Vplivi utišanja genov APOBEC3A in APOBEC3B na izražanje kadherina E, ciklina D1, kaspaze 3, tumor supresorskega gena p53 in protoonkogena c-myc v človeških keratinocitih HFK

Krevel, Anamari (author), Lovšin, Marija Nika (mentor)

Proteini APOBEC so citidin deaminaze, ki katalizirajo deaminacijo citozina v uracil na enoverižni molekuli DNA in RNA. Ker lahko spreminjajo nukleotidno sestavo molekul DNA in RNA, je ta proces eden izmed ključnih obrambnih mehanizmov pri virusnih infekcijah, vendar je odgovoren tudi za pojav mutacij v gostiteljskih celicah in razvoju rakavih bolezni. Znanstveniki so ugotovili, da proteini APOBEC predstavljajo glavni vir somatskih mutacij pri raku materničnega vratu, ki je v veliki meri povezan z okužbo HPV. Geni kadherin E, ciklin D1, p53, kaspaza 3 in C-MYC so v celicah odgovorni za adhezijo, uravnavanje celičnega cikla, proliferacijo in diferenciacijo celic, programirano celično smrt, supresijo tumorjev in uravnavanje transkripcije specifičnih ciljnih genov, zato je še posebej pomembno, da je njihov nivo izražanja uravnan. V našem raziskovalnem delu nas je zanimalo, ali utišanje genov APOBEC vpliva na izražanje teh genov. Z metodo PCR v realnem času smo preverili izražanje tarčnih genov v človeških keratinocitih HFK divjega tipa in HFK16 z utišanimi proteini APOBEC3A in APOBEC3B. Pri keratinocitih HFK divjega tipa nismo ugotovili statističnih razlik v izražanju preiskovanih genov med vzorci. Pri keratinocitih HFK16 so bile statistično značilne razlike prisotne pri genih p53 in C-MYC, pri genih kadherin E, ciklin D1 in kaspaza 3 razlik ni bilo. Preverili smo tudi, ali obstajajo statistične razlike v izražanju tarčnih genov med skupinama HFKWT in HFK16. Prišli smo do podobnih spoznanj, in sicer statistično značilne razlike so bile prisotne pri genih p53 in C-MYC. Prišli smo do zaključkov, da utišanje APOBEC3A in APOBEC3B vpliva na izražanje genov p53 in C-MYC, medtem ko na izražanje genov kadherin E, ciklin D1 in kaspaza 3 nima učinka. Potrebna je previdnost pri interpretaciji teh rezultatov, ker je bila raziskava narejena na majhnem številu vzorcev. V prihodnje bi bilo potrebno raziskavo ponoviti na večjem številu vzorcev.

• Language: Slovenian
• Keywords: APOBEC3A, APOBEC3B, HPV, izražanje genov, qPCR
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-166901
• Publication date in RUL: 30.01.2025

Secondary language

• Language: English
• Title: The effects of APOBEC3A and APOBEC3B gene silencing on the expression of E-cadherin, cyclin D1, caspase 3, tumor suppressor gene p53 and proto-oncogene c-myc in HFK human keratinocytes

Abstract

APOBEC proteins are cytidine deaminases that catalyse the deamination of cytosine to uracil on single-stranded DNA and RNA molecules. Because they can alter the nucleotide composition of DNA and RNA molecules, this process is one of the key defence mechanisms in viral infections, but is also responsible for the occurrence of mutations in host cells and the development of cancers. Researchers have found that APOBEC proteins are a major source of somatic mutations in cervical cancer, which is largely linked to HPV infection. The cadherin E, cyclin D1, p53, caspase 3 and C-MYC genes are responsible in cells for adhesion, cell cycle regulation, cell proliferation and differentiation, programmed cell death, tumour suppression and regulation of transcription of specific target genes, and it is therefore particularly important that their expression levels are regulated. In our research work, we were interested in whether silencing APOBEC genes affects the expression of these genes. We used real-time PCR to examine the expression of target genes in human wild-type and HFK16 keratinocytes with silenced APOBEC3A and APOBEC3B proteins. In wild-type HFK keratinocytes, we found no statistical differences in the expression of the genes examined between samples. In HFK16 keratinocytes, statistically significant differences were present for p53 and C-MYC genes, while no differences were observed for cadherin E, cyclin D1 and caspase 3 genes. We also tested whether there were statistical differences in the expression of target genes between the HFKWT and HFK16 groups. Similar findings were found, with statistically significant differences present for p53 and C-MYC genes. We concluded that silencing of APOBEC3A and APOBEC3B has an effect on the expression of p53 and C-MYC genes, while it has no effect on the expression of cadherin E, cyclin D1 and caspase 3 genes. Caution is needed in the interpretation of these results as the study was performed on a small number of samples. In future, the study should be repeated on a larger number of samples.

• Keywords: APOBEC3A, APOBEC3B, HPV, gene expression, qPCR