EXPRESSION OF AUTOPHAGY AND NECROPTOSIS MARKERS IN BLADDER CARCINOMA IN MICE

Cilj istraživanja: Istražiti postoji li povećan izražaj gena za p62, Beclin 1, LC3A, Atg5 i Ripk3f u tumorskom tkivu u odnosu na zdravo tkivo mokraćnog mjehura miša. Materijali i metode: Eksperimentalno istraživanje provedeno je u Laboratoriju za istraživanje raka Medicinskog fakulteta u Splitu. Miševi C57BL/6J divljeg tipa (wt) stari 6 do 8 tjedana podijeljeni su u 3 eksperimentalne skupine, netretiranu kontrolnu skupinu, ispitivanu skupinu podvrgnutu akutnom tretmanu i ispitivanu skupinu podvrgnutu kroničnom tretmanu. Ispitivane skupine izložene su kemikaliji N-butil-N-(4-hidroksibutil) nitrozaminu (BBN-u) u vodi za piće sa svrhom indukcije tumora mokraćnog mjehura. Akutna skupina miševa bila je izložena 0,05% BBN-u tijekom 2 tjedna, dok je skupina kroničnih miševa BBN-u (0,05%) bila izložena 12 tjedana. Životinje su žrtvovane 2 tjedna (skupina akutnih) i 20 tjedana (skupina kroničnih) nakon početka tretmana. Iz uzoraka je izolirana mRNA, nakon čega je prevedena u cDNA koja je služila kao predložak za qRT-PCR. Kao standard za genski izražaj korišten je gen za aktin. Rezultati: Izražaj gena za p62, Beclin 1, LC3A i Atg5 nije promijenjen u akutnoj skupini u odnosu na kontrolnu netretiranu skupinu. Izražaj gena za Ripk3f statistički je značajno povećan u akutnoj skupini u odnosu na kontrolnu (p<0,0001), kao i u kroničnoj skupini u odnosu na kontrolnu (p<0,001). Zaključak: Geni za autofagijske proteine p62, Beclin 1, LC3A i Atg5 nemaju značajno povišen izražaj u tumorima mokraćnog mjehura miša. Izražaj gena za Ripk2f statistički značajno je povećan te ukazuje na važnost uloge nekroptoze u patogenezi tumora mokraćnog mjehura.Objectives: To determine expression of autophagy related genes p62, Beclin 1, LC3A and Atg5, as well as gene expression of necroptosis related gene Ripk3f, in bladder carcinoma compared to healthy bladder tissue of mice. Materials and methods: Experimental research was conducted at Laboratory for cancer research at University of Split School of Medicine. Wild type C57BL/6J mice 6-8 weeks old were divided into 3 groups: untreated control group, experimental group that undergone acute treatment and experimental group that undergone chronic treatment. Experimental groups were exposed to a chemical carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) administered in drinking water, in order to induce bladder carcinoma. Acute experimental group was exposed to BBN (0.05%) during 2 weeks, while the chronic experimental group was exposed to BBN (0.05%) during 12 weeks. Mice were sacrificed 2 and 20 weeks after commencement of treatment. RNA was isolated from tissue samples and then converted into cDNA by reverse transcription. cDNA was used as a template for qRT-PCR reaction. Actin gene was used as a housekeeping control. Results: Expression of p62, Beclin 1, LC3A and Atg5 was not changed in acute experimental group compared to nontreated control group. Ripk3f expression was significantly increased in both acute and chronic experimental groups compared to nontreated control group (p<0.0001; p