Shuanying Yang

Analysis of the expression protein profiles of lung squamous carcinoma cell using shot-gun proteomics strategy

The aim of this study is to globally screen and identify the expression protein profiles of lung squamous carcinoma cell (SqCC) using shot-gun proteomics strategy and to further analyze function of individual proteins by bioinformatics, which may likely result in the identification of new biomarkers and provide helpful clues for pathogenesis, early diagnosis, and progression of lung SqCC. The specific tumor cells were isolated and collected from the tissues of six patients with lung SqCC by laser capture microdissection (LCM). Total proteins from the LCM cells were extracted, digested with trypsin. The sequence information of resulting peptides was acquired by high-performance liquid chromatography (HPLC) and tandem mass spectrometry (TMS). The global protein profiles of lung SqCC cell were identified with BioworksTM software in IPI human protein database. Cellular component, molecular function, and biological process of the all proteins were analyzed using gene ontology (GO). About 720,000 tumor cells were satisfactorily collected from tissues of six patients with lung SqCC by LCM and the homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. The high resolution profiles including HPLC, full mass spectrum, and tandem mass spectrum were successfully obtained. Database searching of the resulting bimolecular sequence information identified 1982 proteins in all samples. The bioinformatics of these proteins, including amino acids sequence, fraction of coverage, molecular weight, isoelectric point, etc., were analyzed in detail. Among them, the function of most proteins was recognized by using GO. Five candidate proteins, Prohibitin (PHB), Mitogen-activated protein kinase (MAPK), Heat shock protein27 (HSP27), Annexin A1(ANXA1), and High mobility group protein B1 (HMGB1), might play an important role in SqCC genesis, progression, recurrence, and metastasis according to relative literatures. We have successfully isolated the interesting cells and effectively solved the heterogeneous problem of lung SqCC using LCM. The globally expressional proteins of lung SqCC cell were identified by shot-gun proteomics strategy. The five proteins might be hopefully used as markers of lung SqCC.

Use of anchorchip-time-of-flight spectrometry technology to screen tumor biomarker proteins in serum for small cell lung cancer

BackgroundThe purpose of this study is to discover potential biomarkers in serum for the detection of small cell lung cancer (SCLC).Methods74 serum samples including 30 from SCLC patients and 44 from healthy controls were analyzed using ClinProt system combined with matrix-assisted laser desorption/ionization time-of-flight masss spectrometry (MALDI-TOF-MS). ClinProt software and genetic algorithm analysis selected a panel of serum markers that most efficiently predicted which patients had SCLC.ResultsThe diagnostic pattern combined with 5 potential biomarkers could differentiate SCLC patients from healthy persons, with a sensitivity of 90%, specificity of 97.73%. Remarkably, 88.89% of stage I/II patients were accurately assigned to SCLC.ConclusionsAnchorchip-time-of-flight spectrometry technology will provide a highly accurate approach for discovering new biomarkers for the detection of SCLC.

Identification of microRNAs as potential biomarkers for lung adenocarcinoma using integrating genomics analysis

Lung adenocarcinoma (LUAD) is the most common histological subtype of non-small cell lung cancer, but novel biomarkers for early diagnosis are lacking. Extensive effort has been exerted to identify miRNA biomarkers in LUAD. Unfortunately, high inter-lab variability and small sample sizes have produced inconsistent conclusions in this field. To resolve the above-mentioned limitations, we performed a comprehensive analysis based on LUAD miRNome profiling studies using the robust rank aggregation (RRA) method. Moreover, miRNA-gene interaction network, pathway enrichment analysis and Kaplan-Meier survival curves were used to investigate the clinical values and biological functions of the identified miRNAs. A total of six common differentially expressed miRNAs (DEMs) were identified in LUAD. An independent cohort further confirmed that four miRNAs (miR-21-5p, miR-210-3p, miR-182-5p and miR-183-5p) were up-regulated and two miRNAs (miR-126-3p and miR-218-5p) were down-regulated in LUAD tissues. Pathway enrichment analysis also suggested that the above-listed six DEMs may affect LUAD progression via the estrogen signaling pathway. Survival analysis based on the TCGA dataset revealed the potential prognostic values of six DEMs in patients with LUAD (P-value<0.01). In conclusion, we identified a panel of six miRNAs from LUAD using miRNome profiling studies. Our results provide evidence for the use of these six DEMs as novel diagnostic and prognostic biomarkers for LUAD patients.Lung adenocarcinoma (LUAD) is the most common histological subtype of non-small cell lung cancer, but novel biomarkers for early diagnosis are lacking. Extensive effort has been exerted to identify miRNA biomarkers in LUAD. Unfortunately, high inter-lab variability and small sample sizes have produced inconsistent conclusions in this field. To resolve the above-mentioned limitations, we performed a comprehensive analysis based on LUAD miRNome profiling studies using the robust rank aggregation (RRA) method. Moreover, miRNA-gene interaction network, pathway enrichment analysis and Kaplan-Meier survival curves were used to investigate the clinical values and biological functions of the identified miRNAs. A total of six common differentially expressed miRNAs (DEMs) were identified in LUAD. An independent cohort further confirmed that four miRNAs (miR-21-5p, miR-210-3p, miR-182-5p and miR-183-5p) were up-regulated and two miRNAs (miR-126-3p and miR-218-5p) were down-regulated in LUAD tissues. Pathway enrichment analysis also suggested that the above-listed six DEMs may affect LUAD progression via the estrogen signaling pathway. Survival analysis based on the TCGA dataset revealed the potential prognostic values of six DEMs in patients with LUAD (P-value<0.01). In conclusion, we identified a panel of six miRNAs from LUAD using miRNome profiling studies. Our results provide evidence for the use of these six DEMs as novel diagnostic and prognostic biomarkers for LUAD patients.

Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity

Aims: This study aims to analyze the effect of thioredoxin reductase 2 (TrxR2) on lung cancer cell proliferation, apoptosis, invasion and migration in vitro. Main methods: Real‐time PCR was used to measure the expression of TrxR2 in NSCLC tumor tissues. After pAd‐TrxR2 or shRNA‐TrxR2 was transfected into A549 or NCI‐H1299 cells, the cell proliferation was measured by CCK‐8 method; cell apoptosis was measured by flow cytometry; cell invasion and migration was measured by Transwell method. The production of ROS was measured by DCFH‐DA method; the activity of SOD, CAT and GSH‐Px was measured by relative ELISA kit. Key findings: The results showed that TrxR2 was up‐regulated in NSCLC tumor tissues. Inhibition of TrxR2 suppressed NSCLC cell proliferation and induced apoptosis, and inhibited cell invasion and migration. However, overexpression of TrxR2 showed the opposite effect. Furthermore, when cells were transfected with shRNA‐TrxR2, the production of ROS was significantly increased, and SOD, CAT and GSH‐Px activity was decreased. Conversely, pAd‐TrxR2 transfection showed the opposite effect. Significance: Taken together, our results suggest that TrxR2 acts as an oncogenic gene in the context of lung cancer progression. The inhibition of TrxR2 suppressed lung cancer cell proliferation, invasion and migration and induced cell apoptosis by inducing ROS production and decreasing antioxidant activity. TrxR2 may be a potential target for NSCLC treatment. HighlightsTrxR2 was up‐regulated in lung cancer tissues.Inhibition of TrxR2 inhibited cell proliferation, metabolism and induced apoptosis.Inhibition of TrxR2 induced ROS production and decreased antioxidant activity.TrxR2 acts as an oncogenic gene in the context of lung cancer progression.

Sex-determining region of Y chromosome-related high-mobility-group box 2 in malignant tumors: current opinions and anticancer therapy.

Objective: To gain insight into the mechanism by which sex-determining region of Y chromosome (SRY)-related high-mobility-group box 2 (SOX2) involved in carcinogenesis and cancer stem cells (CSCs). Data Sources: The data used in this review were mainly published in English from 2000 to present obtained from PubMed. The search terms were “SOX2”, “cancer”, “tumor” or “CSCs”. Study Selection: Articles studying the mitochondria-related pathologic mechanism and treatment of glaucoma were selected and reviewed. Results: SOX2, a transcription factor that is the key in maintaining pluripotent properties of stem cells, is a member of SRY-related high-mobility group domain proteins. SOX2 participates in many biological processes, such as modulation of cell proliferation, regulation of cell death signaling, cell apoptosis, and most importantly, tumor formation and development. Although SOX2 has been implicated in the biology of various tumors and CSCs, the findings are highly controversial, and information regarding the underlying mechanism remains limited. Moreover, the mechanism by which SOX2 involved in carcinogenesis and tumor progression is rather unclear yet. Conclusions: Here, we review the important biological functions of SOX2 in different tumors and CSCs, and the function of SOX2 signaling in the pathobiology of neoplasia, such as Wnt/&bgr;-catenin signaling pathway, Hippo signaling pathway, Survivin signaling pathway, PI3K/Akt signaling pathway, and so on. Targeting towards SOX2 may be an effective therapeutic strategy for cancer therapy.

Baicalein sensitizes hepatocellular carcinoma cells to 5-FU and Epirubicin by activating apoptosis and ameliorating P-glycoprotein activity

Hepatocellular carcinoma (HCC) has a dismal prognosis in part because of multi-drug resistance (MDR). Baicalein is a flavonoid extracted from Radix Scutellariae with anti-HCC activity. We tested the effects of Baicalein on multi-drug resistant HCC cells (Bel7402/5-FU) known to be resistant to the anticancer drugs 5-FU and Epirubicin. Flow cytometry analysis showed that treatment with 5??g/ml and 10??g/ml Baicalein resulted in increases in the intra-cellular concentrations of Rho123 and Epirubicin in the corresponding group of cells compared to untreated cells, illustrating that Baicalein reverses MDR in Bel7402/5-FU cells. Bel7402/5-FU cells displayed increased P-glycoprotein (P-gp)-mediated drug efflux. However, this efflux was inhibited in cells pre-incubated in Baicalein for 48?h. Moreover, Baicalein induced apoptosis and autophagy and decreased P-gp and Bcl-xl expression levels. All of these results indicate that Baicalein can reverse P-gp-mediated MDR in HCC and may thus be useful for the treatment of drug-resistant HCC.

Bioinformatics analysis of Rab GDP dissociation inhibitor beta and its expression in non-small cell lung cancer

BackgroundLung cancer has been considered as one of the most important causes of cancer-related mortality worldwide. To predict lung cancer, researchers identified several molecular markers. However, many underlying markers of lung cancer remain unclear. One of these markers is Rab GDP dissociation inhibitor beta (GDIβ), which is related to tumorigenicity, development and invasion. This study was designed to analyze the biological characteristics of Rab GDIβ and to detect the mRNA and protein expressions of Rab GDIβ in lung cancer cells; this study also aimed to investigate the functions of this protein in lung cancer.MethodUsing online software from the websites of NCBI, ProtParam and so on, we analyzed the biological characteristics of Rab GDIβ. RT-PCR was performed to detect gene expressions in A549 and 16HBE cell lines and immunohistochemistry (IHC) staining was conducted to detect Rab GDIβ protein expression in 57 cases of human lung cancer tissues and 19 cases of normal lung tissues. The association of protein expression with patient clinical and pathological characteristics was assessed in each dataset.ResultsBioinformatic analysis on Rab GDIβ: The mRNA of human Rab GDIβ contains two transcript variants; the common structural elements of the two proteins are mainly α-helix, random coil, β-turn and extended strand. Three and four transmembrane domains could be found in the entire polypeptide chain of protein variants 1 and 2, respectively; both transcript variants are hydrophilic and soluble proteins. The RT-PCR result: The mRNA expression of Rab GDIβ was down-regulation in A549 cells compared with that in 16HBE cells. The IHC result: The protein expression of Rab GDIβ in lung cancer cells was significantly lower than that in normal lung tissues (P <0.05) but was not correlated with patients’ age, gender, tumor size, pathological type, differentiation, lymph node metastasis, distant metastasis and TNM stage.ConclusionThe expression of Rab GDIβ was low in non-small cell lung cancer (NSCLC). Hence, Rab GDIβ may be a tumor suppressor and could function as an indicator of tumorigenesis in NSCLC; nevertheless, this result should be further studied.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_201

Evolution of the strategies for screening and identifying human tumor antigens.

The prevailing view is that not only can some of the tumor antigens be used as biosensors for cancers, but also they may indeed be used as targets for immunotherapy. The identification of tumor antigens becomes a vital step in oncology research. Both the humoral immune system and the cellular immune system are activated in response to a tumor antigen in vivo of patients with tumor. Immune effector molecules and cells can be used to screen and identify tumor antigens. Specific T cells, including CD8(+) and CD4(+) T cells, can identify T cell epitopes, and specific antibodies in sera can identify B cell epitopes. The researchers have studied this area for decades. Initially, they explored tumor antigens with the use of 1-D SDS-PAGE and sandwich ELISAs. Since 1990s, CTL screening approach and peptide elution approach had been established. After that, SEREX, SERPA and protein microarray technology have become the mainstream highthroughput strategies for identifying tumor antigens. There are some other approaches, such as combinatorial peptide libraries, representational difference analysis of cDNA and bioinformatics methods. This reviews aim is to describe the generation, the theory, the key protocols and the application of some main techniques and provide their benefits and drawbacks.

Discovery of a set of biomarkers of human lung adenocarcinoma through cell-map proteomics and bioinformatics

Carcinogenesis of lung adenocarcinoma remains unclear and very few biomarkers have been accepted for routine clinical use. In order to explore the pathogenesis and screen ideal biomarkers, we conducted cell-map proteomics study in human lung adenocarcinoma. Homogeneous lung adenocarcinoma cells were purified by laser capture microdissection (LCM). A high performance liquid chromatography (HPLC) system was used to separate the total solution proteins. The resulting MS/MS spectra were automatically searched for proteins against IPI human protein database using the TurboSEQUEST searching engine. Physico-chemical properties of the identified proteins, including molecular weight (MW), isoelectric point (PI), were described based on various proteomics web server and statistical analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were used to analyze function of expressed proteins and screen candidate biomarkers according to biological annotation. A total of 843 distinct proteins were identified and were categorized as 10 sorts of molecular function and 17 sorts of biological process based on GO annotation. Further searching against KEGG pathways found that six proteins were involved in WNT signaling pathway, apoptosis pathway, Erb-2 signaling pathway, p53 signaling pathway, ubiquitin-mediated proteolysis and were might be hopefully screened as candidate markers of lung adenocarcinoma. The present study through LCM and cell-map proteomics showed a full view on the expressed protein profiles of lung adenocarcinoma. Several candidate markers are hopeful to be used as molecular targets of diagnosis, treatment and prognosis of lung adenocarcinoma.

Quantitative proteomic analysis of mitochondrial proteins differentially expressed between small cell lung cancer cells and normal human bronchial epithelial cells: Mitochondrial proteins in lung cancer

Small cell lung cancer (SCLC) is highly aggressive and is associated with a dismal prognosis. However, there are no clinically recognized biomarkers for early diagnosis. In this study, we used quantitative proteomics to build differential mitochondrial protein profiles that may be used for early diagnosis and investigated the pathogenesis of lung cancer.