Ocena vplivov mikro RNA hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a-3p in hsa-miR-31-3p na adipogeno diferenciacijo mezenhimskih matičnih celic

Vohar, Anika (author), Marc, Janja (mentor), Vrščaj, Lucija Ana (comentor)

Mezenhimske matične/stromalne celice (MSC) so heterogena populacija odraslih multipotentnih celic, ki lahko in vitro diferencirajo v osteoblaste, adipocite in hondrocite. Pomembno vlogo imajo lahko tudi pri razvoju osteoporoze, kjer se adipogena diferenciacija MSC poveča na račun osteogene. To vodi do znižanja kostne mase, kar je značilno za paciente z osteoporozo. Na diferenciacijo MSC vplivajo različni dejavniki, med katere sodijo tudi molekule mikroRNA (miRNA), ki na posttranskripcijskem nivoju uravnavajo izražanje genov. V sklopu magistrske naloge smo želeli oceniti vlogo petih izbranih miRNA (hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a-3p in hsa-miR-31-3p) pri adipogeni diferenciaciji mezenhimskih matičnih celic. V ta namen smo uporabili spremenjene MSC, ki so imele s pomočjo lentivirusne transdukcije pripravljeno povečano izražanje (angl. overexpression) in druge MSC, ki so imele zmanjšano izražanje (angl. downexpression) naštetih miRNA. Uspešnost adipogene diferenciacije smo ocenili s pomočjo adipogenih označevalcev, adiponektin, vsebujoč C1Q in kolagensko domeno, ADIPOQ, maščobne kisline vezoči protein 4, FABP4 in s peroksisomskim proliferatorjem aktiviran receptor gama, PPARg. Njihovo izražanje smo določili s kvantitativno verižno reakcijo s polimerazo (RT-qPCR) in ga normalizirali glede na izražanje referenčnega gena gliceraldehid-3-fosfat dehidrogenaza, GAPDH. Meritve smo izvajali 1. in na 7. dan adipogene diferenciacije celic MSC. Rezultati so pokazali, da zmanjšano izražanje hsa-miR-363-3p ter povečano izražanje hsa-miR-20b-5p in hsa-miR-133a-3p zavira adipogeno diferenciacijo MSC, medtem ko zmanjšano izražanje hsa-miR-20b-5p in hsa-miR-31-3p spodbuja adipogeno diferenciacijo MSC. Vpliva hsa-miR-375-3p, zmanjšanega izražanja hsa-miR-133a-3p ter povečanega izražanja hsa-miR-363-3p in hsa-miR-31-3p na adipogeno diferenciacijo MSC nismo mogli opredeliti. Glede na rezultate lahko sklepamo, da štiri od petih preiskovanih miRNA zelo verjetno sodelujejo pri usmerjanju MSC v adipogeno diferenciacijo in lahko na ta način posredno zmanjšajo njihovo osteogeno diferenciacijo ter nastajanje osteoblastov v kostnem tkivu. Za vzpodbujanje rasti kostne tvorbe preko vpletenosti miRNA pa so pomembni zlasti nakazani zaviralni učinki na adipogeno diferenciacijo MSC, saj se ti odražajo s pospešeno osteogeno diferenciacijo MSC in nastajanjem osteoblastov v kostnem tkivu. Da bi ugotovili natančen mehanizem teh vplivov, bo potrebno izvesti dodatne raziskave.

• Language: Slovenian
• Keywords: mezenhimske matične/stromalne celice, adipogeneza, adipogeni označevalci, metoda qPCR, miRNA
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-158749
• Publication date in RUL: 20.06.2024

Secondary language

• Language: English
• Title: Assessment of microRNAs hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a-3p, and hsa-miR-31-3p impact on adipogenic differentiation of mesenchymal stem cells

Abstract

Mesenchymal stem/stromal cells (MSCs) are a heterogeneous population of adult multipotent cells capable of in vitro differentiating into osteoblasts, adipocytes, and chondrocytes. They may also play an important role in the development of osteoporosis, where adipogenic differentiation of MSCs increases at the expense of osteogenic differentiation. This leads to a reduction in bone mass, which is characteristic of patients with osteoporosis. Various factors influence the differentiation of MSCs, including microRNA (miRNA) molecules that regulate gene expression at the post-transcriptional level. In this master's thesis, we assessed the role of five selected miRNAs (hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a-3p, and hsa-miR-31-3p) in the adipogenic differentiation of mesenchymal stem cells. For this purpose, we used modified MSCs that had increased expression and decreased expression of the listed miRNAs prepared with lentiviral transduction. We assessed the success of adipogenic differentiation using adipogenic markers adiponectin, containing C1Q and collagen domain (ADIPOQ), fatty acid-binding protein 4 (FABP4), and peroxisome proliferator-activated receptor gamma (PPARγ) as markers. The expression of the markers was determined by quantitative polymerase chain reaction (RT-qPCR) and normalized to the expression of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Measurements were taken on the 1st and 7th day after the initiation of adipogenic differentiation. The results showed that decreased expression of hsa-miR-363-3p and increased expression of hsa-miR-20b-5p and hsa-miR-133a-3p inhibit adipogenic differentiation of MSCs, while decreased expression of hsa-miR-20b-5p and hsa-miR-31-3p promotes adipogenic differentiation of MSCs. The effects of hsa-miR-375-3p, decreased expression of hsa-miR-133a-3p, increased expression of hsa-miR-363-3p and hsa-miR-31-3p on the adipogenic differentiation of MSCs could not be determined. Based on the results, we can conclude that four out of the five investigated miRNAs very likely participate in directing MSCs towards adipogenic differentiation and can indirectly reduce their osteogenic differentiation and the formation of osteoblasts in bone tissue. For promoting bone formation through miRNAs, the indicated inhibitory effects on adipogenic differentiation are particularly important, as they result in accelerated osteogenic differentiation of MSCs and the formation of osteoblasts in bone tissue. To determine the exact mechanism of these influences, further research will be necessary.

• Keywords: mesenchymal stem/stromal cells, adipogenesis, adipogenic markers, qPCR method, miRNA