The role of micro RNA in regulation of gene expression for osteonectin and bone morphenic protein 1 in osteosarcoma cell lines

Pri osteoporozi pride do nastanka osteoporotičnih zlomov, ki so pomemben vzrok bolečin, invalidnosti in smrti, zato je njeno odkrivanje in zdravljenje zelo pomembno. Številne študije so preučile in potrdile vpliv mikro RNA (miRNA) na uravnavanje kostne remodelacije in s tem odkrile nove potencialne tarče za njeno odkrivanje in zdravljenje. Namen naloge je bil preveriti, ali se miR-204-5p veže na mRNA tarčnih genov SPARC in BMPR1A, ali na vezavo miR-204-5p vpliva različna stopnja diferenciacije osteoblastov, ter v nadaljevanju ugotoviti ali miR-204-5p vpliva na izražanje genov SPARC in BMP1. S tehnologijami rekombinantne DNA smo pripravili plazmidne konstrukte pmirGLO, ki so vključevali tarčna (vezavna) zaporedja znotraj 3\u27 regije mRNA genov SPARC in BMPR1A, in nato z dvojnim luciferaznim testom preverili njihovo aktivnost v humanih osteosarkomskih celičnih linijah HOS, MG-63 in saos-2. Preko uporabe različnih osteosarkomskih celic, ki se razlikujejo v stopnji diferenciacije osteoblastov, smo preverili vpliv le-te na vezavo miR-204-5p. Na konec 3\u27 regije gena za kresničkino luciferazo smo klonirali fragmente genov, ki so vsebovali nemutirano in mutirano vezavno mesto za miR-204-5p, in izmerili aktivnost kresničkine (luc) ter renilne luciferaze (Rluc), ki je tudi sestavni del plazmida pmirGLO in izračunali relativne luciferazne aktivnosti (luc/Rluc). Na celicah HOS smo eksperimente izvedli tudi z dodatkom sintetičnih molekul miRNA, hsa-miR-204-5p. P-vrednosti znotraj vseh celičnih linij za vse primerjane plazmide niso bile statistično značilne. Na vezavna mesta, izbrana s pomočjo biostatističnih orodij, se torej miR-204-5p ni vezala, prav tako na to vezavo ne vpliva stopnja diferenciacije osteoblastov, saj ni bilo razlik v luciferazni aktivnosti med različnimi celicami. V drugem delu naloge smo v celicah HOS preverili vpliv mimikov hsa-miR-204-5p na izražanje genov SPARC in BMP1 z verižno reakcijo s polimerazo v realnem času ob uporabi nespecifičnega barvila EvaGreen®. Rezultate smo obdelali s t-testom za neodvisne vzorce in ugotovili, da miR-204-5p ne vpliva na izražanje genov SPARC in BMP1. Z laboratorijskimi eksperimenti nismo potrdili biostatistično izbranih tarč SPARC in BMPR1A kot direktnih tarč za miR-204-5p v osteosarkomskih celičnih linijah. Prav tako nismo dokazali vpliva stopnje diferenciacije osteoblastov na vezavo miR-204-5p in nismo dokazali vpliva miR-204-5p na izražanje genov SPARC in BMP1 v celicah HOS.In osteoporosis, osteoporotic fractures occur. These are a major cause of pain, disability and death, so its detection and treatment are extremely important. Numerous studies have examined and confirmed the effect of micro RNA (miRNA) in regulating bone renewal and thereby discovered new potential targets for its detection and treatment. The purpose of the thesis was to verify whether miR-204-5p binds to binding sites in the mRNA of the target genes SPARC and BMPR1A, whether the degree of differentiation of osteoblasts affects the binding of miR-204-5p, and also to determine whether miR-204-5p affects the expression of the genes SPARC and BMP1. Plasmid constructs of pmirGLO, which included target (binding) sequences within the 3\u27 region of mRNA for the genes SPARC and BMPR1A, were prepared with the technologies of recombinant DNA and their activity was verified in the human osteosarcoma cell lines HOS, saos-2 and MG-63 with a dual luciferase assay. By using the different osteosarcoma cells, which differ in the degree of differentiation of the osteoblasts, we examined the effect of this on the binding of miR-204-5p. Fragments of our genes containing the non-mutated and mutated binding sites for miR-204-5p were cloned at the end of the 3 \u27region of the firefly luciferase gene, and its activity (luc) and the activity of renal luciferase (Rluc), which is also an integral part of plasmid pmirGLO, were then measured. Later the relative luciferase activity (luc/Rluc) was calculated. Experiments were also carried out on HOS cells with the addition of the synthetic miRNA molecules hsa-miR-204-5p. The P-values within all the cell lines and for all the compared plasmids were not statistically significant. Therefore, miR-204-5p did not bind to the binding sites selected using biostatistic tools, and also the degree of differentiation of osteoblasts did not affect its binding since there were no differences in luciferase activity between the different cells. In the second part of the paper, we examined the influence of the mimic hsa-miR-204-5p on the expression of the SPARC and BMP1 gene in HOS cells by real-time polymerase chain reaction using the non-specific EvaGreen® dye. The results were analysed using a t-test for independent samples and showed that miR-204-5p did not affect the gene expression of SPARC and BMP1. Laboratory experiments did not confirm our biostatistically selected targets SPARC and BMPR1A as direct targets for miR-204-5p in osteosarcoma cell lines. They also did not confirm the influence of different degree of differentiation of osteoblasts on binding miR-204-5p and the influence of miR-204-5p on expression of SPARC and BMP1