Regulatory T cell expansion promotes white matter repair after stroke.

Recent research highlights the function of regulatory T cells (Tregs) in white matter integrity in CNS diseases. Approaches that expand the number of Tregs have been utilized to improve stroke recovery. However, it remains unclear if Treg augmentation preserves white matter integrity early after stroke or promotes white matter repair. This study evaluates the effect of Treg augmentation on white matter injury and repair after stroke. Adult male C57/BL6 mice randomly received Treg or splenocyte (2 million, iv) transfer 2 h after transient (60 min) middle cerebral artery occlusion (tMCAO). Immunostaining showed improved white matter recovery after tMCAO in Treg-treated mice compared to mice received splenocytes. In another group of mice, IL-2/IL-2 antibody complexes (IL-2/IL-2Ab) or isotype IgG were administered (i.p) for 3 consecutive days starting 6 h after tMCAO, and repeated on day 10, 20 and 30. The IL-2/IL-2Ab treatment boosted the number of Tregs in blood and spleen and increased Treg infiltration into the ischemic brain. Longitudinal in vivo and ex vivo diffusion tensor imaging analysis revealed an increase in fractional anisotropy 28d and 35d, but not 14d, after stroke in IL-2/IL-2Ab-treated mice compared to isotype-treated mice, suggesting a delayed improvement in white matter integrity. IL-2/IL-2Ab also improved sensorimotor functions (rotarod test and adhesive removal test) 35d after stroke. There were correlations between white matter integrity and behavior performance. Immunostaining confirmed the beneficial effects of IL-2/IL-2Ab on white matter structures 35d after tMCAO. IL-2/IL-2Ab treatment starting as late as 5d after stroke still improved white matter integrity 21d after tMCAO, suggesting long-term salutary effects of Tregs on the late-stage tissue repair. We also found that IL-2/IL-2Ab treatment reduced the number of dead/dying OPCs and oligodendrocytes in the brain 3d after tMCAO. To confirm the direct effect of Tregs on remyelination, Tregs were cocultured with lysophosphatidyl choline (LPC)-treated organotypic cerebella. LPC exposure for 17 h induced demyelination in organotypic cultures, followed by gradual spontaneous remyelination upon removal of LPC. Co-culture with Tregs accelerated remyelination in organotypic cultures 7d after LPC. In conclusion, Boosting the number of Tregs protects oligodendrocyte lineage cells early after stroke and promotes long-term white matter repair and functional recovery. IL-2/IL-2Ab represents a feasible approach of Treg expansion for stroke treatment.

Bexarotene Improve Depression-Like Behaviour in Mice by Protecting Against Neuro-inflammation and Synaptic Damage.

The growing number of evidences suggest that neuroinflammation and synaptic damage are closely related to the onset of depression. Bexarotene (Bex), a retinoid X receptor agonist, is an U.S. Food and Drug Administration-approved drug for the treatment of cutaneous T-cell lymphoma that has recently been reported to have anti-inflammatory and neuroprotective effects in several models of neurological disease including Parkinson's disease, Alzheimer's disease, and so forth. However, the effect of Bex on depression remains unclear. In this study, we investigated effect of Bex on depression-like behaviour in mice induced by lipopolysaccharide (LPS) or corticosterone (CORT). Our results showed that treatment with Bex for 15 days significantly improved LPS-induced depression-like behaviour in social interaction test and CORT-induced depression-like behaviour in forced swimming test and tail suspension test in mice. We found that the Bex treatment depressed the increase in the number of activated microglia and astrocytes in the frontal cortex, and the increase in the levels of inflammatory cytokines TNF-α, IL-1ß and IL-6 in LPS-injected mice. Furthermore, Bex treatment also rescued the decrease in the expression of BDNF, and inhibition of CREB/BDNF/ERK pathway, and improved the expression of synaptic related protein in CORT-induced mice. Based on these results, it is possible that Bex reversed depression-like behaviour in mice by reducing neuroinflammation and protecting against synaptic damage induced by LPS or CORT.

Colorimetric enzymatic determination of glucose based on etching of gold nanorods by iodine and using carbon quantum dots as peroxidase mimics.

Carbon quantum dots (CQDs) with peroxidase-mimicking activity were successfully prepared from litchi rind. A colorimetric method for glucose determination was developed based on etching of gold nanorods (GNRs) using CQDs as peroxidase mimetic. The glucose oxidase-catalyzed oxidation of glucose leads to the generation of H2O2 which oxidizes added iodide under formation of elemental iodine under the catalytic action of CQDs. Iodine then etches the GNRs along the longitudinal direction due to the higher reaction activities at the tips of GNRs. This results in a stepwise decrease in the maximum absorption wavelength of the GNRs, from initially 953 nm to finally 645 nm. Under the optimized conditions, the shift in the maximum absorption wavelength decreases linearly in the 0.01-2.0 mM glucose concentration range, and the detection limit is 3.0 µM. Importantly, this method was applied to the determination of glucose in human serum. It is perceived that the CQDs are valuable peroxidase mimics due to their ease of preparation, low costs and stable catalytic activity. Graphical abstract Carbon quantum dots were prepared from litchi rind. They can induce the oxidation of gold nanorods in the presence of I- ions and H2O2. This finding was applied to design a colorimetric assay for glucose.

Cu(II)-Catalyzed C-N Coupling of (Hetero)aryl Halides and N-Nucleophiles Promoted by α-Benzoin Oxime.

We first reported the new application of a translate metal chelating ligand α-benzoin oxime for improving Cu-catalyzed C-N coupling reactions. The system could catalyse coupling reactions of (hetero)aryl halides with a wide of nucleophiles (e.g., azoles, piperidine, pyrrolidine and amino acids) in moderate to excellent yields. The protocol allows rapid access to the most common scaffolds found in FDA-approved pharmaceuticals.

Cu(II)-Catalyzed Homocouplings of (Hetero)Arylboronic Acids with the Assistance of 2-O-Methyl-d-Glucopyranose.

This is the first report of a natural ligand improving the copper-catalyzed homocouplings of (hetero)arylboronic acids. Various important synthetic biaryl intermediates in organic synthesis could be assembled via this method. To gain insight into this reaction, in situ React IR technology was used to confirm the effectivity of this catalyst system. This protocol could provide important biaryl compounds in high yields within a short time.

Combination of oxaliplatin and S-1 versus sorafenib alone in patients with advanced hepatocellular carcinoma.

Sorafenib and conventional systemic cytotoxicity chemotherapy are currently being used in parallel for the patients with advanced hepatocellular carcinoma (HCC). While sorafenib has been proven to improve the prognosis in patients with this malignant disease, however, the outcome of other newly developed systemic chemotherapeutic regimens remains controversial. We evaluated the outcome and safety of patients treated with the SOX regimen (oxaliplatin + S-1) and those treated with sorafenib in a single-center cohort. This retrospective study involved a total of 46 patients with advanced HCC, 22 of which were treated with SOX regimen (oxaliplatin [130 mg/m2] on day 1 and S-1 [80 mg/m2/day] on day 1-14, every 3 weeks), and 24 were daily treated with sorafenib (400 mg, b.i.d.). The median progression-free survival was 3.6 months (95% confidence interval [CI], 1.7 to 5.6) with SOX and 1.7 months (95% CI, 1.5 to 1.9) with sorafenib, respectively (P = 0.444). The median overall survival in SOX and sorafenib group was 7.6 months (95% CI, 4.3 to 10.9) and 4.7 months (95% CI, 2.7 to 7.3), respectively (P = 0.246). Response rate was 22.2% with SOX and 5.6% with sorafenib, respectively (P = 0.154). The frequent side effects in SOX-treated patients were thrombocytopenia, elevation of transaminase levels and neuropathy, whereas hand-foot syndrome, diarrhea and pruritus were common in sorafenib-treated patients. These preliminary results suggest that the SOX regimen may serve as an effective treatment for patients with advanced HCC, and the treatment-related toxicities were generally well-tolerated.

[Study on preparation of total flavones in Glechoma longituba sustained-release tablets and its in vitro release].

OBJECTIVE: To study the preparation of total flavones in Glechoma longituba sustained-release tablets and evaluate its releasing features in vitro. METHODS: Orthogonal experiment (L(9)3(4)) was used to optimize the process of preparation of total flavones in Glechoma longituba sustained-release tablets. Investigated the release effect in vitro of optimization test result. RESULTS: The optimized prescription was HPMC-K4M and carbopol 934p account for 30% of the total tablet weight, their dosage ratio was 2: 1; Lactose as additives, 20% dosage; 5% PVP ethanol as adhesives, and compressed the wet granule of the materials into the total flavones in Glechoma longituba sustained-release tablets. The released profiles of the sustained-release tablets followed zero-order equation. CONCLUSION: The sustained-release effect of total flavones in Glechoma longituba sustained-release tablets is good, the preparation craft is easy and feasible and worth widely promoting.

Protective effect of 3-n-butylphthalide against intrastriatal injection of malonic acid-induced neurotoxicity and biochemical alteration in rats.

Mitochondrial abnormalities and a defective expression of neurotrophic factors contribute to neuronal damage in Huntington's disease (HD). HD patients showed a reduction in transforming growth factor-ß1 (TGF-ß1) levels in the peripheral blood and in cortical neurons. 3-n-butylphthalide (NBP) is first isolated from the seeds of celery, treats ischemic stroke in China. NBP could attenuate cognitive and motor impairments in the experimental models of Parkinson's disease and Alzheimer's disease, reduce mitochondrial oxidative stress and increase the expression of TGF-ß1 in rats with focal cerebral ischemia. To our knowledge, the effect of NBP on Huntington's disease has not been reported. We proposed the hypothesis that whether NBP could protect mitochondria and regulate TGF-ß1 and its downstream signaling in a HD animal model, further prevents motor dysfunction. Malonic acid is a reversible inhibitor of mitochondrial enzyme complex-II, induces energy crisis and free radical generation. In this study, we used intrastriatal injections of malonic acid in rats to mimic mitochondrial abnormalities and the other HD like symptoms. We found that treatment with NBP significantly attenuated malonic acid-induced motor and cognitive dysfunction in locomotor behaviour test, rotarod test, novel object recognition test and morris water maze test, prevented neurotoxicity and mitochondrial damage, activated TGF-ß1/Akt/Wnt/ß-Catenin pathway in striatum, but didn't regulate mitochondrial fusion and fission. The above effect was partly reversed by a PI3K/Akt inhibitor. Our data support NBP as a potential candidate for the treatment of HD.

IL-33/ST2 Axis Protects Against Traumatic Brain Injury Through Enhancing the Function of Regulatory T Cells.

Traumatic brain injury (TBI) is a devastating condition due to its long-term sequelae on neurological functions. Inflammatory responses after TBI are critical for injury expansion and repair. Recent research in central nervous system (CNS) disorders reveals the importance of IL-33 and its receptor (ST2) as an alarmin system to initiate immune responses. This study explored the role of IL-33/ST2 signaling in TBI. TBI was induced in adult male C57BL/6J mice using a controlled cortical impact (CCI) model. We found that the expression of IL-33 increased in the injured brain and blood, and ST2 was elevated in the circulating and infiltrating regulatory T cells (Tregs) early after TBI. ST2 deficient mice exhibited reduced Treg numbers in the blood and brain 5 days after TBI. The brain lesion size was enlarged in ST2 knockout mice, which was accompanied by deteriorated sensorimotor function 5 days after TBI. In contrast, post-TBI treatment with IL-33 (2 µg/30 g body weight, intranasal) for 3 days significantly reduced brain lesion size and improved neurological functions 5 days after TBI. Meanwhile, IL-33 treatment increased ST2 expression in circulating and brain infiltrating Tregs. To further explore the involvement of Tregs in IL-33/ST2-mediated neuroprotection, Tregs were depleted by CD25 antibody injection. The absence of Tregs significantly reduced the protective effect of IL-33 after TBI. In vitro study confirmed that IL-33 (50 ng/ml) increased the production of IL-10 and TGFß from activated Tregs and boosted the inhibitory effect of Tregs on T effector cell proliferation. Taken together, this study suggests that the activation of IL-33/ST2 signaling reduces brain lesion size and alleviates functional deficits after TBI at least partially through regulating the Treg response. IL-33 may represent a new immune therapeutic strategy to improve TBI outcomes.

Construction of differential expression plasmids of NGF to detect its influence on PC12 cell neuronal differentiation.

Alongside angiogenesis and lymphangiogenesis, neurogenesis also occurs within the cancer microenvironment. Neurogenesis is a complex process involving multiple factors, among which nerve growth factor (NGF) possesses the dual biological roles of neuron nutrition and axon growth promotion. Thus, NGF might be a key molecule involved in regulating cancer-related neurogenesis, which could play a crucial role in the signal transmission system that controls nerve growth in tumors, and enhances the abilities of migration, invasion and metastasis of tumor cells. The present study aimed to construct differential expression plasmids of NGF, in order to detect whether NGF has a vital role in neurogenesis in breast cancer cells. In the present study, 92 clinical cases of breast cancer were collected and immunohistochemical analysis was performed to verify the existence of neurons in the breast cancer microenvironment. Furthermore, recombinant NGF lentiviral overexpression, knockout and silencing plasmids were constructed, and whether NGF has an effect on neuron growth was preliminarily confirmed, indicating that the successfully constructed plasmids could be used to verify the roles of NGF in cancer-associated neurogenesis.

Dual-signal uric acid sensing based on carbon quantum dots and o-phenylenediamine.

Fluorescent carbon quantum dots (CQDs), which showed excitation-dependent emission characteristics, were prepared using a facile hydrothermal method. The structure and optical properties of CQDs were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, UV-Vis spectroscopy, and fluorescence spectroscopy. These CQDs also showed peroxidase-like activity and could catalyze the H2O2-mediated oxidation of o-phenylenediamine (OPD) to form 2,3-diaminophenazine (DAP) with an absorption peak at 420 nm. DAP exhibited an obvious fluorescence emission at 550 nm under the excitation of 360 nm. On the other hand, it decreased the fluorescence of CQDs at 450 nm via inner filter effect. The experimental results indicated that the H2O2 concentration affected the color of DAP and the fluorescence intensity of CQDs and DAP. Thus, a colorimetric and ratiometric fluorescence dual-signal method was established for measuring the concentrations of H2O2 and uric acid (UA). The effects of pH, incubation temperature, incubation time, and OPD concentration on the response were investigated. Under the conditions of pH 7.5, temperature 50 °C, incubation time 30 min, and OPD 1.5 mM, the absorbance and fluorescence intensity ratio responses were linearly dependent on UA concentration ranging from 5.0 µM to 100 µM. The limits of detection were 0.7 and 0.5 µM with a colorimetric method and ratiometric fluorescence method, respectively. More importantly, this dual responsive method has been applied to the determination of UA in urine samples with satisfactory results.

A fluorescein-gold nanoparticles probe based on inner filter effect and aggregation for sensing of biothiols.

Cysteine (Cys), homocysteine (HCys) and glutathione (GSH) are sulfhydryl-containing amino acids known as biothiols being able to bind to gold nanoparticles (AuNPs) via sulfhydryl group, resulting in the aggregation of AuNPs. Owning to their inner filter effect, AuNPs can weaken or even quench the fluorescence of fluorescein. However, the introduction of biothiols to fluorescein-AuNPs leads to the recovery of fluorescein fluorescence. Thus, a simple and reliable turn on fluorescence method was developed for monitoring biothiols with fluorescein-AuNPs as a probe. Several factors, including AuNPs concentration, pH value and incubation time, which might influence the fluorescence reclamation of fluorescein-AuNPs probe, were optimized by taking Cys as an example at room temperature. Under the optimal conditions, sensitive sensing of Cys, HCys and GSH was achieved. The detection limits for Cys, GSH, and HCys were 0.027, 0.023, and 0.030µΜ, respectively. This method was used to the determination of Cys in human serum samples with high precision and accuracy, indicating the potential of the method in practical applications with simple operation, good accuracy and high sensitivity.

Switch on fluorescence mode for determination of l-cysteine with carbon quantum dots and Au nanoparticles as a probe.

Citric acid and urea were used as precursors for the preparation of carbon quantum dots (CQDs) which exhibited a maximum emission wavelength at 515 nm when excited at 410 nm. Upon addition of citrate-stabilized Au nanoparticles (AuNPs) with the maximum absorption wavelength at 520 nm, the fluorescence of the CQDs could be efficiently quenched, attributed to the energy transfer between CQDs and AuNPs. However, the further introduction of l-cysteine (Cys) could cause the aggregation of AuNPs along with a drop in absorption at 520 nm, resulting in the fluorescence recovery of the CQDs-AuNPs system. Therefore, a simple and reliable switch on fluorescence sensing platform for determination of Cys was constructed. The significant factors, such as pH and incubation time, that affected the detection of Cys were optimized with the AuNP concentration set as 2.50 nM at room temperature. Under the optimized conditions, the fluorescence recoveries (ΔF) were strongly correlated with Cys concentration in the 0.20 to 4.0 µM range, and the detection limit is 0.012 µM. More importantly, our CQD-based sensing platform was successfully used for the detection of Cys in milk samples with high precision and accuracy, indicating the potential of the probe in practical applications.

Mechanisms of ebselen as a therapeutic and its pharmacology applications.

Ebselen is a synthetic organoselenium radical scavenger compound that possesses glutathione peroxidase-like activity and its own unique bioactivity by reacting with thiols, hydroperoxides and peroxynitrites. Owing to its high affinity toward several essential reactions, ebselen protects cellular components from oxidative and free radical damage, and it has been employed as a useful tool for studying redox-related mechanisms. Based on numerous in vitro and in vivo research, mechanisms are proposed to understand the biomedical and molecular actions of ebselen in health and disease, and it is currently under clinical trials for the prevention and treatment of various human disorders. Based on these outstanding discoveries, this review summarizes the current understanding of the biochemical and molecular characteristics, pharmacological applications and future directions of ebselen.

Identification and validation of a ten-gene set variation score as a diagnostic and prognostic stratification tool in hepatocellular carcinoma.

We aimed to identify a hepatocellular carcinoma (HCC)-specific gene set during progression. Using the HCC data set from The Cancer Genome Atlas, we found that 10 genes were gradually upregulated with the progression of HCC and associated with survival, classified as HCC-unfavorable genes; 29 genes were gradually downregulated and associated with survival, classified as HCC-favorable genes. Gene set variation analysis (GSVA) was used to score individual samples against the two gene sets. Receiver operating characteristic (ROC) curve analysis showed that both the HCC-unfavorable GSVA score and HCC-favorable GSVA score were reliable biomarkers for diagnosing HCC. Moreover, tROC curve analysis and univariate/multivariate Cox proportional hazards analyses indicated that the HCC-unfavorable GSVA score was an independent prognostic biomarker. The results were validated in an external independent data set. Our results support a ten-gene set variation score as a diagnostic and predictive strategy tool in HCC.

[Studies on purification process of total flavones from Glechoma longituba with macroporous adsorption resin].

OBJECTIVE: To study the purification process of total flavones from Glechoma longituba with macroporous adsorption resin. METHODS: The eluting power and purified ratio of total flavones were selected as indices, the adsorbility and eluting parameters of the process were studied. RESULTS: The best result that the purity of total flavones from Glechoma longituba could reach 68. 94% was based on the followings: total flavones content in the liquid-0.383 mg/mL, feeding volume--20 mL, feeding rate--1.0 mL/min, eluting agent-5BV purify water and 10BV 30% ethanol, eluting rate--1.0 mL/min. CONCLUSION: The process is simple and convenient which can be used in the purification of total flavones from Glechoma longituba, it may has a good application foreground.

Colorimetric detection of hydrogen peroxide and glucose by exploiting the peroxidase-like activity of papain.

Papain, a natural plant protease that exists in the latex of Carica papaya, catalyzes the hydrolysis of peptide, ester and amide bonds. In this work, we found that papain displayed peroxidase-like activity and catalyzed the oxidation of 3,3',5',5'-tetramethylbenzidine (TMB) in the presence of H2O2. This results in the formation of a blue colored product with an absorption maximum at 652 nm. The effects of experimental parameters including pH and reaction temperature on catalytic activity of papain were investigated. The increase of absorbance induced by the catalytic effect of papain offers accurate detection of H2O2 in the range of 5.00-90.0 µM, along with a detection limit of 2.10 µM. A facile colorimetric method for glucose detection was also proposed by combining the glucose oxidase (GOx)-catalyzed glucose oxidation and papain-catalyzed TMB oxidation, which exhibited a linear response in the range of 0.05-0.50 mM with a detection limit of 0.025 mM. The method proposed here displayed excellent selectivity, indicating that common coexisting substances (urea, uric acid, ascorbic acid, maltose, lactose and fructose) in urine did not interfere with detection of glucose. More importantly, the suggested method was successfully used to precisely detect the glucose concentration in human urine samples with recoveries over 96.0%.

Expression and gene regulation network of RBM8A in hepatocellular carcinoma based on data mining.

RNA binding motif protein 8A (RBM8A) is an RNA binding protein in a core component of the exon junction complex. Abnormal RBM8A expression is associated with carcinogenesis. We used sequencing data from the Cancer Genome Atlas database and Gene Expression Omnibus, analyzed RBM8A expression and gene regulation networks in hepatocellular carcinoma (HCC). Expression was analyzed using OncomineTM and Gene Expression Profiling Interactive Analysis tools, while RBM8A alterations and related functional networks were identified using cBioPortal. LinkedOmics was used to identify differential gene expression with RBM8A and to analyze Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Gene enrichment analysis examined target networks of kinases, miRNAs and transcription factors. We found that RBM8A is overexpressed and the RBM8A gene often amplified in HCC. Expression of this gene is linked to functional networks involving the ribosome and RNA metabolic signaling pathways. Functional network analysis suggested that RBM8A regulates the spliceosome, ribosome, DNA replication and cell cycle signaling via pathways involving several cancer-related kinases, miRNAs and E2F Transcription Factor 1. Our results demonstrate that data mining efficiently reveals information about RBM8A expression and potential regulatory networks in HCC, laying a foundation for further study of the role of RBM8A in carcinogenesis.

OAB-14, a bexarotene derivative, improves Alzheimer's disease-related pathologies and cognitive impairments by increasing ß-amyloid clearance in APP/PS1 mice.

The pathogenesis of Alzheimer's disease (AD) is complex, though the clinical failures of anti-AD candidates targeting Aß production (such as ß- and γ-secretase inhibitors) make people suspect the Aß hypothesis, in which the neurotoxicity of Aß is undoubtedly involved. According to studies, >95% of AD patients with sporadic AD are primarily associated with abnormal Aß clearance. Therefore, drugs that increase Aß clearance are becoming new prospects for the treatment of AD. Here, the novel small molecule OAB-14, designed using bexarotene as the lead compound, significantly alleviated cognitive impairments in amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mice after administration for 15 days or 3 months. OAB-14 rapidly cleared 71% of Aß by promoting microglia phagocytosis and increasing IDE and NEP expression. This compound also attenuated the downstream pathological events of Aß accumulation, such as synaptic degeneration, neuronal loss, tau hyperphosphorylation and neuroinflammation in APP/PS1 mice. Moreover, OAB-14 had no significant effect on body weight or liver toxicity after acute and chronic treatment. OAB-14 was well tolerated and its maximum-tolerated dose in mice was >4.0 g/kg. Based on these findings, OAB-14 represents a promising new candidate for AD treatment.

Four-miRNA signature as a prognostic tool for lung adenocarcinoma.

PURPOSE: The aim of this study was to generate a novel miRNA expression signature to accurately predict prognosis for patients with lung adenocarcinoma (LUAD). PATIENTS AND METHODS: Using expression profiles downloaded from The Cancer Genome Atlas database, we identified multiple miRNAs with differential expression between LUAD and paired healthy tissues. We then evaluated the prognostic values of the differentially expressed miRNAs using univariate/multivariate Cox regression analysis. This analysis was ultimately used to construct a four-miRNA signature that effectively predicted patient survival. Finally, we analyzed potential functional roles of the target genes for these four miRNAs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. RESULTS: Based on our cutoff criteria (P<0.05 and |log2FC| >1.0), we identified a total of 187 differentially expressed miRNAs, including 148 that were upregulated in LUAD tissues and 39 that were downregulated. Four miRNAs (miR-148a-5p, miR-31-5p, miR-548v, and miR-550a-5p) were independently associated with survival based on Kaplan-Meier analysis. We generated a signature index based on the expression of these four miRNAs and stratified patients into low- and high-risk groups. Patients in the high-risk group had significantly shorter survival times than those in the low-risk group (P=0.002). A functional enrichment analysis suggested that the target genes of these four miRNAs were involved in protein phosphorylation and the Hippo and sphingolipid signaling pathways. CONCLUSION: Taken together, our results suggest that our four-miRNA signature can be used as a prognostic tool for patients with LUAD.