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Crotonaldehyde (CRA)-one of the major environmental pollutants from tobacco smoke and industrial pollution-is associated with vascular injury (VI). We used proteomics to systematically characterize the presently unclear molecular mechanism of VI and to identify new related targets or signaling pathways after exposure to CRA. Cell survival assays were used to assess DNA damage, whereas oxidative stress was determined using colorimetric assays and by quantitative fluorescence study; additionally, cyclooxygenase-2, mitogen-activated protein kinase pathways, Wnt3a, ß-catenin, phospho-ErbB2, and phospho-ErbB4 were assessed using ELISA. Proteins were quantitated via tandem mass tag-based liquid chromatography-mass spectrometry and bioinformatics analyses, and 34 differentially expressed proteins were confirmed using parallel reaction monitoring, which were defined as new indicators related to the mechanism underlying DNA damage; glutathione perturbation; mitogen-activated protein kinase; and the Wnt and ErbB signaling pathways in VI based on Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction network analyses. Parallel reaction monitoring confirmed significant (p < 0.05) upregulation (> 1.5-fold change) of 23 proteins and downregulation (< 0.667-fold change) of 11. The mechanisms of DNA interstrand crosslinks; glutathione perturbation; mitogen-activated protein kinase; cyclooxygenase-2; and the Wnt and ErbB signaling pathways may contribute to VI through their roles in DNA damage, oxidative stress, inflammation, vascular dysfunction, endothelial dysfunction, vascular remodeling, coagulation cascade, and the newly determined signaling pathways. Moreover, the Wnt and ErbB signaling pathways were identified as new disease pathways involved in VI. Taken together, the elucidated underlying mechanisms may help broaden existing understanding of the molecular mechanisms of VI induced by CRA.
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BACKGROUND: Protein biomarkers play important roles in cancer diagnosis. Many efforts have been made on measuring abnormal expression intensity in biological samples to identity cancer types and stages. However, the change of subcellular location of proteins, which is also critical for understanding and detecting diseases, has been rarely studied. RESULTS: In this work, we developed a machine learning model to classify protein subcellular locations based on immunohistochemistry images of human colon tissues, and validated the ability of the model to detect subcellular location changes of biomarker proteins related to colon cancer. The model uses representative image patches as inputs, and integrates feature engineering and deep learning methods. It achieves 92.69% accuracy in classification of new proteins. Two validation datasets of colon cancer biomarkers derived from published literatures and the human protein atlas database respectively are employed. It turns out that 81.82 and 65.66% of the biomarker proteins can be identified to change locations. CONCLUSIONS: Our results demonstrate that using image patches and combining predefined and deep features can improve the performance of protein subcellular localization, and our model can effectively detect biomarkers based on protein subcellular translocations. This study is anticipated to be useful in annotating unknown subcellular localization for proteins and discovering new potential location biomarkers.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/patologia , Proteínas/metabolismo , Neoplasias do Colo/metabolismo , Bases de Dados de Proteínas , Humanos , Imuno-Histoquímica , Aprendizado de Máquina , Proteínas/classificaçãoRESUMO
INTRODUCTION: MicroRNAs (miRNAs) play important roles in tumorigenesis. In this study, we investigated the role of miR-221 in the development and progression of clear cell renal cell carcinoma (ccRCC). METHODS: Quantitative real-time PCR (qRT-PCR) was used to measure the expression level of miR-221 in ccRCC tissues and cell lines. Then, we investigated the role of miR-221 to determine its potential roles on renal cancer cell proliferation, migration and invasion in vitro. A luciferase reporter assay was conducted to confirm the target gene of miR-221 and the results were validated in renal cancer cells. RESULTS: In the present study, we found that miR-221 was significantly increased in ccRCC tissues and cell lines. Knocked-down expression of miR-221 remarkably inhibited cell proliferation, migration and invasion of renal cancer cells. Moreover, at the molecular level, our results suggested that TIMP2 as a direct target of miR-221 through which miR-221 promoted tumor cell proliferation, migration and invasion. CONCLUSIONS: These findings suggested that miR-221 play an oncogenic role in the renal cancer cell proliferation, migration and invasion by directly inhibiting the tumor suppressor TIMP2, indicating miR-221 act as a potential new therapeutic target for the treatment of ccRCC.