Project description:MicroRNAs (miRNAs) are intrinsic regulators in the various cellular processes, and their abnormalities are considered to be involved in the onset of human disorders, including cancer. Circulating miRNA is focused as new cancer biomarker however it is regarded that circulating RNA are released not only from tumor but also by various pathways. Recently, exosomes, small membrane vesicles, have been a major interest in cancer research field, because of their unique biological properties. Exosomes are secreted from various cells and the components (Lipids, mRNAs, miRNAs and proteins) reflect origin of the cells secreting them. Identification of exosomal miRNAs from cancer cells is expected to provide useful biomarkers of cancer. To identify specific exosomal miRNAs as candidate biomarkers for colorectal cancer, we profiled exosomal miRNAs in sera of colon cancer patients (n=88) at various TNM stages (I to IV) and healthy controls (n=11) and selected significantly higher microRNAs in serum exosomes of colorectal cancer patients than that of healthy controls. Moreover, we tried to detect their serum exosome levels of using samples from patients after surgical resection of primary tumors (n=24). Serum exosomes were prepared by step-wise ultra-centrifugation methods in 11 healthy controls and 88 colorectal cancer patients with various TNM stages (I; n=20, stage II; n=20, stage IIIa; n=20, stage IIIb: n=16, stage IV; n=12) (age; 35-65) .Exosome fraction was mixed with Trizol-LS reagent (Invitrogen), and aqueous phase was collected by adding chloroform. After addition of ethanol to the aqueous phase, it was placed on to an RNeasy mini spin column (Qiagen) for the purification of total RNAs. The total RNAs were analyzed by Agilent Human miRNA V3 Microarray (G4470C) according to the manufacturer's instructions.
Project description:MicroRNAs (miRNAs) are intrinsic regulators in the various cellular processes, and their abnormalities are considered to be involved in the onset of human disorders, including cancer. Circulating miRNA is focused as new cancer biomarker however it is regarded that circulating RNA are released not only from tumor but also by various pathways. Recently, exosomes, small membrane vesicles, have been a major interest in cancer research field, because of their unique biological properties. Exosomes are secreted from various cells and the components (Lipids, mRNAs, miRNAs and proteins) reflect origin of the cells secreting them. Identification of exosomal miRNAs from cancer cells is expected to provide useful biomarkers of cancer. To identify specific exosomal miRNAs as candidate biomarkers for colorectal cancer, we profiled exosomal miRNAs in sera of colon cancer patients (n=88) at various TNM stages (I to IV) and healthy controls (n=11) and selected significantly higher microRNAs in serum exosomes of colorectal cancer patients than that of healthy controls. Moreover, we tried to detect their serum exosome levels of using samples from patients after surgical resection of primary tumors (n=24). Serum exosomes were prepared by step-wise ultra-centrifugation methods in 24 colorectal cancer patients (age; 35-65) after surgical resection of primary tumor (TNM stage I; n=6, stage II; n=5, stage IIIa; n=5, stage IIIb; n=5, stage IV; n=3) .Exosome fraction was mixed with Trizol-LS reagent (Invitrogen), and aqueous phase was collected by adding chloroform. After addition of ethanol to the aqueous phase, it was placed on to an RNeasy mini spin column (Qiagen) for the purification of total RNAs. The total RNAs were analyzed by Agilent Human miRNA V3 Microarray (G4470C) according to the manufacturer's instructions.
Project description:Colorectal cancer (CRC) remains a major cause of cancer related-death in developed countries. The risk of death is correlated with the stage of CRC determined at the primary diagnosis and early diagnosis is associated with enhanced survival rate. Consequently, there is an interest in using proteomics technologies to identify specific markers of adenomatous polyps as well as advanced stages of CRC.This study supports the concept that serum proteins can discriminate adenoma and CRC patients from unaffected patients and highlights the value of the SERPIN family as potential biomarkers of CRC.
Project description:Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.
Project description:Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.
Project description:Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.
Project description:Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.
Project description:Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.