of slowing the disease progression in a phase 2 trial. The present study aims to understand the effect of GNE mutation on its enzymatic activity and identification of potential small effector molecules. We characterized different GNE mutations (p.Asp207Val, p.Val603Leu, p.Val727Met, p.Ile618Thr and p.Arg193Cys) prevalent in Asian population that were cloned, expressed and purified from Escherichia coli as full-length recombinant proteins. Our study demonstrates that full length GNE can be expressed in E. coli in its active form and analysed for the functional activity. Each mutation showed variation in epimerase and kinase activity and responded to the small effector molecules (metformin, BGP-15 kaempferol, catechin, quercetin) in a differential manner. Our study opens an area for futuristic structural determination of full length GNE and identification of potential therapeutic molecules.

High production of d-psicose from d-fructose by immobilized whole recombinant Bacillus subtilis cells expressing d-psicose 3-epimerase from Agrobacterium tumefaciens.

d-Psicose 3-epimerase (DPEase) can catalyze the isomerization of d-fructose to be rare sugar d-psicose, which has wide application prospects in the food and medical fields. In this study, the DPEase gene from Agrobacterium tumefaciens was constructed into plasmid pMA5, and was successfully expressed in the host Bacillus subtilis WB600 (B. subtilis). After optimization of the fermentation conditions, whole recombinant B. subtilis WB600/pMA5-At-DEPase(O) cells produced d-psicose from d-fructose with a conversion rate of 29.01 ± 0.19%, which could be used for the efficient synthesis of d-psicose. To further improve the whole recombinant B. subtilis application, B. subtilis cells were immobilized onto a gel bead biocatalyst by Ca-alginate. After optimization of the biotransformation conditions, the conversion rate of the immobilized biocatalyst reached 20.74 ± 0.39%, which was lower than the free cells. However, the results showed that the immobilized biocatalyst had higher thermal/pH stability and storability, and the gel beads could be recycled for at least six batches. The results showed that the amount of d-psicose generated reached 32.83 ± 2.56 g/L with the immobilized biocatalyst after six times biotransformation, whereas the free cells produced only approximately 10.44 ± 0.07 g/L. The results showed that immobilized recombinant B. subtilis cells are promising to use for the efficient synthesis of d-psicose.

A New Sugar for an Old Phage: a c-di-GMP-Dependent Polysaccharide Pathway Sensitizes Escherichia coli for Bacteriophage Infection.

Bacteriophages are ubiquitous parasites of bacteria and major drivers of bacterial ecology and evolution. Despite an ever-growing interest in their biotechnological and therapeutic applications, detailed knowledge of the molecular mechanisms underlying phage-host interactions remains scarce. Here, we show that bacteriophage N4 exploits a novel surface glycan (NGR) as a receptor to infect its host Escherichia coli. We demonstrate that this process is regulated by the second messenger c-di-GMP and that N4 infection is specifically stimulated by the diguanylate cyclase DgcJ, while the phosphodiesterase PdeL effectively protects E. coli from N4-mediated killing. PdeL-mediated protection requires its catalytic activity to reduce c-di-GMP and includes a secondary role as a transcriptional repressor. We demonstrate that PdeL binds to and represses the promoter of the wec operon, which encodes components of the enterobacterial common antigen (ECA) exopolysaccharide pathway. However, only the acetylglucosamine epimerase WecB but none of the other ECA components is required for N4 infection. Based on this, we postulate that NGR is an N-acetylmannosamine-based carbohydrate polymer that is produced and exported to the cell surface of E. coli in a c-di-GMP-dependent manner, where it serves as a receptor for N4. This novel carbohydrate pathway is conserved in E. coli and other bacterial pathogens, serves as the primary receptor for various bacteriophages, and is induced at elevated temperature and by specific amino acid-based nutrients. These studies provide an entry point into understanding how bacteria use specific regulatory mechanisms to balance costs and benefits of highly conserved surface structures. IMPORTANCE Because bacterial surface glycans are in direct contact with the environment they can provide essential protective functions during infections or against competing bacteria. But such structures are also "Achilles' heels" since they can serve as primary receptors for bacteriophages. Bacteria thus need to carefully control the exposure of conserved surface glycans to balance costs and benefits. Here, we identify a novel exopolysaccharide that is widely conserved in E. coli and is used by N4 and related bacteriophages as primary receptor. We demonstrate that the synthesis of NGR (N4 glycan receptor) is tightly controlled by the second messenger c-di-GMP in a highly specific manner and by a single diguanylate cyclase. These studies provide an example of how bacteria can alleviate the strong selective pressure imposed on them by bacteriophages entering through conserved surface structures by carefully regulating their synthesis and secretion.

Phytophthora sojae apoplastic effector AEP1 mediates sugar uptake by mutarotation of extracellular aldose and is recognized as a MAMP.

Diseases caused by Phytophthora pathogens devastate many crops worldwide. During infection, Phytophthora pathogens secrete effectors, which are central molecules for understanding the complex plant-Phytophthora interactions. In this study, we profiled the effector repertoire secreted by Phytophthora sojae into the soybean (Glycine max) apoplast during infection using liquid chromatography-mass spectrometry. A secreted aldose 1-epimerase (AEP1) was shown to induce cell death in Nicotiana benthamiana, as did the other two AEP1s from different Phytophthora species. AEP1 could also trigger immune responses in N. benthamiana, other Solanaceae plants, and Arabidopsis (Arabidopsis thaliana). A glucose dehydrogenase assay revealed AEP1 encodes an active AEP1. The enzyme activity of AEP1 is dispensable for AEP1-triggered cell death and immune responses, while AEP-triggered immune signaling in N. benthamiana requires the central immune regulator BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1. In addition, AEP1 acts as a virulence factor that mediates P. sojae extracellular sugar uptake by mutarotation of extracellular aldose from the α-anomer to the ß-anomer. Taken together, these results revealed the function of a microbial apoplastic effector, highlighting the importance of extracellular sugar uptake for Phytophthora infection. To counteract, the key effector for sugar conversion can be recognized by the plant membrane receptor complex to activate plant immunity.

N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization.

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the ß-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.

Overexpression of arogenate dehydratase reveals an upstream point of metabolic control in phenylalanine biosynthesis.

Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the d-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell.

Glycolysis- and immune-related novel prognostic biomarkers of Ewing's sarcoma: glucuronic acid epimerase and triosephosphate isomerase 1.

INTRODUCTION: Owing to the poor prognosis of Ewing's sarcoma, reliable prognostic biomarkers are highly warranted for clinical diagnosis of the disease. MATERIALS AND METHODS: A combination of the weighted correlation network analysis and differentially expression analysis was used for initial screening; glycolysis-related genes were extracted and subjected to univariate Cox, LASSO regression, and multivariate Cox analyses to construct prognostic models. The immune cell composition of each sample was analysed using CIBERSORT software. Immunohistochemical analysis was performed for assessing the differential expression of modelled genes in Ewing's sarcoma and paraneoplastic tissues. RESULTS: A logistic regression model constructed for the prognosis of Ewing's sarcoma exhibited that the patient survival rate in the high-risk group is much lower than in the low-risk group. CIBERSORT analysis exhibited a strong correlation of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages (P < 0.05). Immunohistochemical analysis confirmed the study findings. CONCLUSIONS: GLCE and TPI1 can be used as prognostic biomarkers to predict the prognosis of Ewing's sarcoma, and a close association of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages provides a novel approach to the disease immunotherapy.

Efficient biosynthesis of D-allulose in Bacillus subtilis through D-psicose 3-epimerase translation modification.

The combined catalysis of glucose isomerase (GI) and D-psicose 3-epimerase (DPEase) provided a convenient route for the direct synthesis of D-allulose from d-glucose, whose cost is lower than d-fructose. In the present research, the weak activity of DPEase was the key rate-limiting step and resulted in the accumulation of d-fructose in engineered Bacillus subtilis. Then, the 5'-untranslated region (5'-UTR) structure of the mRNA translational initiation region was optimized for the precise control of DPEase expression. The manipulation of the 5'-UTR region promoted the accessibility to ribosome binding and the stability of mRNA, resulting in a maximum of 1.73- and 1.98-fold increase in DPEase activity and intracellular mRNA amount, respectively. Under the optimal catalytic conditions of 75 °C, pH 6.5, 110 g/L d-glucose, and 1 mmol/L Co2+, the reaction equilibrium time was reduced from 7.6 h to 6.1 h. We hope that our results could provide a facilitated strategy for large-scale production of D-allulose at low-cost.

Multiple evolutionary origins reflect the importance of sialic acid transporters in the colonization potential of bacterial pathogens and commensals.

Located at the tip of cell surface glycoconjugates, sialic acids are at the forefront of host-microbe interactions and, being easily liberated by sialidase enzymes, are used as metabolites by numerous bacteria, particularly by pathogens and commensals living on or near diverse mucosal surfaces. These bacteria rely on specific transporters for the acquisition of host-derived sialic acids. Here, we present the first comprehensive genomic and phylogenetic analysis of bacterial sialic acid transporters, leading to the identification of multiple new families and subfamilies. Our phylogenetic analysis suggests that sialic acid-specific transport has evolved independently at least eight times during the evolution of bacteria, from within four of the major families/superfamilies of bacterial transporters, and we propose a robust classification scheme to bring together a myriad of different nomenclatures that exist to date. The new transporters discovered occur in diverse bacteria, including Spirochaetes, Bacteroidetes, Planctomycetes and Verrucomicrobia, many of which are species that have not been previously recognized to have sialometabolic capacities. Two subfamilies of transporters stand out in being fused to the sialic acid mutarotase enzyme, NanM, and these transporter fusions are enriched in bacteria present in gut microbial communities. Our analysis supports the increasing experimental evidence that competition for host-derived sialic acid is a key phenotype for successful colonization of complex mucosal microbiomes, such that a strong evolutionary selection has occurred for the emergence of sialic acid specificity within existing transporter architectures.

Identification of hub genes with prognostic values in multiple myeloma by bioinformatics analysis.

PURPOSE: Multiple myeloma (MM) is a malignant disease with abnormal proliferation of clonal plasma cells. Hypoxia is an important factor in the pathogenesis and development of MM. However, the underlying mechanisms are not fully understood. MATERIAL & METHODS: To determine hub genes related to hypoxia in MM, this study took integrated bioinformatics analysis with two expression datasets (GSE80140 and GSE80545) downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were filtrated under the condition of both p-value < 0.05 and [log2FoldChange (log2FC)] > 1. Then, gene ontology (GO) and Kyoto encyclopedia of genes and genomes enrichment (KEGG) analysis, and protein-protein interaction (PPI) network construction were utilized to further explore these DEGs. PrognoScan evaluated all the candidate hub genes for survival analysis. RESULTS: In total, three hub genes, including FH, TSTA3, and POLR3G, were screened out to be related to hypoxia in MM. Patients with the lower expression level of FH, TSTA3, and POLR3G have statistically significantly longer disease- specific survival (Cox p < 0.05). CONCLUSION: We identified FH, TSTA3, and POLR3G as hub genes which can affect MM patients'outcome and new biomarkers for diagnosis and prognosis of MM. Further functional and mechanistic studies are need to develop in order to make them as potential target for clinical treatment.

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-D-glucose epimerization in Pseudomonas putida KT2440.

L-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-ß-L-rhamnose (dTDP-L-Rha). dTDP-L-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-L-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-L-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-keto-6-deoxy-L-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-L-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

Characterization of a Recombinant D-Allulose 3-epimerase from Thermoclostridium caenicola with Potential Application in D-Allulose Production.

In recent years, with the increasing public health awareness, low-calorie rare sugars have received more attention on a global scale. D-Allulose, the C-3 epimer of D-fructose, is a representative rare sugar. It displays high sweetness and excellent physiological functions, but only provides a caloric value of 0.4 kcal/g. D-Allulose 3-epimerase (DAEase) is indispensable in D-allulose production. In this study, a putative DAEase from Thermoclostridium caenicola was identified and characterized. The novel T. caenicola DAEase displayed maximum activity at pH 7.5 and 65 °C in the presence of 1 mM Co2+. The half-life (t1/2) at 50 °C was 13.6 h, and the melting temperature (Tm) was 62.4 °C. It was strictly metal-dependent, and the addition of Co2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t1/2 value at 55 °C and 4.8 °C increase in Tm. Furthermore, DAEase displayed high relative activity (89.0%) at a weakly acidic pH 6.5 and produced 139.8 g/L D-allulose from 500 g/L D-fructose, achieving a conversion ratio of 28.0%. These findings suggest that T. caenicola DAEase is a promising biocatalyst for the production of D-allulose.

D-Allulose 3-epimerase of Bacillus sp. origin manifests profuse heat-stability and noteworthy potential of D-fructose epimerization.

BACKGROUND: D-Allulose is an ultra-low calorie sugar of multifarious health benefits, including anti-diabetic and anti-obesity potential. D-Allulose 3-epimerase family enzymes catalyze biosynthesis of D-allulose via epimerization of D-fructose. RESULTS: A novel D-allulose 3-epimerase (DaeB) was cloned from a plant probiotic strain, Bacillus sp. KCTC 13219, and expressed in Bacillus subtilis cells. The purified protein exhibited substantial epimerization activity in a broad pH spectrum, 6.0-11.0. DaeB was able to catalyze D-fructose to D-allulose bioconversion at the temperature range of 35 °C to 70 °C, exhibiting at least 50 % activity. It displaced excessive heat stability, with the half-life of 25 days at 50 °C, and high turnover number (kcat 367 s- 1). The coupling of DaeB treatment and yeast fermentation of 700 g L- 1 D-fructose solution yielded approximately 200 g L- 1 D-allulose, and 214 g L- 1 ethanol. CONCLUSIONS: The novel D-allulose 3-epimerase of Bacillus sp. origin discerned a high magnitude of heat stability along with exorbitant epimerization ability. This biocatalyst has enormous potential for the large-scale production of D-allulose.

Tissue-specific transplantation antigen P35B functions as an oncogene and is regulated by microRNA-125a-5p in lung cancer.

Tissue­specific transplantation antigen P35B (TSTA3) expression is upregulated in esophageal squamous cell carcinoma and breast cancer, and functions as an oncogene in breast cancer. However, the roles and underlying mechanisms of TSTA3 in lung cancer have not been fully elucidated. The current study aimed to reveal the role of TSTA3 in lung cancer and explore whether TSTA3 may be modulated by microRNA (miR)­125a­5p to activate ß­catenin signaling. Immunohistochemical staining and western blotting were used to analyze TSTA3 expression in lung cancer tissues and cells. Cell functions were assessed via Cell Counting Kit­8, flow cytometry, wound­healing, Transwell and in vivo tumor formation assays. The effect of TSTA3 on the activation of ß­catenin signaling was determined using western blot and immunofluorescence analyses. The association between miR­125a­5p and TSTA3 was determined by western blotting and luciferase gene reporter assay. The present study revealed that, compared with normal tissues and cells, TSTA3 expression was significantly increased in lung cancer tissues and cell lines, and high TSTA3 expression predicted a poor prognosis and more malignant clinical features in patients with lung cancer. TSTA3 upregulation significantly enhanced ß­catenin expression and promoted its nuclear accumulation. In addition, TSTA3 expression was negatively regulated by miR­125a­5p, which was downregulated in lung cancer. Furthermore, TSTA3 overexpression markedly promoted cell proliferation, migration, invasion and tumorigenesis, and suppressed cell apoptosis. TSTA3 downregulation abolished the effects of miR­125a­5p downregulation on promoting lung cancer cell malignant transformation. Overall, the current study demonstrates that TSTA3 is regulated by miR­125a­5p and functions as an oncogene in lung cancer via promoting the activation of ß­catenin signaling.