Geni zunajceličnega matriksa kot označevalci invazije v neoplazmah debelega črevesa in danke

Žlajpah, Margareta (author), Zidar, Nina (mentor), Boštjančič, Emanuela (comentor)

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.

• Language: Slovenian
• Keywords: rak debelega črevesa in danke, adenom, adenom s psevdoinvazijo, zunajcelični matriks, kvantitativna verižna reakcija s polimerazo, imunohistokemija
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2021
• PID: 20.500.12556/RUL-129066
• COBISS.SI-ID: 77703683
• Publication date in RUL: 25.08.2021

Secondary language

• Language: English
• Title: Genes of extracelullar matrix as markers of invasion in colorectal neoplasms

Abstract

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.

• Keywords: colorectal carcinoma, colorectal adenoma, adenoma with epithelial misplacement, extracellular matrix, quantitative polymerase chain reaction, immunohistochemistry