Efekat mutacije gena VHL na nivo ekspresije proteina mTOR signalnog puta u svetloćelijskom karcinomu bubrega
The effect of VHL gene mutation on the expression levels of mTOR signaling pathway proteins in clear cell renal cell carcinoma
Author
Ilić, Bojana B.Mentor
Damjanović, Svetozar
Committee members
Matić, Gordana
Brajušković, Goran

Đorđević, Ana

Marković-Lipkovski, Jasmina

Metadata
Show full item recordAbstract
Sporadični svetloćelijski karcinomi bubrega asocirani su sa mutacijama tumor
supresorskog gena VHL kod 60-80 % pacijenata. U odsustvu funkcionalnog proteina
VHL dolazi do akumulacije faktora indukovanih hipoksijom koji regulišu ekspresiju
gena uključenih u procese angiogeneze, eritropoeze, ćelijskog metabolizma i
preživljavanja. Pored specifičnog genetičkog profila i povišenog nivoa HIF-2α
svetloćelijske karcinome bubrega karakteriše glikolitički metabolički put i povišena
aktivnost mTOR signalnog puta. Nepovoljan energetski status i hipoksija blokiraju
aktivnost mTOR signalnog puta preko proteina AMPK i REDD1.
Ova studija imala je za cilj da utvrdi mogću asocijaciju mutacionog statusa gena
VHL sa patohistološkim parametrima, nivoom HIF-2α iRNK i nivoom proteina HIF-2α,
mTOR, TSC1, TSC2, eIF4E-BP1, p70S6K1, AMPK, REDD1 i PDK3 u svetloćelijskom
karcinomu bubrega.
Studija je obuhvatala 47 pacijenata kojima je urađena totalna nefrektomija zbog
prisustva svetloćelijskog karcinoma bubrega. ...Genomska DNK izolovana iz uzoraka
venske krvi i somatska DNK izolovana iz uzoraka tumorskog i neizmenjenog tkiva
bubrega upotrebljene su za određivanje mutacionog statusa gena VHL i gubitka
heterozigotnosti 3p lokusa sekvenciranjem, metodom amplifikacije višestruko ligiranih
proba i fragmentnom analizom mikrosatelitnih markera. Metilacioni status promotora
gena VHL ispitan je metil specifičnom PCR reakcijom nakon bisulfitne modifikacije
DNK. Kvantitativni RT-PCR primenjen je za relativnu kvantifikaciju HIF-2α iRNK u
tumorskom i neizmenjenog tkivu bubrega. Kvantitafikacija ekspresije proteina HIF-2α,
mTOR, TSC1, eIF4E-BP1, p70S6K1, AMPK, REDD1 i PDK3 utvrđena je Western blot
metodom. Semikvantitativna imunohistohemijska analiza korišćena je za utvrđivanje
nivoa ekpresije proteina TSC2...
Sporadic clear cell renal carcinoma is associated with mutations of the VHL gene
in 60-80 % patients. The absence of a functional VHL protein leads to accumulation of
hypoxia-inducible factors which regulate expression of genes involved in angiogenesis,
erythropoiesis, cell metabolism and survival. Specific genetic background,
overexpression of HIF-2α transcription factor as well as glycolytic metabolism and
upregulated activity of mTOR pathway are the main characteristics of clear cell renal
carcinoma. The lack of energy and hypoxia inhibit mTOR signaling through activity of
AMPK and REDD1 proteins.
The aim of present study was to determine association of VHL gene mutational
status with pathohistological parameters, expression level of mRNA HIF-2α and
expression levels of mTOR, TSC1, TSC2, eIF4E-BP1, p70S6K1, AMPK, REDD1 and
PDK3 proteins in clear cell renal carcinoma.
The study included 47 patients who underwent radical nephrectomy due to
unilateral kidney tumor. DNA was extracted fr...om peripheral venous blood, tumorous
and corresponding healthy renal tissue. The VHL gene mutational status and loss of
heterozigosity of 3p loci were detected using DNA sequencing, MLPA and analysis of
microsatellite markers. The VHL gene promoter methylation analysis was examined by
MS-PCR after bisulphite modification of DNA. Quantitative real time PCR was
performed for relative quantification of HIF-2 alpha mRNA. Expression levels of HIF-
2α, mTOR, TSC1, eIF4E-BP1, p70S6K1, AMPK, REDD1 and PDK3 proteins was
determined using Western blot method. Immunohistochemistry was performed for semiquantitative
analysis of TSC2 protein.
Alteration of VHL gene and/or changes of 3p loci were detected in 37/47 (78.7
%) analysed tumorous samples...