Screening of bio-markers for diagnosis and monitoring of Hepatocellular carcinoma

Abnormalities of tissues or organs caused by uncontrolled and irregular proliferation of cells are defined as cancer. Hepatocellular carcinoma, which constitutes 80-90% of primary liver tumors, is caused by chronic Hepatitis B (80%) and chronic Hepatitis C infection. Nowadays, biomarker analyzes are conducted by the researchers for early diagnosis of diseases. One of them is cell free DNA analysis. Cell free DNAs are released into body fluids as a result of cell necrosis or apoptosis. DNA that is released into body fluids after cell necrosis represents the tumor tissue. Cell free DNAs can be genomic DNA fragments as well as mtDNA. It is known that mtDNA, which has a weak repair mechanism, is hypersensitive to environmental factors and has an important role in the molecular mechanism of cancer. It has been reported that the number of mtDNA copies increased in some diseases and decreased in some diseases. MicroRNAs, which are considered to be prognostic and predictive biomarkers, are small RNA fragments without coding. They are involved in many events in the cell. Tumors with PD-1 ligand can interact with PD-L1 in T cells to inhibit immunological defenses that develop against them. Because of this feature, PD-L1 is known to play an active role in various types of cancer. In this thesis, to determine the change of mtDNA copy number in hepatocellular carcinoma, DNA samples isolated from 34 patients and 34 healthy volunteers were quantified by digital PCR and qPCR for the first time. According to the results of digital PCR, the ratio between the patient and the control group was 2.8 times, and the result of qPCR analysis was 1.7 times. In the same patient and control group (hsa-mir-33a 7.3 times, hsa-mir-203b 4.6 times, hsa-mir361-3p 5.1 times less expressed and it was determined that the level of expression of hsa-mir-424 did not show any significant change between the patient and control group. In this study, copy number change analysis was performed between PD-L1 gene and Beta Actin gene. There was no significant difference in the number of copies between the two genes in the patient and control groups. In addition, new method was developed for cell free DNA isolation from serum / plasma samples. For this purpose, 9 different isolation methods, two of which are commercial kit, were compared with qPCR by reference to both nuclear DNA and mtDNA. As a result of this study, significant difference was found between the number of mtDNA copies, hsa-mir-33a, hsa-mir-203b and hsa-mir361-3p in HCC patients compared to healthy individuals.