الفهرس 
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Abstract
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancers with cirrhosis being the major risk factor for its development. Worldwide, Egypt has the highest prevalence of HCV and therefore HCC representing a major health problem in Egypt. The alterations in cancer driver genes and associated pathways are the major triggers for HCC. So, the identification and targeting of these genes are beneficial to understand HCC and to develop a new therapy.
RNAi (RNA interference) is a gene editing technique that has been used to manipulate gene expression. RNAi can bind to complementary mRNA and degrades or regulates the mRNA. Subsequently, a decrease in protein synthesis occurs.
Based on bioinformatics analysis tool (www.proteinatlas.org) and genome data from the previous studies deposited in The Cancer Genome Atlas (TCGA) (http:// cancergenome.nih.gov), CTNNB1 and RABIA, HCC associated genes, was retrieved.
This study was done at the Medical Biochemistry Department, Faculty of Medicine, Ain Shams University during the period from February 2018 – April 2019.
The objectives of the current study were: to target CTNNB1 and RABIA mRNA in HepG2 cell lines by using two different RNAi for each, then evaluate the effect of their Knocking down on the cell viability and proliferative activity of HepG2 cells. Also, we aimed to validate that interference by
measuring the expression level of these genes by real-time PCR. Finally, the consequence of
CTNNB1 interference on HepG2 proliferation was confirmed through immunofluorescence staining with a
specific polyclonal antibody against Ki67 (a marker of proliferation). Also, the consequence of
RAB1A interference on HepG2 cells was evaluated by microscopic examination of autophagosomes.
Cell culture results revealed that there was no significant difference between mock and untreated
HepG2 cell lines either in viable cell counts or viability percent detected by Trypan blue
exclusion test (p >0.05). Both viable cell counts an viability percent were significantly
lower in HepG2 cell lines transfected with siCTNNB1 or siRAB1A compared to mock HepG2 cell lines
(p<0.05).
Number of viable cells and viability percent were significantly affected in HepG2 cells transfected
with siCTNNB1 compared to cell lines transfected with siRAB1A
(p<0.05).
To ensure that knockdown analysis of CTNNB1 and
RAB1A worked optimally; HepG2 cells were transfected with a positive control siRNA. It provides
high knockdown of ubiquitous human cell survival genes resulting in a high degree of cell death.
We found that HepG2 cells transfected with
siCTNNB1 or siRAB1A had significant differences in viable cell count and viability percent compared to cells transfected with positive control siRNA.
To determine if changes in phenotype or gene expression are nonspecific; HepG2 cells was transfected with negative control siRNA. It is a non-silencing siRNA with no homology to any known mammalian gene. HepG2 cells transfected with either siCTNNB1 or siRAB1A had significant differences in viable cell counts and viability percent when compared to those transfected with negative control siRNA.
Regarding cell proliferation assay, no significant differences between mock and untreated cell lines were found in number of active proliferative cells. The active proliferative cell count was calculated from CellTiter 96® AQueous One Solution Cell Proliferation standard curve for HepG2. But, active proliferative cell counts were significantly affected in HepG2 cells transfected with (siCTNNB1& siRAB1A) (p<0.05) compared to mock HepG2 cells. While number of active proliferative cells was significantly affected in transfected cells with siCTNNB1 compared to those transfected with siRAB1A.
Microscopic examination of different HepG2 cells revealed that there were huge condensed colonies of cells, with high nuclear-cytoplasmic ratio in untreated HepG2 cells. While in HepG2 cells transfected with siCTNNB1, there were moderate condensed colonies with moderate-sized cells. In HepG2 cells transfected with siRAB1A, there were discrete colonies with moderate-sized cells.
To validate the interference of candidate genes, real-time PCR was carried out in different HepG2 cells. The relative expression levels of CTNNB1 and RAB1A were not significantly differed between mock and untreated HepG2 cells. However, the cells transfected with siCTNNB1 and siRAB1A had lower expression levels of CTNNB1 and RAB1A respectively compared to mock HepG2 cells (p <0.05).
The relative expression of CTNNB1 and RAB1A were positively correlated with cell culture results (namely, viable cell count detected by Trypan blue exclusion test, viability percent, and active proliferative cells by MTT assay) (p <0.05).
The consequence of CTNNB1 interference on HepG2 cells proliferation was done using immunofluorescence staining against Ki67 (a marker of proliferation). Mock HepG2 cells had a homogenous high expression of Ki67 with high fluorescence intensity and the expression was localized to the nucleus and cytoplasm reflecting high proliferation rate. While HepG2 cells transfected with siCTNNB1 shows a discrete spread out hepatic cells with moderate faint expression of Ki67 and the fluorescence intensity was lower compared to mock HepG2 cells. These findings reflect the effect of CTNNB1 silencing on inhibiting cell proliferation in HepG2 cells.Concerning the consequence of RAB1A interference on HepG2 cells, microscopic examination for autophagosomes was performed. HepG2 cells transfected with siRAB1A had few autophagosomes in comparison to mock HepG2 cells. This finding reflects the effect of RAB1A knockdown on autophagosome formation in HepG2 cells.