Cloning of mikroRNA sequence and analysis of their biological activity in glioblastoma cell line

Nekodirajuće RNA (ncRNA) važna su skupina regulatornih molekula koja se sastoji od transkripata dijelova genoma koji ne kodiraju za proteine. Sudjeluju u brojnim procesima u eukariotskim stanicama, poput regulacije ekspresije gena i interferencije s RNA. Jedna skupina malih jednolančanih ncRNA su mikroRNA (miRNA). Geni miRNA uobičajeno se nalaze unutar introna i transkribira ih RNA polimeraza II, nakon čega se transkript cijepa RNazama Drosha i Dicer. Novonastala jednolančana RNA, duga 19 do 25 nukleotida, u citoplazmi formira kompleks s proteinima zvan miRISC (kompleks za utišavanje induciran s miRNA). miRISC regulira ekspresiju gena tako što se veže na ciljanu mRNA koja ima sekvencu komplementarnu miRNA koja se nalazi u kompleksu, čime vodi do degradacije mRNA ili sprječavanja inicijacije translacije. Mnoge miRNA imaju ulogu tumorskih supresora ili onkogena, te se pokazalo da su miR-34a i miR-7 važni tumorski supresori čija je ekspresija utišana u brojnim tumorima čovjeka. Cilj diplomskog rada bio je konstrukcija plazmidnih vektora koji su eksprimirali sekvencu za prekursore miR-34a i miR-7 te proučavanje bioloških promjena u staničnoj liniji glioblastoma A1235 transficiranoj plazmidnim konstruktima. Uzgojene su stanične linije s povećanom ekspresijom miR-34a i miR-7, te je praćena njihova sposobnost proliferacije, otpornost na citostatike i ekspresija proteina koji su potencijalna meta miRNA.Non-coding RNAs (ncRNA) belong to an important group of regulatory molecules which consist of transcripts from non-protein coding genomic regions. They have a role in many biological processes in eukariotic cells, such as gene expression regulation and RNA interference. One group of small single stranded ncRNAs are microRNAs (miRNA). miRNA genes are usually located inside introns and are transcribed by RNA polymerase II, after which the transcipt is processed by Drosha and Dicer RNase. Newly formed single stranded RNA, 19 to 25 nucleotides long, forms a complex with proteins called miRISC (miRNA induced silencing complex) in the cytoplasm. miRISC regulates gene expression by binding with the target mRNA which has a sequence complementary to the miRNA in the complex, leading to mRNA degradation or translation initiation inhibition. Many miRNAs act as tumor supressors or oncogenes, and it was shown that miR-7 and miR-34a are important tumor supressors whose expression is silenced in multiple human cancers. The aim of this thesis was to construct plasmid vectors which expressed precursor sequences of miR-34a and miR-7, and to study biological changes in glioblastoma A1235 cell line transfected with plasmid constructs. Cell lines overexpressing miR-7 and miR-34a were produced, and cell proliferation, resistance to cytostatics and expression of potential protein targets were analyzed