%0 Journal Article %T Effect of 5-Azacytidine as A Demethylation Agent on PCNA and RB1 Genes in Hepatocellular Carcinoma Cell Model “HepG2” %J Egyptian Academic Journal of Biological Sciences. C, Physiology and Molecular Biology %I Egyptian Society of Biological Sciences %Z 2090-0767 %A El Nobey, Muhamed A. %A El Hamidy, Salim M. %A Ullah, Ihsan %A Omar, Abdulkader S. %A Alkenani, Naser A. %A Zari, Ali %A Batawi, Ashwaq H. %A El-Araby, Rady E. %D 2022 %\ 09/21/2022 %V 14 %N 2 %P 73-83 %! Effect of 5-Azacytidine as A Demethylation Agent on PCNA and RB1 Genes in Hepatocellular Carcinoma Cell Model “HepG2” %K 5-azacytidine %K Hepatocellular carcinoma %K PCNA gene %K RB1 gene %K HepG2 cells, DNA methylation, Gene expression, Real-time PCR, Methylation-specific PCR %R 10.21608/eajbsc.2022.260474 %X Epigenetic alterations are associated with human cancer development and the inactivation of tumor suppressor genes. Two such tumor suppressor genes, PCNA and RB1, showed an aberrant gene expression in HCC. DNA methylation alters the expression of genes and is one of the processes that transform normal cells into cancer cells. PCNA and RB1 genes screened methylation in the promoter regions in HepG2 cells. The current study examined the effect of DNA methyltransferase inhibitor (5-Azacytidine) on gene expression of PCNA and RB1 genes after treatment and showed the impact of 5-aza-CR on the Methylation degree of HepG2 cells. HepG2 cell line originated from hepatocellular carcinoma (HCC). Since HepG2 exhibits the characteristics of human liver carcinoma, it was a good model for detecting the changes in methylation patterns and the gene expression level that was detectable in a clinical setting. The human HepG2 cell line was treated with 5, 10, and 25 µM of 5-aza-CR for 24 h, 48 h, and 72 h. Methylation of PCNA and RB1 was detected by methylation-specific polymerase chain reaction (MSP). PCNA and RB1 gene expression detected by reverse transcription-polymerase chain reaction. The influence of 5-aza-CR on Cell viability was assessed by SRB assay for 24 h, 48 h, and 72 h. The IC50 is 20.52 µM for 24 h, 12.6117 µM for 48 h, and 10.63 µM for 72 h after exposure to 5-aza-CR, which showed that 5-aza-CR inhibited the growth of HepG2 cells in a time, and dose-dependent manner. Although other genes may be demethylated due to the 5-aza-CR treatment, we concentrated on the PCNA and RB1 genes. In HepG2 cells, PCNA and RB1 gene methylation were found before 5-aza-CR treatment. In contrast, no PCNA or RB1 gene expression was detected. Treatment with different concentrations of 5-aza-CR significantly decreased the methylation degree of the PCNA and RB1. 5-aza-CR at 25 µM for 72h showed the highest induction activity of PCNA and RB1 gene expression. Methylation-specific PCR results showed that 5-aza-CR promoted the expression of PCNA and RB1 by demethylation. Our results illustrate that 5-aza-CR could reverse the abnormal methylation degree of the PCNA and RB1 genes that are hypermethylated in HepG2 cells and induces the expression of the PCNA and RB1 genes by demethylation. %U https://eajbsc.journals.ekb.eg/article_260474_d73a4050343fca034f28bbab0f0dcf87.pdf