Analiza ekspresije i uloge humanog gena SOX14 u neuralnoj diferencijaciji in vitro i regulaciji malignog fenotipa

Abstract

Visok stepen očuvanosti tokom evolucije i činjenica da do danas nije opisan nijedan klinički sindrom povezan sa mutacijama u genu SOX14, ukazuju na značajnu ulogu ovog gena tokom embrionalnog razvića. Iako je prošlo dvadeset godina od otkrića gena SOX14, funkcija ovog gena još uvek nije dovoljno istražena. U ovoj doktorskoj disertaciji analizirana je ekspresija i uloga gena SOX14 u procesu neuralne diferencijacije in vitro i regulaciji malignog fenotipa ćelija poreklom od karcinoma grlića materice. Tokom neuralnog razvića kod miša i pileta, gen Sox14 je eksprimiran u određenom setu neuralnih progenitorskih ćelija i u određenim tipovima zrelih neurona u mozgu. Za članove SOXB1 podgrupe, kojoj pripadaju geni SOX1, SOX2 i SOX3, poznato je da doprinose održavanju neuralnih progenitorskih ćelija u stanju proliferacije, sprečavajući proces neuralne diferencijacije, dok se za članove SOXB2 podgrupe, koja obuhvata gene SOX14 i SOX21, pretpostavlja da imaju ulogu u određivanju sudbine neuralnih progenitora, indukujući proces neurogeneze. Smatra se da nivo ekspresije SOX14 i SOX21 proteina tokom neuralnog razvića određuje da li će ćelija ostati neuralni progenitor ili će započeti proces neurogeneze. Iako je okarakterisan kao marker neurona, u ovoj tezi je pokazano da se gen SOX14 eksprimira u pluripotentnim ćelijama, neuronima, kao i u drugim tipovima ćelija koje se dobijaju nakon neuralne diferencijacije pluripotentnih NT2/D1 i P19 ćelija in vitro. Takođe, u ovoj tezi je pokazano da je profil ekspresije SOX14 proteina tokom neuralne diferencijacije sličan profilu ekspresije markera postmitotičkih neurona β-III-Tubulina kod NT2/D1 i P19 ćelija. Rezultati prikazani u ovoj doktorskoj disertaciji su pokazali da izlaskom ćelija iz stanja pluripotentnosti i ulaskom u proces neuralne diferencijacije dolazi do povećanja nivoa ekspresije SOX14 proteina. Pokazano je da SOX14 protein ima preklapajući profil ekspresije sa SOXB1 proteinima tokom procesa neuralne diferencijacije in vitro, a dobijeni rezultati su ukazali na specifičnu regulaciju ekspresije gena SOX14 na post-transkripcionom nivou u pluripotentnim ćelijama. Novija istraživanja ukazuju da je ekspresija gena SOX14 izmenjena u maligno transformisanim ćelijama. Naime, istraživanja su pokazala da je gen SOX14 metilovan u tkivima poreklom od tumora, i to naročito u tkivima poreklom od karcinoma grlića materice. Dok studije metilacionog statusa na uzorcima tkiva pacijentkinja obolelih od ovog karcinoma ukazuju na odsustvo ekspresije gena SOX14 i njegovu potencijalnu ulogu tumor supresora, in vitro studije na ćelijama poreklom od karcinoma grlića materice ukazuju na onkogeni potencijal ovog gena. Upravo je kontradiktornost ovih podataka otvorila nove pravce istraživanja usmerene na detaljnu analizu ekspresije i uloge gena SOX14 u regulaciji malignog fenotipa ćelija poreklom od karcinoma grlića materice. Rezultati prikazani u ovoj doktorskoj disertaciji su pokazali da SOX14 ostvaruje funkciju tumor supresora u HeLa ćelijama aktivacijom p53 signalnog puta koji se smatra jednim od najvažnijih signalnih puteva u ćeliji...

SOX14 might have an essential role during development due to the high evolutionary conservation and lack of any known mutated phenotype associated with this gene. Although the first SOX14 gene in vertebrates was cloned and characterized two decades ago, the function of this gene is largely unknown. This doctoral dissertation provides an insight into SOX14 expression and its roles during neural differentiation in vitro and in regulation of malignant phenotype of cervical carcinoma cells. During the neural development, the expression of Sox14 gene is restricted to a defined set of neural progenitors and the precise subset of neurons in the brain. Because of its specific expression in the brain, SOX14 is recognised as a marker of neurons. While SOXB1 subgroup of genes, including SOX1, SOX2 and SOX3 genes, maintains neural progenitors' identity, it is proposed that SOXB2 subgroup, comprising SOX14 and SOX21 genes, are important for induction of neurogenesis. It is suggested that the fine balance between expressions of these proteins during early stages of neural development determines neural progenitor identity. Results presented in this thesis show that SOX14 is expressed in pluripotent cells, as well as in neurons and non-neuronal differentiated cells. Also, it is shown that the expression profile of SOX14 correlates to the expression profile of neuronal marker β-III-Tubulin and that the exit of cells from the pluripotent state toward neural differentiation is accompanied with the increased expression of SOX14. It is also shown that the expression of SOX14 overlaps with the expression of SOXB1 proteins during neural differentiation in vitro. The presented results also imply the specific post-transcriptional mechanism of regulation of SOX14 expression in pluripotent cells. Recently it became evident that SOX14 has a role during malignant transformation of the cells. Previous studies showed that SOX14 is methylated in tissues originating from several tumors, including cervical carcinoma. Results obtained by the analysis of the methylation status of SOX14 gene in tissues from cervical carcinoma suggested its potential role as a tumor suppressor, while, on the other hand, in vitro studies suggested that SOX14 exerts oncogenic potential in cervical carcinoma cells. These contradictory data opened up the possibility for further study of the role of SOX14 gene in cervical carcinoma. Results presented in this thesis show that SOX14 exerts tumor suppressor activity in HeLa cells through activation of p53 signaling pathway, one of the most important signaling pathways in cell. In particular, increased expression of SOX14 leads to stabilization of p53 protein by increasing the level of phosphorylated form of p53 protein in HeLa cells. Phosphorylated p53 acts as a transcriptional regulator of target genes that have an important role in tumor growth prevention. The increased expression level of SOX14 and stabilization of p53 are followed by induction of the expression of p53 target genes, mainly involved in apoptosis and cell cycle regulation, such as pro-apoptotic BAX gene and cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1. Increased expression of SOX14, through activation of a p53 signaling pathway, leads to decreased proliferation, cell cycle arrest and induction of apoptosis...