Izražaj cirkulirajućih mikronaRNA (miR-125a, miR-125b, miR-126, miR-99b, miR-let7a) u bolesnika s mijelodisplastičnim sindromom [Expression of circulating microRNA (miR-125a, miR-125b, miR-126, miR-99b, miR-let7a) in myelodysplastic syndrome patients]

Mandac Rogulj, Inga (2020) Izražaj cirkulirajućih mikronaRNA (miR-125a, miR-125b, miR-126, miR-99b, miR-let7a) u bolesnika s mijelodisplastičnim sindromom [Expression of circulating microRNA (miR-125a, miR-125b, miR-126, miR-99b, miR-let7a) in myelodysplastic syndrome patients]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Myelodysplastic syndrome (MDS) is a group of heterogeneous clonal hematologic disorders of hematopoietic stem cells, followed by ineffective hematopoiesis of one or more cell lines with the onset of consequent cytopenia with an increased risk of progression to acute myeloid leukemia. According to the World Health Organization classification, the diagnosis of MDS is based on morphological, clinical, cytogenetic, immunophenotypic and biological criteria. In everyday clinical practice, the diagnosis of MDS is based on invasive cytomorphological analysis of peripheral blood and bone marrow cells, determination of blast percent, type and degree of dysplasia, presence of ring sideroblasts, and cytogenetic analysis of bone marrow cells. Micro Messenger Ribonucleic Acids (miRNAs) are short, non-coding molecules of 18 to 25 nucleotides in length that play an important role in regulating cell development and metabolism, their differentiation and proliferation, regulation of the cell cycle and cell death. Tumor cells release miRNAs into the circulation (plasma, serum) where they remain relatively stable. Although their discovery allowed linking of disease and miRNA gene expression, a precondition for their clinical application was the determination of gene expression by real-time quantitative polymerase chain reaction with satisfactory efficacy and specificity. In the literature, gene expression of miRNAs has been linked to the diagnosis, classification and progression of various diseases. Many studies have been conducted so far about the molecular mechanisms and epigenetic pathways in MDS and their prognostic and therapeutic significance, but few studies have analyzed the importance of miRNAs in MDS. The aim of this study was to examine the level of change of gene expression of specific mRNAs (miR-125a, miR-99b, miR-126, miR-125b, miR-let-7a) in plasma of healthy volunteers and subjects diagnosed with MDS. Gene expression of these specific mRNAs was determined in plasma samples from healthy volunteers (18) and subjects with MDS (41). This paper describes for the first time the expression of a selected miRNA cluster (125a, 125b, 99b, let-7a) in the plasma of untreated MDS patients. A significant difference was found between the study group and healthy control in miR-99b level, where at normalized values relative to miR-126, an increased level in subjects compared to control was observed 4,521 times (P = 0.004). Diagnostic and prognostic significance of miR-125a was observed and correlated negatively with erythrocyte count and hemoglobin level in the diagnosis of MDS.The results of the study suggest that gene expression of miRNA (125a, 125b, 99b, let7a) could be regulated by the same mechanism and may be clinically relevant in subjects with MDS.

Abstract in Croatian

Mijelodisplastični sindrom (MDS) je skupina heterogenih klonskih hematoloških poremećaja hematopoetskih matičnih stanica, praćenih neučinkovitom hematopoezom jedne ili više staničnih linija uz pojavu posljedičnih citopenija s povišenim rizikom od progresije u akutnu mijeloičnu leukemiju. Prema klasifikaciji Svjetske zdravstvene organizacije, dijagnoza MDS temelji se na morfološkim, kliničkim, citogenetičkim, imunofenotipskim i biološkim kriterijima. U svakodnevnoj kliničkoj praksi dijagnoza MDS čini se invazivnom citomorfološkom analizom stanica periferne krvi i koštane srži, određivanjem postotka blasta, tipa i stupnja displazije, prisutnosti prstenastih sideroblasta i citogenetičkoj analizi stanica koštane srži. Mikroglasničke ribonukleinske kiseline (miRNA) kratke su, nekodirajuće molekule duljine 18 do 25 nukleotida, koje imaju važnu ulogu u regulaciji razvoja i metabolizma stanica, njihovoj diferencijaciji i proliferaciji, regulaciji staničnog ciklusa i stanične smrti. Tumorske stanice oslobađaju miRNA u cirkulaciju (plazmu, serum) gdje ostaju relativno stabilni. Iako je njihovo otkriće omogućilo povezivanje bolesti i genske ekspresije miRNA, preduvjet za njihovu kliničku primjenu jest određivanje genske ekspresije kvantitativnom lančanom reakcijom polimerazom u stvarnom vremenu uz zadovoljavajuću učinkovitost i specifičnost. U literaturi se genska ekspresija miRNA dovodi u vezu s dijagnozom, klasifikacijom i progresijom različitih bolesti. Provedena su istraživanja o molekularnim mehanizmima i epigenetičkim putovima u MDS te njihovu prognostičkom i terapijskom značenju, ali je ograničen broj studija dokazao važnost miRNA u dijagnostici MDS. Cilj rada bilo je ispitivanje razine promjene genskih ekspresija specifičnih miRNA (miR-125a, miR-99b,miR-126, miR-125b, miR-let-7a) u plazmi zdravih dobrovoljaca i ispitanika s dijagnozom MDS. Genske ekspresije navedenih, specifičnih miRNA određene su u uzorcima plazme zdravih dobrovoljaca (18) i ispitanika s MDS-om (41). U ovom radu je prvi put opisan izražaj odabranog klastera miRNA (125a, 125b, 99b, let-7a) u plazmi neliječenih MDS bolesnika. Dokazana je značajna razlika između ispitivane skupine i zdrave kontrole u razini miR-99b, gdje se kod normaliziranih vrijednosti u odnosu na miR-126, uočava povećana razina kod ispitanika u odnosu na kontrolu za 4,521 puta (P=0,004). Uočena je dijagnostička, ali i prognostička važnost miR-125a koji negativno korelira s brojem eritrocita i razinom hemoglobina kod dijagnoze MDS. Rezultati istraživanja upućuju na to da bi genske ekspresije miR (125a, 125b, 99b, let7a) mogle biti regulirane istim mehanizmom i da bi mogle biti klinički važne u ispitanika s MDS.

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