Drugs/agents for the treatment of ischemic stroke: Advances and perspectives.

Ischemic stroke (IS) poses a significant threat to global human health and life. In recent decades, we have witnessed unprecedented progresses against IS, including thrombolysis, thrombectomy, and a few medicines that can assist in reopening the blocked brain vessels or serve as standalone treatments for patients who are not eligible for thrombolysis/thrombectomy therapies. However, the narrow time windows of thrombolysis/thrombectomy, coupled with the risk of hemorrhagic transformation, as well as the lack of highly effective and safe medications, continue to present big challenges in the acute treatment and long-term recovery of IS. In the past 3 years, several excellent articles have reviewed pathophysiology of IS and therapeutic medicines for the treatment of IS based on the pathophysiology. Regretfully, there is no comprehensive overview to summarize all categories of anti-IS drugs/agents designed and synthesized based on molecular mechanisms of IS pathophysiology. From medicinal chemistry view of point, this article reviews a multitude of anti-IS drugs/agents, including small molecule compounds, natural products, peptides, and others, which have been developed based on the molecular mechanism of IS pathophysiology, such as excitotoxicity, oxidative/nitrosative stresses, cell death pathways, and neuroinflammation, and so forth. In addition, several emerging medicines and strategies, including nanomedicines, stem cell therapy and noncoding RNAs, which recently appeared for the treatment of IS, are shortly introduced. Finally, the perspectives on the associated challenges and future directions of anti-IS drugs/agents are briefly provided to move the field forward.

Loss of PTPRS elicits potent metastatic capability and resistance to temozolomide in glioblastoma.

Glioblastoma (GBM) is the most aggressive brain tumor type with worse clinical outcome due to the hallmarks of strong invasiveness, high rate of recurrence, and therapeutic resistance to temozolomide (TMZ), the first-line drug for GBM, representing a major challenge for successful GBM therapeutics. Understanding the underlying mechanisms that drive GBM progression will shed novel insight into therapeutic strategies. Receptor-type tyrosine-protein phosphatase S (PTPRS) is a frequently mutated gene in human cancers, including GBM. Its role in GBM has not yet been clarified. Here, inactivating PTPRS mutation or deficiency was frequently found in GBM, and deficiency in PTPRS significantly induced defects in the G2M checkpoint and limited GBM cells proliferation, leading to potent resistance to TMZ treatment in vitro and in vivo. Surprisingly, loss of PTPRS triggered an unexpected mesenchymal phenotype that markedly enhances the migratory capabilities of GBM cells through upregulating numerous matrix metalloproteinases via MAPK-MEK-ERK signaling. Therefore, this work provides a therapeutic window for precisely excluding PTPRS-mutated patients who do not respond to TMZ.

Calcinosis universalis accompanied by symptomatic systemic sclerosis.

Calcinosis cutis is represented by the deposition of insoluble calcium salts in the skin and subcutaneous tissue. Calcinosis can lead to repeated episodes of local inflammation and repeated infections, resulting in pain and functional disability, and even death. Here, we present a case of a patient with SSc who experienced calcinosis universalis and eventually died from recurrent infections at the sacrococcygeal calcification.

Integrating Metabolomics and Network Pharmacology to Decipher the Hepatoprotective Effect Mechanisms of Magnesium Isoglycyrrhizinate Injection.

This study aimed to explore the liver protective effects of a fourth-generation glycyrrhizic acid product (magnesium isoglycyrrhizinate injection, MII) in the treatment of mice with drug-induced liver injury-specifically, to determine its effects on plasma metabolites. Moreover, the possible mechanism of its intervention in lipid metabolism and amino acid metabolism through the liver protective effect was preliminarily explored, combined with network pharmacology. The liver injury model of mice was established using acetaminophen (APAP). The protective effect of MII on the mice model was evaluated using pathological tissue sections and biochemical indices such as alanine transaminase (ALT), aspartate aminotransferase (AST), and superoxide dismutase (SOD). Metabolomics analysis of plasma was performed using the UHPLC-QTOF/MS technique to screen for potential biomarkers and enriched metabolic pathways. The potential targets and pathways of MII were predicted by network pharmacology, and the mechanism was verified by Western blot analysis. MII significantly improved the pathological liver changes in mice with liver injury. The content of ALT and AST was decreased, and the activity of SOD was increased significantly (p < 0.05, 0.01). A total of 29 potential biomarkers were identified in the metabolomics analysis, mainly involving seven pathways, such as lipid metabolism and amino acid metabolism. A total of 44 intersection targets of MII in the treatment of liver injury were obtained by network pharmacology, involving lipid metabolism and other related pathways. Western blot analysis results showed that MII could significantly reduce the expression of JAK2 and STAT3. MII can effectively ameliorate liver injury in modeled mice through related pathways such as lipid metabolism and amino acid metabolism. This study could provide not only a scientific basis for the elucidation of the mechanism of action of MII in exerting a hepatoprotective effect, but also a reference for its rational clinical application.

Simultaneous Detection of Two Extracellular Vesicle Subpopulations in Saliva Assisting Tumor T Staging of Oral Squamous Cell Carcinoma.

Extracellular vesicles (EVs), acting as important mediators of intercellular communication, play an essential role in physiological processes, which have unique potential in the medical field. However, the heterogeneity of EVs limits their development for disease diagnosis and therapy, making the EV subpopulation analysis extremely valuable. In this article, a simple microfluidic approach was presented for the on-chip specific isolation and detection of two phenotypes of EVs (Annexin V+ EGFR+ EVs and Annexin V- EGFR+ EVs) based on different biomolecule-modified magnetic nanospheres and a fluorescence labeling technique. Combined with the control of the magnetic field in the microzone and fluid flow, it was easy to form two separate functional regions in the chip to capture different EV subpopulations. This method was successfully applied to the tests of clinical saliva samples in 75 oral squamous cell carcinoma (OSCC) patients and 10 healthy people. The results showed that the total level of EGFR+ EVs was much higher in OSCC patients that in healthy people. Meantime, the ratio of Annexin V+ EGFR+ EVs to Annexin V- EGFR+ EVs was found to be negatively correlated with tumor T stage of OSCC patients with a statistical difference, which suggested the ratio as a clinical index for monitoring the progression of OSCC in real time based on a noninvasive method. The approach provided a novel idea for evaluating the tumor T stage of OSCC and a powerful tool for clinical application.

Helicobacter pylori CagA Interacts with SHP-1 to Suppress the Immune Response by Targeting TRAF6 for K63-Linked Ubiquitination.

Helicobacter pylori is the major etiological agent for most gastric cancer. CagA has been reported to be an important virulence factor of H. pylori, but its effect on the immune response is not yet clear. In this study, wild-type C57BL/6 mice and Ptpn6me-v/me-v mice were randomly assigned for infection with H. pylori We demonstrated that CagA suppressed H. pylori-stimulated expression of proinflammatory cytokines in vivo. Besides, we infected mouse peritoneal macrophages RAW264.7 and AGS with H. pylori Our results showed that CagA suppressed expression of proinflammatory cytokines through inhibiting the MAPKs and NF-κB pathways activation in vitro. Mechanistically, we found that CagA interacted with the host cellular tyrosine phosphatase SHP-1, which facilitated the recruitment of SHP-1 to TRAF6 and inhibited the K63-linked ubiquitination of TRAF6, which obstructed the transmission of signal downstream. Taken together, these findings reveal a previously unknown mechanism by which CagA negatively regulates the posttranslational modification of TRAF6 in innate antibacterial immune response and provide molecular basis for new therapeutics to treat microbial infection.

Hydrogen Sulfide Alleviates Liver Injury Through the S-Sulfhydrated-Kelch-Like ECH-Associated Protein 1/Nuclear Erythroid 2-Related Factor 2/Low-Density Lipoprotein Receptor-Related Protein 1 Pathway.

BACKGROUND AND AIMS: Protein S-sulfhydration mediated by H2 S has been shown to play important roles in several diseases. However, its precise role in liver disease and the related mechanism remain unclear. APPROACH AND RESULTS: We showed that in streptozotocin (STZ)-treated and high-fat diet (HFD)-treated low-density lipoprotein receptor-negative (LDLr-/- ) mice, the H2 S donor GYY4137 ameliorated liver injury, decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, mitigated lipid deposition, and reduced hepatocyte death. Strikingly, S-sulfhydration of Kelch-like ECH-associated protein 1 (Keap1) was decreased in the livers of patients with fatty liver under diabetic conditions. In STZ+HFD-treated LDLr-/- mice and in high glucose-treated and oxidized low-density lipoprotein (ox-LDL)-treated primary mouse hepatocytes, the GYY4137-mediated increase in Keap1 S-sulfhydration induced nuclear erythroid 2-related factor 2 (Nrf2) dissociation from Keap1, which enhanced the nuclear translocation of Nrf2 itself and the consequent expression of antioxidant proteins. Keap1 Cys151 mutation significantly reduced Keap1 S-sulfhydration and abolished the hepatoprotective effects of H2 S both in vivo and in vitro. Nrf2 deficiency inhibited the H2 S-induced beneficial impacts in Nrf2-/- mice. Similarly, in CCl4 -stimulated mice, GYY4137 increased Keap1 S-sulfhydration, improved liver function, alleviated liver fibrosis, decreased hepatic oxidative stress, and activated the Nrf2 signaling pathway; and these effects were abrogated after Keap1 Cys151 mutation. Moreover, H2 S increased the binding of Nrf2 to the promoter region of LDLr-related protein 1 (Lrp1) and consequently up-regulated LRP1 expression, but these effects were disrupted by Keap1 Cys151 mutation. CONCLUSIONS: H2 S-mediated Keap1 S-sulfhydration alleviates liver damage through activation of Nrf2. Hence, administration of exogenous H2 S in the form of the H2 S donor GYY4137 may be of therapeutic benefit in the context of concurrent hyperlipidemia and hyperglycemia-induced or CCl4 -stimulated liver dysfunction.

A synthesized olean-28,13ß-lactam targets YTHDF1-GLS1 axis to induce ROS-dependent metabolic crisis and cell death in pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is a severe malignant with a 5-year survival rate of approximately 9%. Oleanolic acid is a well-known natural triterpenoid which exhibits pharmacological activities. We previously synthesized a series of oleanolic acid derivatives and evaluated the tumor-suppressive activity of olean-28,13ß-lactam (B28) in prostate cancer. However, the detailed mechanism remains to be understood. METHODS: The anti-tumor activity of B28 in PAAD was confirmed by RTCA, colony formation assay and flow cytometry. GO and KEGG enrichment analyses were performed to analyze the differentially expressed genes (DEGs) obtained by RNA sequencing. The effects of B28 on cell bioenergetics were evaluated by seahorse analyzer. Lenti-virus packaged plasmids were performed to knockdown or overexpress target genes. Alteration of mitochondrial membrane potential, ROS and GSH/GSSG were measured by corresponding detection kits according to the manufacturer's protocol. RESULTS: We evaluated and confirmed the promising anti-tumor activity of B28 in vitro. RNA-seq profile indicated that multiple metabolic pathways were interrupted in B28 treated PAAD cells. Next, we demonstrated that B28 induces cellular bioenergetics crisis to inhibit PAAD cells growth and induce cell death. We further validated that cell cycle arrest, inhibition of cell growth, cell apoptosis and cell bioenergetics disruption were functionally rescued by ROS scavenger NAC. Mechanistically, we found glutamine metabolism was inhibited due to B28 administration. Moreover, we validated that down-regulation of GLS1 contributes to ROS generation and bioenergetics interruption induced by B28. Furthermore, we elucidated that YTHDF1-GLS1 axis is the potential downstream target of B28 to induce PAAD cell metabolic crisis and cell death. Finally, we also confirmed the anti-tumor activity of B28 in vivo. CONCLUSIONS: Current study demonstrates B28 disrupts YTDFH1-GLS1 axis to induce ROS-dependent cell bioenergetics crisis and cell death which finally suppress PAAD cell growth, indicating that this synthesized olean-28,13ß-lactam maybe a potent agent for PAAD intervention.

Role of store-operated Ca2+ entry in cardiovascular disease.

Store-operated channels (SOCs) are highly selective Ca2+ channels that mediate Ca2+ influx in non-excitable and excitable (i.e., skeletal and cardiac muscle) cells. These channels are triggered by Ca2+ depletion of the endoplasmic reticulum and sarcoplasmic reticulum, independently of inositol 1,4,5-trisphosphate (InsP3), which is involved in cell growth, differentiation, and gene transcription. When the Ca2+ store is depleted, stromal interaction molecule1 (STIM1) as Ca2+ sensor redistributes into discrete puncta near the plasma membrane and activates the protein Ca2+ release activated Ca2+ channel protein 1 (Orai1). Accumulating evidence suggests that SOC is associated with several physiological roles in endothelial dysfunction and vascular smooth muscle proliferation that contribute to the progression of cardiovascular disease. This review mainly elaborates on the contribution of SOC in the vasculature (endothelial cells and vascular smooth muscle cells). We will further retrospect the literature implicating a critical role for these proteins in cardiovascular disease. Video Abstract.

Synthesis and biological evaluation of hybrids from optically active ring-opened 3-N-butylphthalide derivatives and 4-fluro-edaravone as potential anti-acute ischemic stroke agents.

The treatment of acute ischemic stroke (AIS) remains a tough challenge in clinic. Here, we report the anti-AIS activity of R- and S-FMPB generated from hybridization of ring-opened R- and S-3-N-butylphthalide (R- and S-NBP) derivatives (R- and S-APB) with 4-fluro-edaravone (4-F-Eda), respectively. S-FMPB (10 mg/kg, iv) significantly improved the neurological score and alleviated cerebral infarction and edema of rats suffered from transient middle cerebral artery occlusion (tMCAO), superior to RS- and R-FMPB, as well as better than RS-FMPB by oral administration in previous studies. Importantly, S-FMPB is more active not only than the equimolar S-APB and 4-F-Eda alone or in combination but also than the clinical drugs NBP and edaravone (Eda) in combination at the equimolar doses. Furthermore, S-FMPB showed relative stability in plasma or liver microsome of rats but could be converted into two active metabolites (S-NBP and 4-F-Eda) in rats with good pharmacokinetic properties in terms of longer half-life period (t1/2) and mean residence time (MRT) as well as larger area under the concentration-time curve (AUC) of S-NBP than those from S-NBP and 4-F-Eda single or in combination by iv administration, suggesting that S-NBP and 4-F-Eda may synergistically play the anti-AIS activity. Our findings suggest that S-FMPB may be used as a potential anti-AIS agent to further study.

A novel naphthalimide-based fluorescent probe for the colorimetric and ratiometric detection of SO2 derivatives in biological imaging.

SO2 is a well-known signal molecule and one of reactive sulfur species, which closely participates in many metabolic processes. While unbalanced metabolism of sulfur dioxide can lead to serious complications of various diseases. Therefore, a rapid and accurate monitoring of SO2 derivatives with high selectivity and sensitivity would be beneficial for their bio-analytic studies. Herein, a novel ratiometric fluorescent probe (NG-TCF) based on ICT mechanism for monitoring SO2 was developed. The probe underwent a nucleophilic addition of HSO3-/SO32- to give rise to a 120 nm blue-shift dual-emission signal changes in enhanced green channel and subdued red channel under a single wavelength excitation. The probe showed fast response rate (within 7 min), good sensitivity (the detection limit is 1.53 µM), and specific response toward HSO3-/SO32- over other bio-species, including H2S and ClO-. Moreover, the probe can be applied for visual ratio imaging of exogenous and endogenous SO2 derivatives in living cells.

NSun2 promotes cell migration through methylating autotaxin mRNA.

NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3'-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.

Transcriptome analysis of the transdifferentiation of canine BMSCs into insulin producing cells.

BACKGROUND: Bone marrow mesenchymal stem cells are a potential resource for the clinical therapy of certain diseases. Canine, as a companion animal, living in the same space with human, is an ideal new model for human diseases research. Because of the high prevalence of diabetes, alternative transplantation islets resource (i.e. insulin producing cells) for diabetes treatment will be in urgent need, which makes our research on the transdifferentiation of Bone marrow mesenchymal stem cells into insulin producing cells become more important. RESULT: In this study, we completed the transdifferentiation process and achieved the transcriptome profiling of five samples with two biological duplicates, namely, "BMSCs", "islets", "stage 1", "stage 2" and "stage 3", and the latter three samples were achieved on the second, fifth and eighth day of induction. A total of 11,530 differentially expressed transcripts were revealed in the profiling data. The enrichment analysis of differentially expressed genes revealed several signaling pathways that are essential for regulating proliferation and transdifferentiation, including focal adhesion, ECM-receptor interaction, tight junction, protein digestion and absorption, and the Rap1 signaling pathway. Meanwhile, the obtained protein-protein interaction network and functional identification indicating involvement of three genes, SSTR2, RPS6KA6, and VIP could act as a foundation for further research. CONCLUSION: In conclusion, to the best of our knowledge, this is the first survey of the transdifferentiation of canine BMSCs into insulin-producing cells according with the timeline using next-generation sequencing technology. The three key genes we pick out may regulate decisive genes during the development of transdifferentiation of insulin producing cells.

SHP-1 suppresses the antiviral innate immune response by targeting TRAF3.

Type I interferons play a pivotal role in innate immune response to virus infection. The protein tyrosine phosphatase SHP-1 was reported to function as a negative regulator of inflammatory cytokine production by inhibiting activation of NF-κB and MAPKs during bacterial infection, however, the role of SHP-1 in regulating type I interferons remains unknown. Here, we demonstrated that knockout or knockdown of SHP-1 in macrophages promoted both HSV-1- and VSV-induced antiviral immune response. Conversely, overexpression of SHP-1 in L929 cells suppressed the HSV-1- and VSV-induced immune response; suppression was directly dependent on phosphatase activity. We identified a direct interaction between SHP-1 and TRAF3; the association between these two proteins resulted in diminished recruitment of CK1ε to TRAF3 and inhibited its K63-linked ubiquitination; SHP-1 inhibited K63-linked ubiquitination of TRAF3 by promoting dephosphorylation at Tyr116 and Tyr446. Taken together, our results identify SHP-1 as a negative regulator of antiviral immunity and suggest that SHP-1 may be a target for intervention in acute virus infection.

Study on the Dynamic Proliferation of JEV in BHK-21 Cells.

INTRODUCTION: Epidemic Japanese encephalitis is one of the most important zoonotic diseases that cause central nervous system damage. The vaccination has become the most effective and economical measure for its control. Hence, real-time monitoring of Japanese encephalitis virus (JEV) proliferation is crucial to optimize virus inoculation, culturing conditions, and virus harvest time. METHODS: The proliferation dynamics of JEV in BHK-21 cells was studied by combining the established quantitative PCR method with the conventional TCID50 assay in this study. RESULTS: The proliferation curve determined by the 2 methods has a definite parallel relationship, but the quantitative real-time PCR method (4 h) is faster and more sensitive than the TCID50 method (3-4 days). The determination results of TCID50 showed that the highest viral titer was 105.44 TCID50/0.1 mL and 104.86 TCID50/0.1 mL in cell suspension and culture supernate, respectively, while the virus RNA copies reached the peak at 1.0 × 107.5 copies/µL and 1.0 × 105.6 copies/µL in cell suspension and culture supernate, respectively. CONCLUSION: The comprehensive analysis showed that the best time for JEV proliferation in BHK-21 cell was 60 h post infection.

Correction: Synthesis and biological evaluation of hybrids from farnesylthiosalicylic acid and hydroxylcinnamic acid with dual inhibitory activities of Ras-related signaling and phosphorylated NF-κB.

Correction for 'Synthesis and biological evaluation of hybrids from farnesylthiosalicylic acid and hydroxylcinnamic acid with dual inhibitory activities of Ras-related signaling and phosphorylated NF-κB' by Yong Ling et al., Org. Biomol. Chem., 2014, 12, 4517-4530, DOI: 10.1039/C4OB00023D.

Purification of cell-derived Japanese encephalitis virus by dual-mode chromatography.

Purification of the enveloped virus poses a challenge as one must retain viral infectivity to preserve immunogenicity. The traditional process of virus purification is time-consuming, laborious and hard to scale up. Here, a rapid, simple and extensible laboratory program for the purification of Japanese encephalitis virus (JEV) was developed by using differential centrifugation, ultrafiltration, Sepharose 4 fast flow gel chromatography, and CaptoTM Core 700 chromatography. The entire process recovered 61.64% of the original virus, and the purified virus particles maintained good activity and immunogenicity. The purification process described has potential application in large-scale production of high-purity JEV.

Candidate gene prioritization for chronic obstructive pulmonary disease using expression information in protein-protein interaction networks.

BACKGROUND: Identifying or prioritizing genes for chronic obstructive pulmonary disease (COPD), one type of complex disease, is particularly important for its prevention and treatment. METHODS: In this paper, a novel method was proposed to Prioritize genes using Expression information in Protein-protein interaction networks with disease risks transferred between genes (abbreviated as PEP). A weighted COPD PPI network was constructed using expression information and then COPD candidate genes were prioritized based on their corresponding disease risk scores in descending order. RESULTS: Further analysis demonstrated that the PEP method was robust in prioritizing disease candidate genes, and superior to other existing prioritization methods exploiting either topological or functional information. Top-ranked COPD candidate genes and their significantly enriched functions were verified to be related to COPD. The top 200 candidate genes might be potential disease genes in the diagnosis and treatment of COPD. CONCLUSIONS: The proposed method could provide new insights to the research of prioritizing candidate genes of COPD or other complex diseases with expression information from sequencing or microarray data.

Variation in textural parameters of hepatic lesions during contrast medium injection.

BACKGROUND: Textural parameters extracted using quantitative imaging techniques have been shown to have prognostic value for hepatocellular carcinoma (HCC). PURPOSE: To evaluate whether the contrast medium timing of the image acquisition affects the reproducibility of textural parameters in HCC and hepatic tissue. MATERIAL AND METHODS: This retrospective study included 17 patients with 37 HCC lesions. Perfusion computed tomography (CT) was obtained after 50 mL contrast medium injection. HCC lesions were segmented for analysis. The gray-level co-occurrence (GLCM) textural analysis parameters, homogeneity, energy, entropy, inertia, and correlation were calculated. Variation was quantified by calculating the SD of each parameter during respective perfusion series and the inter lesion variation as the SD among the lesions. RESULTS: The average change in texture parameters in both HCC and hepatic tissue per second after injection was 0.01% to 0.3% of the respective texture parameter. In HCC, the average variation in homogeneity, energy, and entropy within each lesion after contrast medium injection was significantly less than the variation observed among the lesions (23% to 74%, P < 0.001). Significant differences in energy, entropy, inertia, and correlation between hepatic tissue and HCC were observed. However, when considering the intra-individual variation of hepatic tissue over time, only the HCC parameter energy was significantly outside that 95% confidence interval (P < 0.02). CONCLUSION: The contrast medium timing does not affect the reproducibility of textural parameters in HCC and hepatic tissue. Thus, contrast medium timing should not be an issue at CT texture analysis of HCC.

Network-based integration method for potential breast cancer gene identification.

Breast cancer is the most common female death-causing cancer worldwide. A network-based integration method was proposed to identify potential breast cancer genes. First, genes were prioritized using a gene prioritization algorithm by the strategy of disease risks transferred between genes in a network with weighted vertexes and edges. Our prioritization algorithm was effectives and robust for top-ranked seed gene number and higher area under the curve values compared to ToppGene and ToppNet. Then, 20 potential breast cancer genes were identified as common genes of the top 50 candidate genes for their robustness in multiple prioritizations. These genes could accurately classify tumor and normal samples of all and paired sample sets and three independent datasets. Of potential breast cancer genes, 18 were verified by literature and 2 were novel genes that need further study. This study would contribute to the understanding of the genetic architecture for the diagnosis and treatment of breast cancer.