Genes of extracelullar matrix as markers of invasion in colorectal neoplasms

Rak debelega črevesa in danke (RDČD) je posledica kopičenja morfoloških in (epi)genetskih sprememb. Kljub poznavanju histopatoloških značilnosti je pri mnogih lezijah postavitev pravilne diagnoze zaradi netipične histopatološke slike zahtevna. V pomoč bi lahko bili označevalci, specifični za različne stopnje razvoja RDČD. Opredelili smo vlogo šestih genov zunajceličnega matriksa (ECM), katerih izražanje se je glede na bioinformatske analize statistično značilno razlikovalo med adenomom in RDČD. V raziskavo smo vključili 105 biopsijskih tkivnih vzorcev 62 bolnikov, pri katerih so bili endoskopsko ali operativno odstranjeni adenom, adenom s psevdoinvazijo, maligniziran adenom ali RDČD. Eksperimentalno validacijo smo naredili na mRNA in proteinskem nivoju, analizo (epi)genetske regulacije pa na RNA in DNA nivoju. Na proteinskem in mRNA nivoju smo ugotovili, da je pri genih DCN, SPON2, SPARC in SPP1 izražanje premo-sorazmerno z malignostjo neoplazme. Na nivoju mRNA je bilo izražanje EPHA4 povišano pri adenomih s psevdoinvazijo, medtem ko je bilo izražanje FN1 v razvoju RDČD podobno kot v normalni sluznici. Ugotavljali smo tudi (epi)genetsko regulacijo genov, katerih izražanje se je spreminjalo v razvoju RDČD. Izražanje gena DCN je bilo soodvisno od miR-200c, medtem ko je bilo izražanje gena SPARC soodvisno od metilacijskega statusa promotorja. Naša analiza ni pokazala nobene statistično značilne povezave med izbranimi načini regulacije in izražanjem gena SPON2. Analiza (epi)genetske regulacije gena SPP1 pa je pokazala, da je izražanje gena SPP1 soodvisno tako od miR-146 kot od metilacijskega statusa promotorja in spremembe števila kopij gena. Z eksperimentalno validacijo izbranih genov in proteinov ECM smo ugotovili, da imajo pomembno vlogo v razvoju RDČD. Čeprav so vzorci njihovega izražanja preveč zapleteni, da bi jih lahko uporabili kot diagnostične označevalce pri vsakdanjem delu, pa prispevajo k boljšemu razumevanju sprememb ECM v razvoju RDČD, kar nam bo lahko v prihodnosti v pomoč pri odkrivanju novih označevalcev in načinov zdravljenja.Colorectal carcinoma (CRC) arises due to the accumulation of morphological and (epi)genetic changes. Despite well-defined histopathological features, there are many lesions with ambiguous histopathological features making diagnosing difficult. For this reason, markers specific for each stage of CRC development could be helpful. We experimentally validated six genes of the extracellular matrix (ECM), whose expression was identified in the bioinformatics analysis as significantly different between adenoma and CRC. Our study included 105 biopsy tissue samples from 62 patients who had endoscopically or surgically removed adenoma, adenoma with epithelial misplacement, malignant adenoma, or CRC. Experimental validation of expression was performed on mRNA and protein level and (epi)genetic regulation on DNA and RNA level. We showed that expression of DCN, SPON2, SPARC in SPP1 on mRNA and protein level increased with level of malignancy. Expression of EPHA4 was up-regulated in adenomas with epithelial misplacement, whereas expression of FN1 was similar to healthy mucosa throughout CRC development. Furthermore, we were interested in the (epi)genetic regulation of genes whose expression increased with level of malignancy. Expression of DCN was inversely proportional to miR-200c, while expression of SPARC was correlated to the methylation status of its promotor region. Our analysis did not show any significant associations between selected types of regulation and expression of SPON2. In contrast, expression of SPP1 was associated with the expression of miR-146a, the methylation status of the promotor region, and the copy number variation. Experimental validation of selected ECM genes and proteins provided further evidence of their important role in the development of CRC. Though the expression patterns of analysed genes and proteins are too complex to be used directly in diagnostic work as markers, they might contribute to a better understanding of ECM changes in CRC development and help in search for new marker(s) and treatment modalities in the future

Expression of extracellular matrix-related genes and their regulatory microRNAs in problematic colorectal polyps

Colorectal carcinoma usually evolves gradually, forming a spectrum of lesions, due to accumulation of genetic mutations and epigenetic alterations. Many early lesions are detected since the introduction of screening programs. The greatest challenge is to distinguish between adenomas with epithelial misplacement (AEM) and adenomas with early carcinoma (AEC), considering the diagnosis affects prognosis and treatment. We analyzed the expression of selected extracellular matrix (ECM)-related genes and proteins, and their regulatory microRNAs using RT-qPCR and immunohistochemistry in biopsies from 44 patients. Differences were observed in AEM in comparison to AEC for DCN, EPHA4, FN1, SPON2, and SPP1, reflecting inflammatory stromal reaction to traumatisation and misplacement of dysplastic glands in the submucosa in the former, and desmoplastic stromal reaction to true invasion of dysplastic glands in the submucosa in the latter. Expression of regulatory microRNAs hsa-miR-200c and hsa-miR-146a significantly negatively correlated with the expression of their regulated genes, while significant difference between AEM and AEC was observed only for hsa-miR-29c. The described expression patterns are too complex to be used in diagnostic work, but might contribute to better understanding ECM changes in colorectal carcinoma development, helping to find new markers in the future

Expression of Extracellular Matrix-Related Genes and Their Regulatory microRNAs in Problematic Colorectal Polyps

Colorectal carcinoma usually evolves gradually, forming a spectrum of lesions, due to accumulation of genetic mutations and epigenetic alterations. Many early lesions are detected since the introduction of screening programs. The greatest challenge is to distinguish between adenomas with epithelial misplacement (AEM) and adenomas with early carcinoma (AEC), considering the diagnosis affects prognosis and treatment. We analyzed the expression of selected extracellular matrix (ECM)-related genes and proteins, and their regulatory microRNAs using RT-qPCR and immunohistochemistry in biopsies from 44 patients. Differences were observed in AEM in comparison to AEC for DCN, EPHA4, FN1, SPON2, and SPP1, reflecting inflammatory stromal reaction to traumatisation and misplacement of dysplastic glands in the submucosa in the former, and desmoplastic stromal reaction to true invasion of dysplastic glands in the submucosa in the latter. Expression of regulatory microRNAs hsa-miR-200c and hsa-miR-146a significantly negatively correlated with the expression of their regulated genes, while significant difference between AEM and AEC was observed only for hsa-miR-29c. The described expression patterns are too complex to be used in diagnostic work, but might contribute to better understanding ECM changes in colorectal carcinoma development, helping to find new markers in the future

Identification and validation of new cancer stem cell-related genes and their regulatory microRNAs in colorectal cancerogenesis

Significant progress has been made in the last decade in our understanding of the pathogenetic mechanisms of colorectal cancer (CRC). Cancer stem cells (CSC) have gained much attention and are now believed to play a crucial role in the pathogenesis of various cancers, including CRC. In the current study, we validated gene expression of four genes related to CSC, L1TD1, SLITRK6, ST6GALNAC1 and TCEA3, identified in a previous bioinformatics analysis. Using bioinformatics, potential miRNA-target gene correlations were prioritized. In total, 70 formalin-fixed paraffin-embedded biopsy samples from 47 patients with adenoma, adenoma with early carcinoma and CRC without and with lymph node metastases were included. The expression of selected genes and microRNAs (miRNAs) was evaluated using quantitative PCR. Differential expression of all investigated genes and four of six prioritized miRNAs (hsa-miR-199a-3p, hsa-miR-335-5p, hsa-miR-425-5p, hsa-miR-1225-3p, hsa-miR-1233-3p and hsa-miR-1303) was found in at least one group of CRC cancerogenesis. L1TD1, SLITRK6, miR-1233-3p and miR-1225-3p were correlated to the level of malignancy. A negative correlation between miR-199a-3p and its predicted target SLITRK6 was observed, showing potential for further experimental validation in CRC. Our results provide further evidence that CSC-related genes and their regulatory miRNAs are involved in CRC development and progression and suggest that some them, particularly miR-199a-3p and its SLITRK6 target gene, are promising for further validation in CRC

Epithelial-mesenchymal transition-related microRNAs and their target genes in colorectal cancerogenesis

MicroRNAs of the miR-200 family have been shown experimentally to regulate epithelial-mesenchymal transition (EMT). Although EMT is the postulated mechanism of development and progression of colorectal cancer (CRC), there are still limited and controversial data on expression of miR-200 family and their target genes during CRC cancerogenesis. Our study included formalin-fixed paraffin-embedded biopsy samples of 40 patients (10 adenomas and 30 cases of CRC with corresponding normal mucosa). Expression of miR-141, miR-200a/b/c and miR-429 and their target genes (CDKN1B, ONECUT2, PTPN13, RND3, SOX2, TGFB2 and ZEB2) was analysed using quantitative real-time PCR. Expression of E-cadherin was analysed using immunohistochemistry. All miRNAs were down-regulated and their target genes showed the opposite expression in CRC compared to adenoma. Down-regulation of the miR-200 family at the invasive front in comparison to the central part of tumour was observed as well as a correlation of expression of miR-200b, CDKN1B, ONECUT2 and ZEB2 expression to nodal metastases. Expression of the miR-200 family and SOX2 also correlated with E-cadherin staining. These results suggest that the miR-200 family and their target genes contribute to progression of adenoma to CRC, invasive properties and development of metastases. Our results strongly support the postulated hypotheses of partial EMT and intra-tumour heterogeneity during CRC cancerogenesis

miR-200b, ZEB2 and PTPN13 Are Downregulated in Colorectal Carcinoma with Serosal Invasion

Serosal invasion is an independent negative prognostic factor in certain cancers, including CRC. However, the mechanisms behind serosal invasion are poorly understood. We therefore assumed that epithelial-mesenchymal transition (EMT) might be involved. Our study included 34 patients with CRC, 3 stage pT2, 14 stage pT3 and 17 showing serosal invasion (stage pT4a according to TNM staging system). RNA isolated from formalin-fixed paraffin-embedded tissue samples was analysed for expression of the miR-200 family and their target genes CDKN1B, ONECUT2, PTPN13, RND3, SOX2, TGFB2 and ZEB2 using real-time PCR. We found upregulation of miR-200b and ONECUT2 in CRC pT3 and pT4a compared to normal mucosa, and downregulation of CDKN1B in CRC pT3. Moreover, we observed, downregulation of miR-200b, PTPN13 and ZEB2 in CRC with serosal invasion (pT4a) compared to pT3. Our results suggest the involvement of partial EMT in serosal invasion of CRC. In addition, PTPN13 seems to be one of the important regulators involved in serosal invasion, and ONECUT2 in tumour growth

Differential expression of decorin in metastasising colorectal carcinoma is regulated by miR-200c and long non-coding RNAs

Decorin (DCN) is one of the matricellular proteins that participate in normal cells’ function as well as in cancerogenesis. While its expression in primary tumours is well known, there is limited data about its expression in metastases. Furthermore, the post-transcriptional regulation of DCN is still questionable, although it is well accepted that it is an important mechanism of developing metastatic cancer. The aim of our study was to analyse the expression of DCN and its potential regulatory ncRNAs in metastatic colorectal carcinoma (CRC). Nineteen patients with metastatic CRC were included. Using qPCR, we analysed the expression of DCN, miR-200c and five lncRNAs (LUCAT1, MALAT1, lncTCF7, XIST, and ZFAS1) in lymph node and liver metastases in comparison to the invasive front and central part of a primary tumour. Our results showed insignificant upregulation of DCN and significant upregulation for miR-200c, MALAT1, lncTCF7 and ZFAS1 in metastases compared to the primary tumour. miR-200c showed a positive correlation with DCN, and the aforementioned lncRNAs exhibited a significant positive correlation with miR-200c expression in metastatic CRC. Our results suggest that DCN as well as miR-200c, MALAT1, lncTCF7 and ZFAS1 contribute to the development of metastases in CRC and that regulation of DCN expression in CRC by ncRNAs is accomplished in an indirect manner