Efekat mutacije gena VHL na nivo ekspresije proteina mTOR signalnog puta u svetloćelijskom karcinomu bubrega

Abstract

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 from 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...