Evaluation of microrna's as biomarker in hepatocellular carcinoma patients infected with Hepatitis B virus

his study investigates the potential use of 6 pre-determined human microRNAs (hsa-mir-16, hsa-mir-28, hsa-miR-122, hsa-miR-221, hsa-mir-224 and hsa-mir-501) as diagnostic biomarkers in hepatocellular carcinoma. In order to evaluate this potential, miRNA expression levels on HBV positive serum samples from subjects without hepatocellular carcinoma (HCC) are compared with HBV positive serum and liver pathology samples from subjects with HCC, using Real-Time PCR and relative quantitation .All patient groups represent the epidemiology in Turkey. The correlation between the expression levels of these 6 miRNAs with HBV DNA log value is evaluated. Two snRNAs and one mRNA were analyzed for normalization in serum and tissue samples within this research. Expression of the given miRNAs were also compared with lesion size on tissue samples. Real-Time PCR results showed; 8,3 fold increase in mir-16 expression on tissue samples of HBV DNA positive patients with HCC; 2,3 fold decrease in mir-28 expression on serum samples of HBV DNA positive patients with HCC; 9,6 fold decrease in mir-28 expression on tissue samples of HBV DNA positive patients with HCC; 5,9 fold decrease in miR-122 expression on serum samples of HBV-HCC positive patients, 6,9 fold decrease in miR-122 expression on tissue samples of HBV-HCC positive patients, 5,4 fold decrease in miR-122 expression on serum samples of HBV positive patients without HCC, 8 fold increase in miR-221 expression on tissue samples of HBV-HCC positive patients, 3,3 fold increase in miR-224 expression of serum samples of HBV-HCC positive patients, 4,7 fold increase in miR-224 expression on tissue samples of HBV-HCC positive patients, 7,6 fold increase in miR-224 expression on serum samples of HBV positive patients without HCC, 4,2 fold increase in miR-501 expression on serum samples of HBV-HCC positive patients, 12,6 fold increase in miR-501 expression of HBV-HCC positive patients, 4,4 fold increase in miR-501 expression on serum samples of HBV positive patients without HCC diagnosis. Based on HBV DNA log levels along with the expression values of the pre-determined miRNAs, no significant correlation has been detected. Data derived from Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH-reference gene) mRNA and two snRNAs (small nuclear RNA/snRNA U44 and snRNA U6) are used for the normalization of relative quantitation results. Average cycle threshold (Ct) values in serum were found to be 31,18±5,98 for GAPDH, 26,49±1,75 for snRNA U6 and 25,94±4,3 for snRNA U44. Average Cycle Threshold (Ct) values in tissue samples were found to be 23,83±4,36 for GAPDH, 18,10±1,54 for snRNA U6 and 20,41±1,13 for snRNA U44. The correlation between lesion size and the expressions of 6 pre-determined miRNAs is also evaluated. As a result of this analysis, a positive correlation is observed for the expressions of the following miRNAs; miR-16 on lesions larger than 2 cm, miR-224 on lesions larger than 4,2 cm and miR-501 on lesions larger than 2.1 cm. Contrarily, miR-122 and miR-28 expressions were decreased on lesions larger than 3.6 cm and 0.8 cm respectively. miR-122 on samples with a size of 3,6-4,5 cm and miR-224 on samples larger than 4,5 cm in size. Results do not indicate any correlation between the lesion size and the expression of miR-221. This study indicates that miR-28 can be used as a diagnostic biomarker for HCC especially on serum screening, the use of snRNA U6 is appropriate for normalization in Real-Time PCR (relative quantitation) applications and 4 of these pre-determined miRNAs (miR-16, miR-28, miR-122 and miR-224) show increased expression patterns with different lesion sizes while no such correlation is seen with HBV DNA levels, miR-501 was found to be increased almost 2-fold on lesions with size from 2.1 to 14 cm. As a result of this study, it was concluded that miR-28 provided preliminary data for the evaluation of a non-invasive screening test for the early diagnosis of HCC in the clinical validation phase. The correlation observed between the lesion sizes and miRNA expression suggests that after further scientific studies are carried out by diversifying the number of clinical specimens and lesion sizes, these types of miRNA screening methods may present a preliminary information about the size of the lesion with a statistical analysis software.