الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma (HCC) is the main primary liver cancer and represents the fifth cause of cancer in males, the seventh in females, and is considered the third reason of cancer-related death around the world. The last decade has witnessed a huge ascent in the frequency of HCC, with particularly high incidence reported in Egypt.
Chemotherapy is an important therapeutic option for the treatment of tumors. Doxorubicin is the most effective cytotoxic drug for treatment of HCC. Nevertheless, the clinical outcome of HCC is still poor, which can be attributed to the frequent occurrence of resistance to various chemotherapeutic agents that permits cancerous cells to proliferate uncontrollably and to become more aggressive with a greater ability to metastasize to other organs. Alterations in drug efflux were considered one of the primary causes of multidrug resistance (MDR). Especially, the overexpression of ABC (ATP-binding cassette) super family of transporters is firmly connected to chemoresistance. Likewise, activation of mitogen-activated protein kinase (MAPK) pathway was accompanied with MDR in HCC.
Many studies have affirmed the significance of microRNAs (miRNAs) in the management of tumor cell sensitivity to drugs and modifying the expression of genes contributed to drug transport. MiR-122 is often down-regulated in primary HCC. It has been demonstrated that loss of miR-122 expression is firmly associated with dysregulation of differentiation of hepatocytes, poor prognosis, and metastasis of liver cancer that is resulted from MDR progression.
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Summary
The aim of the present study is to evaluate the role of mir-122 restoration as molecular targeted therapy affecting alpha-fetoprotein (AFP) and genes/proteins contributed to MDR phenomena and MAPK pathway. As well as, study the effect of doxorubicin treatment on MDR and MAPK pathway and estimate the relationship between them. In addition, compare the therapeutic effects of doxorubicin and mir-122 individually and in combination.
This study was conducted on Human HCC-cell line, HepG2 cells that were treated with different treatments of doxorubicin and/or mir-122 mimics; compared to the corresponding control cells. These cells were subjected to the following:
1- Total microRNAs were isolated from all transfected cells with mir-122 mimic or negative control, to evaluate the mir-122 level by miSCript miRNA PCR system; using qRT-PCR.
2- Total RNAs were isolated from all cells to quantify the level of MDR genes, MAPK genes, and AFP gene; using qRT-PCR.
3- Total protein extracts were isolated from all cells to estimate the level of MDR protein, ABCB1; using western blotting technique.
4- Cell culture media were isolated from all treatments to quantify the level of AFP; using ELISA.
5- DNA content of the cells was stained with propidium iodide to analyze cell cycle distribution; using flowcytometry.
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The results of this study revealed the following:
1- Statistically significant increases in ABCB1 and ABCF2 mRNAs expression levels were detected in HepG2 cells treated with acute high doxorubicin dosage, but this increase was reduced with continuous treatment with a chronic low doxorubicin dose.
2- Treatment of HepG2 cells with mir-122 mimics resulted in a statistically significant decrease in ABCB1 mRNA and protein expression levels.
3- Treatment of HepG2 cells with a chronic low dose of doxorubicin caused a statistically significant decrease in ABCC1 expression level.
4- Restoration of mir-122 mimics after treatment of HepG2 cells with a chronic low dose of doxorubicin, resulted in a statistically significant decrease of ABCF2 gene expression level.
5- Restoration of mir-122 expression in HCC cells that were then treated with doxorubicin diminished their tumorigenic properties, specifically growth, proliferation, invasion, and migration. This was achieved through the accumulation of cells in G0/G1 phase with 85% and G2/M with 9%.
6- There were no significant changes in AFP expression levels in cells treated with acute/chronic doses of doxorubicin or even transfected with mir-122 mimics.
7- A highly statistically significant increase of RAC1 gene expression level was detected in cells treated with chronic low doxorubicin dose; this increase was less than that recorded for cells treated with an acute high dose.
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8- Treatment with chronic low doses of doxorubicin resulted in a reduction of MAP3K2 gene expression level.
9- A statistically positive correlation was found between gene expression levels of MAPK11 and ABCB1 in wild HepG2 cells transfected with mir-122 mimics.
10- A statistically positive correlation between RAC1 and ABCB1 genes in cells treated with a chronic low doxorubicin dose followed/unfollowed with mir-122 transfection.