| Abstract | Epigenetiku definiramo kao znanstvenu disciplinu koja proučava nasljednu i reverzibilnu promjenu funkcije gena neovisno o slijedu baza u molekuli DNA. Epigenomična mreža signala nastaje interakcijom DNA metilacija i post-translacijskih histonskih modifikacije, djelovanjem regulatornih proteina i nekodirajuće RNA. Dolazi do remodelacije kromatina i promjene u ekspresiji gena. MikroRNA su male, nekodirajuće, oko 22 nukleotida duge RNA molekule. Sparivanjem s mRNA, miRNA modulira translacijsku represiju ili mRNA degradaciju. Epigenomičke promjene u somatskim stanicama mogu biti preokrenute upotrebom epigenetičkih lijekova. Za očekivati je da multifaktorski poremećaji s kasnim nastupom, kao što je osteoporoza i osteoartritis, imaju snažnu epigenetičku komponentu. U žarištu patologije svih skeletnih bolesti je disregulacija remodelacije kosti. miRNA-195 u neobjavljenoj studiji pokazala je visoku ekspresiju u osteoporoznim kostima u odnosu na zdrave kontrole. Za navedenu miRNA traženi su ciljni geni, jedan od njih je gen myb. c-Myb pripada u skupinu transkripcijskih faktora, najveća prevalencija c-Myb-a je u koštanoj srži, tu obavlja ulogu u hematopoezi. Cilj istraživanja je bio procijeniti veže li se miRNA-195, prisutna u stanicama osteosarkoma, na mRNA kloniranog gena, uz kontrolnu reakciju. Drugim riječima utječe li miRNA potencijalno epigenetičkim mehanizmom na smanjenu ekspresiju proteina c-Myb te tako sudjeluje u patogenezi osteoporoze. Vektor pmirGLO smo prvo umnožili u bakterijskim stanicama nakon transfekcije, zatim smo ga izolirali iz stanica i pokidali restrikcijskim enzimima XboI i XhoI. Ljepljive krajeve, kakve stvaraju enzimi, dodali smo na sekvencu miRNA vezujućeg mjesta. Pomoću T4 DNA ligaze smo spojili oligonukleotide s vektorom te napravili transfekciju stanica E.coli ligacijskom smjesom. Iz jedne stanice koja je narasla na selektivnoj podlozi izolirali smo plazmid i provjerili pomoću PCR-a duljinu inserta u plazmidu. Plazmid je bio očekivane veličine te smo ga poslali na sekvencioniranje. Kloniranje gena je bilo uspješno, međutim zbog vremenskog ograničenja eksperiment na stanicama humanog osteosarkoma još nije učinjen. |
| Abstract (english) | Epigenetics refers to heritable variations in gene expression that are not caused by changes in DNA sequence. Key epigenetic players are DNA methylation and histone post-translational modifications, which interplay with each other, with regulatory proteins and with non-coding RNAs, to remodel chromatin and change gene expresion. MicroRNAs constitute a large family of small, approximately 22-nucleotide-long, non-coding RNAs that have emerged as key post-transcriptional regulators of gene expression in metazoans and plants. By base pairing to mRNAs, microRNAs mediate translational repression or mRNA degradation. Epigenomic changes in somatic cells, can be reversed by the use of epigenetic drugs. It is, therefore, reasonable to expect that several complex multi-factorial late-onset disorders, like osteoporosis and osteoarthritis, could have a strong epigenetic component. The focal point of all skeletal pathologies is the deregulation of bone remodeling, mediated by bone-forming osteoblasts and bone resorbing osteoclasts. In unpublished study miRNA-195 showed high expression in osteoporotic bones compared to healthy controls. One of miRNA-195 target genes is myb gene. c-Myb is transcription factor, with the highest prevalence in the bone marrow, there it plays a role in hematopoiesis. Our aim was to evaluate whether miRNA-195, present in the cells of human osteosarkoma, is binding MYBs mRNA, and maybe in that way reducing expression of protein c-myb. That epigenetic mechanism could participate in the pathogenesis of osteoporosis. We amplified vector pmirGLO in bacterial cells, after that we isolated plasmid from one cell and cleaved it with restriction enzyme XboI and XhoI. We have added same sticky ends, that enzyme produced, to the sequence of miRNA binding sites. Using T4 DNA ligase we coupled oligonucleotides with vector and transfect cells of E.coli with the ligation mixture. From a single cell, that was grown on selective medium, we isolated the plasmid and checked the length of his insert with PCR. The length was as we expected so we sent it on sequencing. Gene cloning has been successful, however, due to time constraints, experiment on human osteosarkoma cells has not yet been made. |
