Izbor kontrolnih celičnih linij za analizo izražanja kanabinoidnih receptorjev pri raku dojke

Keber, Eva (avtor), Debeljak, Nataša (mentor)

Rak dojk ima visoko incidenco in smrtnost. V Sloveniji vsako leto zboli več kot 1500 bolnikov. V zadnjih letih je bilo narejenih veliko raziskav o spremenjenem izražanju kanabinoidnih receptorjev v vzorcih raka dojke z nasprotujočimi si rezultati o vplivu kanabinoidov na izid zdravljenja. Nasprotujoče si raziskave so lahko posledica uporabe različnih bioloških vzorcev raka dojk (npr. tkiv, celičnih linij). Namen moje naloge je bil pokazati, da so podatkovne zbirke ustrezno orodje za določitev bioloških vzorcev, ki jih v nadaljnjih analizah izbranih genov lahko uporabimo kot notranjo kontrolo ravni izražanja; tj. pozitivno kontrolo z visoko ravnjo izražanja in negativno kontrolo z nizko ravnjo/brez izražanja. V prvem delu naloge sem na osnovi analize različnih baz podatkov (CCLE, GTEx, GeneCards, Human Protein Atlas, itd.) opredelila celične linije, ki predstavljajo pozitivno in negativno kontrolo izražanja genov za kanabinoidne receptorje CNR1 in CNR2. Analiza je pokazala, da se oba gena izražata skoraj v vseh tkivih, a da je raven izražanja CNR1 zmeraj višja kot pri CNR2. Raven izražanja gena CNR1 je višja od ravni izražanja CNR2 tudi v celičnih linijah. Kot kontrolo izražanja CNR1 sem izbrala celično linijo U-251-MG z ravnjo izražanja 8,32 log2(TPM+1). Kot negativno kontrolo za izražanje CNR1 in CNR2 sem izbrala celično linijo SK-BR-3 z ravnjo izražanja 0,0 log2(TPM+1). V drugem, laboratorijskem delu naloge, sem na izbranih celičnih linijah eksperimentalno določila teoretično opredeljeno raven izražanja. Iz celičnih linij sem izolirala celotno RNA, jo reverzno prepisala v cDNA in kvantitativno analizo izražanja genov CNR1 in CNR2 izvedla s kvantitativnim PCR. Za normalizacijo sem uporabila hišna gena RPLP0 in HPRT1. Rezultati eksperimentalne analize so pokazali, da imajo teoretično izbrane kontrolne celične linije pričakovano (visoko oz. nizko) raven izražanja genov CNR1 in CNR2. S tem sem potrdila hipotezo, da je izbor celičnih linij, ki temelji na analizi podatkovnih zbirk, verodostojen in zanesljiv.

• Jezik: Slovenski jezik
• Ključne besede: kanabinoidni receptorji, rak dojke, raven izražanja, baze podatkov
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-150229
• COBISS.SI-ID: 172457475
• Datum objave v RUL: 15.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Determination of controls cell lines for expression analysis of cannabinoid receptors in breast cancer

Izvleček

Breast cancer has a high incidence and mortality. More than 1500 patients are diagnosed with breast cancer every year in Slovenia. In recent years, many studies have been carried out on the altered expression of cannabinoid receptors in breast cancer samples, with conflicting results on the impact of cannabinoids on treatment outcome. The conflicting research may be due to the use of different breast cancer biological samples (e.g. tissues, cell lines). The aim of my thesis was to show that databases are an appropriate tool to identify biological samples that can be used as an internal control of expression levels in downstream analyses of selected genes; i.e. a positive control with a high level of expression and a negative control with a low level/no expression. In the first part of the thesis, I identified cell lines representing positive and negative expression controls for the cannabinoid receptor genes CNR1 and CNR2 based on the analysis of different databases (CCLE, GTEx, GeneCards, Human Protein Atlas, etc.). The analysis showed that both genes are expressed in almost all tissues, but that the expression level of CNR1 is always higher than that of CNR2. The expression level of the CNR1 gene is also higher than that of CNR2 in the cell lines. As a control for CNR1 expression, I selected the U-251-MG cell line with an expression level of 8.32 log2(TPM+1). As a negative control for CNR1 and CNR2 expression, I selected the SK-BR-3 cell line with an expression level of 0.0 log2(TPM+1). In the second, laboratory part of the thesis, I experimentally determined the theoretically defined expression level in the selected cell lines. I isolated total RNA from the cell lines, reverse transcribed it into cDNA and quantitatively analysed the expression of the CNR1 and CNR2 genes by quantitative PCR. I used the housekeeping genes RPLP0 and HPRT1 for normalisation. The results of the experimental analysis showed that the theoretically selected control cell lines have the expected (high and low) expression levels of CNR1 and CNR2 genes. This confirmed my hypothesis that the selection of cell lines based on the database analysis is plausible and reliable.

• Ključne besede: cannabinoid receptors, breast cancer, expression levels, database