Role of HCV Core gene of genotype 1a and 3a and host gene Cox-2 in HCV-induced pathogenesis
Virology Journal
Role of HCV Core gene of genotype 1a and 3a and host gene Cox-2 in HCV-induced pathogenesis
Shah Jahan 0
Saba Khaliq 0
Bushra Ijaz 0
Waqar Ahmad 0
Sajida Hassan 0
0 Applied and Functional Genomics Laboratory, National Centre of Excellence in Molecular Biology, University of Punjab , Lahore 53700 , Pakistan
Background: Hepatitis C virus (HCV) Core protein is thought to trigger activation of multiple signaling pathways and play a significant role in the alteration of cellular gene expression responsible for HCV pathogenesis leading to hepatocellular carcinoma (HCC). However, the exact molecular mechanism of HCV genome specific pathogenesis remains unclear. We examined the in vitro effects of HCV Core protein of HCV genotype 3a and 1a on the cellular genes involved in oxidative stress and angiogenesis. We also studied the ability of HCV Core and Cox-2 siRNA either alone or in combination to inhibit viral replication and cell proliferation in HCV serum infected Huh-7 cells. Results: Over expression of Core gene of HCV 3a genotype showed stronger effect in regulating RNA and protein levels of Cox-2, iNOS, VEGF, p-Akt as compared to HCV-1a Core in hepatocellular carcinoma cell line Huh-7 accompanied by enhanced PGE2 release and cell proliferation. We also observed higher expression levels of above genes in HCV 3a patient's blood and biopsy samples. Interestingly, the Core and Cox-2-specific siRNAs down regulated the Core 3a-enhanced expression of Cox-2, iNOS, VEGF, p-Akt. Furthermore, the combined siRNA treatment also showed a dramatic reduction in viral titer and expression of these genes in HCV serum-infected Huh-7 cells. Taken together, these results demonstrated a differential response by HCV 3a genotype in HCVinduced pathogenesis, which may be due to Core and host factor Cox-2 individually or in combination. Conclusions: Collectively, these studies not only suggest a genotype-specific interaction between key players of HCV pathogenesis but also may represent combined viral and host gene silencing as a potential therapeutic strategy.
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Background
An estimated 3% of the world’s population is chronically
infected with Hepatitis C virus (HCV) which is the main
cause of liver fibrosis and cirrhosis, often leading to
HCC (hepatocellular carcinoma) in a substantial number
of patients. Almost 10 million people in Pakistan are
living with HCV [1,2]. The most prevalent HCV genotype
in Pakistan is 3a followed by 1a [3]. HCV virion is
enveloped and has a positive strand genome comprising
9.6 kb RNA which is processed by cellular and viral
proteases into 10 viral proteins, Core, E1, E2, p7 (structural
proteins), NS2, NS3, NS4a, NS4b, NS5a and NS5b
(nonstructural proteins). Although specific mechanisms by
which HCV disease progresses remains unknown, direct
interaction of specific viral proteins with host cell
system has shown to be accounted for some of its
pathophysiological profile of HCV patients [4].
Besides nucleocaspid formation, HCV Core protein, in
particular, also modulates gene transcription, cell
proliferation, cell death and interferes with metabolism
leading to oxidative stress, liver steatosis and eventually
HCC. Oxidative stress has emerged as a key contributor
in the development and progression of HCV-induced
pathogenesis of liver [5,6]. Several factors might
contribute to oxidative stress associated with HCC, as HCV
Core and NS5a proteins, which are able to up-regulate
Cox-2 expression; a key player of oxidative stress in
hepatocytes derived cells [7]. The expression of Cox-2
in HCC was found to correlate with the levels of several
key molecules implicated in carcinogenesis such as
iNOS (induced nitric oxide synthase), VEGF (vascular
endothelial growth factor) and p-Akt [8,9]. iNOS and
Cox-2 have carcinogenic effects achieved either directly
or by producing mediator that regulate cellular growth
[10]. Cox-2 can induce angiogenesis growth factors via
VEGF in HCV associated HCC [9,11,12]. A number of
other inflammatory mediators including nitric oxide
(NO), Cox-2, and prostaglandin E2 (PGE2) in cultured
hepatocellular carcinoma cells also stimulate VEGF [11].
Cox-2 activates Akt in human HCC via a
p13-kinasedependent mechanism [13], it acts as an important
signal mediator, which regulates cell survival and
proliferation [14,15]. Despite several studies, mechanisms
involved in HCV-associated pathogenesis are not
completely understood; it may vary as a function of viral
genotype [16].
Very few studies have been undertaken to evaluate the
role of HCV Core protein of genotype 3a and 1a in
HCV induced pathogenesis. In this study, we selected
Huh-7 cells as culture model system for the transient
transfection using HCV 3a and 1a Core genes and viral
load analysis using HCV-infected serum as inoculum.
We found a greater effect of HCV Core of genotype 3a
on the expression levels of Cox-2, iNOS, VEGF, p-Akt
in transiently expressing Core protein and in
seruminfected Huh-7 cells as well as i (...truncated)