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' 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 'region 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.
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