Cripts, except COL12A1, ten (namely IL8, MMP3, IL1B, CHI3L1, GREM1, IL1RN, CXCL1, CXCL2, CA7 and SLC7A5) are thought to be associated with colorectal carcinogenesis and progression. In accordance with our findings, 7 of them, such as IL8, CHI3L1, CXCL1, CXCL2, MMP3, SLC7A5 and CA7, were found to be differentially expressed in CRC compared to normal tissue in previous microarray studies [5?,9?0,12,26?1]. CA7 [29] was also found to be downregulated not only in carcinoma, but in adenoma samples. Interleukin 8 (IL8) promotes cell proliferation and migration of human colon carcinoma cells through metalloproteinase-cleavage proHB-EGF [32]. The expression of SLC7A5 cationic amino acid transporter was also found to be significantly associated with cell proliferation and angiogenesis [33], moreover it seems to play an important role in enhancing the tumor growth in vivo [34]. The secreted interleukin-like Gro-alpha oncogene (CXCL1) and matrix-metalloproteinase 3 (MMP3) promote tumor initiation and growth (21?2), while chitinase 3 like-1 (CHI3L1) can protect cancer or/and stromal cells against apoptosis [35]. Elevated expression of interleukin 1 beta (IL1B) mRNA increases the risk of non-small cell lung cancer [36]. Although, it is known that IL1B polymorphisms are associated with tumor recurrence in stage II colon cancers [37], the function of this gene has not been clarified in CRC. Gremlin 1 (GREM1) as an antagonist of bone morphogenic proteins, has been shown to regulate early development and tumorigenesis. It was overexpressed in various human tumors and plays an oncogenic role especially in carcinomaspurchase TA-01 MedChemExpress 80-49-9 Biomarkers for Dysplasia-Carcinoma TransitionFigure 3. Separation of high-grade dysplastic adenoma and early cancer samples using the set of 11 transcripts. A. Microarray B. Realtime PCR C. Heat map of real-time PCR D. Real-time PCR considering the changes in the diagnosis E. Heat map of real time PCR considering the changes in the diagnosis; Adenoma HGD = high-grade dysplastic adenoma, CRC early stage = colorectal cancer early stage. doi:10.1371/journal.pone.0048547.gincluding CRC [38]. In previous studies, a highly significant upregulation of CXCL2 chemokine was found in CRC compared to normal colonic mucosa which could be already detected also in benign adenoma referring to the involvement of CXCL2 in the dysplasia-carcinoma transition [39]. In summary, this study identified a set of 11 discriminatory transcripts which could correctly classify not just normal, adenoma and CRC biopsies, but high-grade dysplastic adenoma and early stage CRC samples, even if using a large independent sample set.Although 10 of the 11 discriminatory genes are already known to be associated with CRC, these markers as a combined discriminative set are firstly applied in this study. The identified set of 11 markers was proved to be a highly specific and sensitive discriminator of the colorectal dysplasia-carcinoma transition which is of great clinical importance regarding the early diagnosis of CRC. These markers can establish the basis of gene expression based diagnostic classification of benign and malignant colorectal diseases and of development of diagnostic real-time PCR cards,Biomarkers for Dysplasia-Carcinoma Transitionfurthermore they are to be utilized for prospective biopsy screening both at mRNA and protein levels.AcknowledgmentsWe would like to thank Tim Allen MSc. from Cranfield University, UK for reviewing the manuscript.Supporting InformationTable S1.Cripts, except COL12A1, ten (namely IL8, MMP3, IL1B, CHI3L1, GREM1, IL1RN, CXCL1, CXCL2, CA7 and SLC7A5) are thought to be associated with colorectal carcinogenesis and progression. In accordance with our findings, 7 of them, such as IL8, CHI3L1, CXCL1, CXCL2, MMP3, SLC7A5 and CA7, were found to be differentially expressed in CRC compared to normal tissue in previous microarray studies [5?,9?0,12,26?1]. CA7 [29] was also found to be downregulated not only in carcinoma, but in adenoma samples. Interleukin 8 (IL8) promotes cell proliferation and migration of human colon carcinoma cells through metalloproteinase-cleavage proHB-EGF [32]. The expression of SLC7A5 cationic amino acid transporter was also found to be significantly associated with cell proliferation and angiogenesis [33], moreover it seems to play an important role in enhancing the tumor growth in vivo [34]. The secreted interleukin-like Gro-alpha oncogene (CXCL1) and matrix-metalloproteinase 3 (MMP3) promote tumor initiation and growth (21?2), while chitinase 3 like-1 (CHI3L1) can protect cancer or/and stromal cells against apoptosis [35]. Elevated expression of interleukin 1 beta (IL1B) mRNA increases the risk of non-small cell lung cancer [36]. Although, it is known that IL1B polymorphisms are associated with tumor recurrence in stage II colon cancers [37], the function of this gene has not been clarified in CRC. Gremlin 1 (GREM1) as an antagonist of bone morphogenic proteins, has been shown to regulate early development and tumorigenesis. It was overexpressed in various human tumors and plays an oncogenic role especially in carcinomasBiomarkers for Dysplasia-Carcinoma TransitionFigure 3. Separation of high-grade dysplastic adenoma and early cancer samples using the set of 11 transcripts. A. Microarray B. Realtime PCR C. Heat map of real-time PCR D. Real-time PCR considering the changes in the diagnosis E. Heat map of real time PCR considering the changes in the diagnosis; Adenoma HGD = high-grade dysplastic adenoma, CRC early stage = colorectal cancer early stage. doi:10.1371/journal.pone.0048547.gincluding CRC [38]. In previous studies, a highly significant upregulation of CXCL2 chemokine was found in CRC compared to normal colonic mucosa which could be already detected also in benign adenoma referring to the involvement of CXCL2 in the dysplasia-carcinoma transition [39]. In summary, this study identified a set of 11 discriminatory transcripts which could correctly classify not just normal, adenoma and CRC biopsies, but high-grade dysplastic adenoma and early stage CRC samples, even if using a large independent sample set.Although 10 of the 11 discriminatory genes are already known to be associated with CRC, these markers as a combined discriminative set are firstly applied in this study. The identified set of 11 markers was proved to be a highly specific and sensitive discriminator of the colorectal dysplasia-carcinoma transition which is of great clinical importance regarding the early diagnosis of CRC. These markers can establish the basis of gene expression based diagnostic classification of benign and malignant colorectal diseases and of development of diagnostic real-time PCR cards,Biomarkers for Dysplasia-Carcinoma Transitionfurthermore they are to be utilized for prospective biopsy screening both at mRNA and protein levels.AcknowledgmentsWe would like to thank Tim Allen MSc. from Cranfield University, UK for reviewing the manuscript.Supporting InformationTable S1.
