Supervisors info:
Ανδρέας Σκορίλας, Καθηγητής, Τμήμα Βιολογίας ΕΚΠΑ
Μαργαρίτης Αυγέρης, Αναπληρωτής Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Χρήστος Κοντός, Επίκουρος Καθηγητής, Τμήμα Βιολογίας ΕΚΠΑ
Summary:
Bladder cancer is the second most common type of urinary tract cancer, after prostate cancer. It is the sixth most common form of cancer in the male population and the ninth leading cause of death. The incidence of the disease increases significantly with age, while men have a 3 to 4 times greater risk of developing the disease. Bladder cancer presents a strong clinical heterogeneity, in terms of prognosis and progression of the disease, patient survival and response to treatment. The prognosis of the disease is important for the individualized treatment of the patient and for the creation of a post-therapeutic follow-up protocol. The prognostic indicators that exist today are unable to predict with certainty the course of urothelial cancer, because of long-term post-treatment follow-up of patients is necessary through invasive cystoscopies, urine cytological tests and CT/MRI (Computed tomography scan/Magnetic Resonance Imaging). Cost for treating and monitoring patients is very high. Decoding the function of the genes involved in the disease and finding new regulatory molecules that interact with these genes is a field of intense research interest that aims to find new biomarkers for better patient prognosis and treatment planning.
Non-coding RNAs are called RNAs that do not translate to any protein, while many of them have regulatory roles or participate in protein synthesis. This category includes small RNAs (micro RNAs/miRNAs), as well as tRNA-derived fragments/tRFs.
miRNAs are defined as single-stranded non-coding RNA molecules, ∼20 nucleotides in size that are produced endogenously and can regulate expression at the post-transcriptional level. miR-146a has been associated with bladder cancer many times. In many studies it has been found that it has an increased expression in the cancerous tissues of patients, as well as in their urine.
5΄-tRF-LysCTT is a tRNA fragment (tRNA-derived fragments/ tRFs), specifically derived from tRNALysCCT. The specific tRF in histological samples of patients with bladder cancer, when it has a high expression, seems to be associated with an unfavorable prognosis of the disease, with muscle invasive type of cancer, with tumors of high malignancy grade according to WHO 2004 and high risk according to the EORTC classification. At the same time regarding the progression after treatment, its high levels were associated with the progression of non-myoinvasive disease to myoinvasive disease, as well as worse overall survival and disease-free survival in patients with muscle-invasive type of cancer.
The purpose of this work is to develop a protocol for measuring the levels of non-coding RNAs, miR-146a and 5΄-tRF-LysCTT, in the blood serum of patients with bladder cancer and their clinical evaluation as liquid biopsy biomarkers.
For this work, whole blood was sampled from patients (n=41) with bladder cancer before treatment. Afterwards, the appropriate processing was carried out to isolate the serum from the whole blood. Subsequently, the development of a quantitative real-time PCR protocol was carried out to determine the levels of non-coding RNAs, miR-146a and 5΄-tRF-LysCTT, in the serum of patients with bladder cancer, with the help of of SYBR Green I and relative quantification of 2-ΔΔCT. Finally, an extensive biostatistical analysis was performed to assess the value of the studied molecules at a clinical level and the possibility of using them as prognostic indicators for a better risk stratification of patients with urothelial cancer and to improve the prediction of the post-therapeutic course of the disease.
Among the two non-coding RNAs studied, miR-146a and 5΄-tRF-LysCTT, miR-146a appeared to tend to be elevated in patients with high WHO 2004 grade malignancy and in patients with T1 stage disease characterized as high-grade malignancy. In the sample of patients we examined, there did not appear to be a statistically significant difference in the levels of the two non-coding RNAs in the serum of patients with bladder cancer, between the various stages of the disease, as well as between patients with non-muscle invasive and muscle invasive types of cancer. Also, no difference was detected between high- and low-grade malignancy patients, except for the trend of increased miR-146a in patients with high-grade malignancy tumor. Finally, none of the two non-coding RNAs was associated with better or worse disease-free survival and progression-free survival in patients with non-muscle invasive type of cancer, while the same was observed for the overall survival of patients with muscle-invasive cancer.
Therefore, the two non-coding RNAs we examined did not show differences in our group of patients. With the sole exception of miR-146a which tended to have elevated levels in patients with WHO 2004 high grade malignancy and in patients with stage T1 and high-grade malignancy. Based on the previous results of experiments done in the laboratory, but also the existing literature, the collection, isolation, and analysis of more patient samples would be decisive for the use of these molecules in the clinical evaluation of patients through liquid biopsy.
