Молекулно профилиране при рака на простатата
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Дата
2015-12-01
Автори
Качакова/Kachakova, Дарина Людмилова/Darina Lyudmilova
Заглавие на списанието
ISSN на списанието
Заглавие на тома
Издател
Резюме
ЦЕЛ И ЗАДАЧИ *****
2.1. ЦЕЛ *****
Изследване на генетични фактори асоциирани с рака на простатата при български пациенти и анализ на промоторното хиперметилиране, нивата на експресия на иРНК и микроРНК в различни биологични материали за определяне на тяхната роля като диагностични и прогностични биомаркери. ******
2.2. ЗАДАЧИ *****
За изпълнението на поставената цел си възложихме следните задачи:
2.2.1. Подбор на пациенти с РП и контроли с български етнически произход (пациенти с доброкачествена простатна хиперплазия - ДПХ, популационни контроли съответстващи по пол, възраст и етническа принадлежност на пациентите, млади асимптоматични мъже - МАМ) и събиране на различни биологични материали.
2.2.2. Оптимизиране на протоколите за изолиране на ДНК/РНК от изследваните биологични материали, където е необходимо.
2.2.3. Провеждане на асоциативно проучване с подбрани от GWAS показали асоциация с РП еднонуклеотидни полиморфни варианти разположени в различни хромозомни локуси (7q21, 8q24, 10q11, 11p15, 12q13, 19q13) и финно картиране на хромозомен локус 11p15 за прецизиране на асоциацията с РП получена за този локус.
2.2.4. Провеждане на асоциативно проучване с подбрани от литературата полиморфизми в AMACR и в гени, свързани с метаболизма на андроген (CYP1B1, AR).
2.2.5 Провеждане на генно експресионен анализ на PCA3 (DD3), PSA (KLK3), AMACR, GSTP1, COLPH2 (GOLM1) в урина на пациенти с РП и контроли с ДПХ чрез PCR в реално време, TaqMan технология.
2.2.6. Изследване на промоторното хиперметилиране на GSTP1, HIST1H4K и RASSF2 в урина на пациенти с РП и контроли (ДПХ и МАМ) чрез HRM, MethyLight.
2.2.7. Изследване на нивата на експресия на let-7c, miR-30c, miR-141 и miR-375 в плазма от пациенти с РП и контроли (ДПХ и МАМ) чрез PCR в реално време, SybrGreen технологията.
2.2.8. микроРНК експресионно профилиране в туморни и нормални простатни тъкани чрез микрочипов анализ.
2.2.9. Анализ на получените резултати. ******** SUMMARY ***
Prostate cancer (PC) is a multifactorial, heterogeneous and multifocal disease. It is the most frequently diagnosed malignancy in men worldwide. Despite the advance in science, medicine and early diagnosis PC remains the second leading cause of cancer deaths among men in developed countries.
Despite their low specificity and sensitivity the serum levels of PSA and digital rectal examination are so far the standard methods used to determine the need for prostatic biopsy for the diagnosis of PC. Finding new noninvasive diagnostic biomarkers in order to reduce the number of unnecessary needle biopsies, and also new prognostic biomarkers for differentiating aggressive from indolent forms for improvement of therapy are needed.
PC is a genetic, genomic and epigenetic disease. Therefore the goal of the current dissertation is to study the genetic factors associated with the disease in Bulgarian patients and to analyze promoter hypermethylation, and expression levels of mRNAs and miRNAs in different biological materials in order to evaluate their roles as diagnostic and prognostic biomarkers.
Polymorphic variants found in GWAS and located on different chromosomes loci (7q21, 8q24, 10q11, 11p15, 12q13, 19q13) were studied. Fine mapping of the 11p15 chromosome region was also performed in order to elucidate the association with PC observed for this locus. The association of rs1016343, rs7841060 and rs4871008 in 8q24, and rs7920517 in 10q11 with PC in Bulgarian patients was confirmed. An association of the polymorphisms in 8q24 and some haplotype combinations with the development of aggressive tumours was estimated for the first time but these results should be validated in larger samples. We found a new associated polymorphic variant - rs11564710 in 11p15 which leads to two-fold increase risk for disease development. It is more strongly associated with PC in comparison with rs7127900 which was initially disovered.
The studied polymorphic variants in CYP1B1 and AR genes related to the metabolism of androgens and AMACR gene related to metabolism of branched fatty acids did not show statistically significant association with PC in Bulgarian patients.
The analyzed promoter hypermethylated genes GSTP1, HIST1H4K and RASSF2 in urine were rejected as good diagnostic and prognostic biomarkers. The methylation of GSTP1 and HIST1H4K showed correlation with age but not with diagnosis. Methylation of RASSF2 was not found in patients nor in controls.
Expression levels of PCA3, PSA, AMACR, GSTP1 and COLPH2 (GOLM1) in urine were analyzed. PCA3 and AMACR most reliably distinguish patients with PC from controls with BPH. The combination of these two biomarkers with PSA in urine showed better diagnostic accuracy than their use separately and in comparison with serum PSA levels.
From the studied miRNAs in plasma (let-7c, miR-30c, miR-141 and miR-375) miR-375 levels discriminate between PC patients and BPH controls more precisely than serum PSA levels and expression levels of PCA3 in urine. Combinations of analyzed miRNAs in plasma and serum PSA levels distinguish patients with PC from BPH controls even better than their use separately or in comparison with the combinations of studied biomarkers in urine.
In order to find new potential biomarkers for PC miRNA expression profiling in tumour and normal prostatic tissues by microarray analysis was performed. Twenty eight miRNAs showed increased and 14 miRNAs decreased expression in tumour prostatic tissues. Twenty eight of them are not reported in the literature for association with PC. Gene ontology analysis showed that the most of mRNAs (genes) regulated by diferentialy expressed miRNAs participate in regulation of gene expression, apoptosis, cell prolifieration, cellular response to growth factors and DNA damage stimulus, and others. The most influenced pathways by genes with changed expression are MAPK signaling pathway, pathways involved in regulation of apoptosis, cell adhesion, cell cycle, DNA repair, Wnt, EGF-EGFR, p53, NOTCH signaling pathways, telomere maintanence and others. Cluster analysis showed that 27 from miRNAs with elevated expression and 5 of the miRNAs with decreased expression most reliably discriminate patients from controls and these miRNAs could be analyzed in subsequent validation studies as potential diagnostic biomarkers.
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Our study shows that combinations of noninvasive biomarkers lead to better diagnostic accuracy in comparison with their use separately. Estimated combinations outperform serum PSA levels and have potential to enter clinical practice after their validation in larger sample cohorts. The conducted whole miRNA expression profiling complements the knowledge about miRNAs related to prostate carcinogenesis. Most of the studied polymorphic variants selected from literature data have low penetrance and the association with PC in patients with Bulgarian ethnicity was not confirmed. In order to evaluate their role a larger case-control study should be performed. A new polymorphic variant in previously associated with PC chromosome locus 11p15 was found. We showed for first time that expression levels of miR-375 in plasma could be used as diagnostic biomarker in PC.
Описание
Дисертационен труд за присъждане на
Образователна и научна степен „ДОКТОР“; - 325 с. + Автореферат /CD/;
Научна специалност: „Молекулярна генетика”;
Професионално направление 4.3. Биологически науки;
Област на висше образование 4. Природни науки, математика и информатика; Научни ръководители:
Акад. проф. д-р Ваньо Митев, дм, дбн,
Доц. д-р Радка Кънева, дб
Научен консултант:
Гл. ас. д-р Атанаска Миткова, дб; София, 2015
Ключови думи
Молекулярна биология - дисертации; Простата - тумори - дисертации; Тумори - дисертации; Генитални тумори - мъже - дисертации , Molecular Biology - dissertations; Prostatic Neoplasms - dissertations; Neoplasms - dissertations; Genital Neoplasms, Male - dissertations