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Krožne ribonukleinske kisline (circRNA) so skupina endogenskih ribonukleinskih kislin z enoverižno kovalentno zaprto strukturo. Z razvojem visokozmogljivih tehnologij določevanja zaporedja RNA in bioinformatskih orodij so ugotovili, da so zelo številčne, raznolike in ohranjene med vrstami, njihovi vzorci izražanja pa so specifični v različnih vrstah celic in tkiv kot tudi v različnih fazah razvoja. Odkrili so, da imajo raznolike molekularne funkcije, ki se odražajo v številnih fizioloških procesih (tako na celični kot na sistemski ravni) in posledično tudi v patoloških procesih. Čedalje več raziskav ugotavlja, da so nekatere krožne RNA povezane z razvojem hepatocelularnega karcinoma (HCC), ki je najpogostejša oblika raka jeter. Spremenjena raven izražanja krožnih RNA v HCC ni le posledica sprememb v celicah oziroma tkivu (zaradi česar imajo krožne RNA velik potencial kot diagnostični biološki označevalci), ampak imajo krožne RNA tudi same pomembno vlogo pri razvoju patološkega stanja preko vpliva na različne procese, kot so celični cikel, angiogeneza, apoptoza, metabolizem in epitelijsko-mezenhimski prehod. Preučevali smo vlogo heterogenega jedrnega ribonukleoproteina K (hnRNPK) in RNA-vezavnega proteina 4 (RBM4) pri uravnavanju izražanja izbranih krožnih RNA. Proteina hnRNPK in RBM4 imata pomembno vlogo pri procesiranju prekurzorske RNA. Za hnRNPK so pokazali, da se pri HCC čezmerno izraža, za RBM4 pa so ugotovili, da višja raven izražanja pri pacientih s HCC pozitivno korelira z večjimi možnostmi preživetja. Tekom diplomskega dela smo pripravili vektor za čezmerno izražanje proteina RBM4 v sesalskem sistemu. Ta vektor in vektor za čezmerno izražanje proteina hnRNPK smo s pomočjo transfekcije vnesli v celične kulture modelne celične linije za HCC Huh-7 in s prenosom western in imunodetekcijo dokazali čezmerno izražanje obeh proteinov. Z metodo kvantitativne verižne reakcije s polimerazo smo merili raven izražanja izbranih mRNA in circRNA v celičnih kulturah. Naši rezultati nakazujejo možnost, da čezmerno izražanje proteina hnRNPK vpliva na povišanje ravni izražanja krožnih RNA hsa_circ_0040921 in hsa_circ_0062682, čezmerno izražanje proteina RBM4 pa vpliva na povišano raven izražanja krožnih RNA hsa_circ_0048492 in hsa_circ_0028196. Za statistično značilnost rezultatov je bilo število ponovitev prenizko. Circular ribonucleic acids (circRNAs) are a group of endogenic ribonucleic acids with a single-stranded covalently closed structure. With the development of high throughput RNA-sequencing methods and bioinformatic tools it has been found that circRNAs are abundant, diverse and conserved, and that their expression patterns are spatiotemporal specific. Their diverse molecular functions result in numerous physiological processes (on a cell as well as on a systemic level) and, consequently, also in pathological processes. Numerous studies have showed that certain circRNAs are associated with the development of the hepatocellular carcinoma (HCC), which is the most common liver cancer type. The modulated circRNA expression level in HCC is not simply a consequence of changes in cells or tissue (on account for what they are considered as potential biomarkers), but rather that circRNA on their own play a salient role in the pathologies development by effecting diverse processes, e.g. cell cycle, angiogenesis, apoptosis, metabolism and epithelial-mesenchymal transition. We studied the role of the heterogenous nuclear ribonucleoprotein K (hnRNPK) and the RNA binding protein 4 (RBM4) in the expression regulation of selected circRNAs. These proteins exhibit an essential role in the precursor RNA processing. For hnRNPK it has been proved that it is overexpressed in HCC, whereas for RBM4 it has been discovered that the higher expression level in HCC patients positively correlates with a higher survival rate. During the course of the bachelor’s thesis, we succeeded to prepare a vector for RBM4 overexpression in a mammal system. With this vector and a vector for hnRNPK overexpression we transfected cell cultures of the HCC model cell line Huh-7 and with the means of western blot and immunodetection proved the overexpression of the two proteins. With the method of quantitative polymerase chain reaction, we measured the expression level of selected mRNAs and circRNAs in cell cultures. Our results indicate that the overexpression of hnRNPK increases the expression level of circRNAs hsa_circ_0040921 and hsa_circ_0062682 and that the overexpression of RBM4 increases the expression level of circRNAs RNA hsa_circ_0048492 and hsa_circ_0028196. For our findings to be statistically significant a bigger number of experiment repeats would be needed. |
