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Page 8 of 10 Farghaly et al. Hepatoma Res 2018;4:41 I http://dx.doi.org/10.20517/2394-5079.2018.30

understood. Recent studies have demonstrated that Alu RNA plays a major role in post transcriptional
regulation of gene expression for example by affecting alternative splicing, mRNA stability and protein
synthesis [33,34] . One of the recent identified targets that is modulated by Alu-RNA is the RPL22-mRNA .
[20]
Thus, here our findings further confirm the regulatory effect of Alu-RNA molecules on gene expression of
RPL22 that activated upon HS treatment. Interestingly, regulation of RPL22 in Alu-RNA dependent manner
leads to significant interruption of HCV replication in HepG2 cells. Therefore, the current data provide a
new technique that can prevent HCV replication in host cells without a harmful effect on treated cells as
compared with non-treated cells. HS refers to cellular exposure to rapid stress changes such as temperature,
toxins, oxidative stress, heavy metals, and pathogenic infections. Specifically temperature induced HS, even
of a few degrees, has the ability to disturb protein folding. Other cellular damages have been reported in
response to HS stress including rearrangement of cytoskeleton, alternation of organelle location, decreasing
of ATP production, decreasing of proteins translation, changes in RNA splicing and gene silencing .
[35]
The present data indicate the possible regulation of RPL22 expression in infected HepG2 cells that were
subjected to HS. RPL22 is an RNA-binding protein with 60S large ribosomal subunit that plays a crucial
role of macrolide resistance in bacteria . In vertebrates, RPL22 mutation might increase the proliferation of
[36]
cells and then increase cancer risk. However, RPL22 has not been implicated in any lung diseases, especially
in lung cancer [36,37] . Other study demonstrated that human RPL22 protein interact with HCV-NS5A and
support viral RNA translation . NS5A protein is the most common HCV research regarding its potential
[38]
regulation of cellular immune response following infection. NS5A protein is translated from HCV genome
as one of a large number of ploy-proteins that processed by NS3 protease . NS5A protein modulates host
[39]
interferon signaling via direct interaction with the cellular factor retinoic acid-inducible gene-I (RIG-I)
protein resulted in blocking of interferon signaling in infected cells . Additionally, NS5A plays the key role
[40]
during HCV replication cycle and viral particles assembling through interaction with several viral and host
proteins to insure viral replication. Several evidences indicate that NS5A is localized in certain modified
cytoplasmic membrane during HCV replication that facilitates its significant role in HCV replication
complex and replicase [41,42] . Here, the relative expression of NS5A has been detected by q-RT-PCR using
newly designed specific oligonucleotides. Our results showed that NS5A relative expression was significantly
reduced in infected cells that were subjected to HS in comparison with control infected cells. On the other
hand, flowcytometry has been used to investigate the clearance status of NS5A protein in HepG2 cells that
were treated with HS. Interestingly, in comparison with control infected cells, our findings reveal that the
percentage of NS5A positive cells was 30% of infected cells that were treated HS. Meanwhile, the percentage
of NS5A positive cells was up to 70% regarding the control infected cells. These data demonstrate that HCV
replication is potentially interrupted in HepG2 cells that were subjected to 5 min of HS. Taken together,
these data provide an evidence for the possible inhibition of HCV infection via HS treatment affecting the
expression of RPL22 through activation of Alu non-coding repeated element in HepG2 cells.

DECLARATIONS
Acknowledgments
The authors thank the chemist Rofida Refaai, Ain Shames Specialized Hospital for providing the blood
samples derived from patient infected with HCV genotype 4. Special thanks to Prof. Dr. Mahmud Elmeteany,
Faculty of Medicine, Ain Shames University, for his medical supervision.

Authors’ contributions
Planned and designed the study: Khalil H
Did the experiments assessing and developments: Farghaly H, Guirgis AA, Khalil H
Wrote the manuscript: Khalil H

Availability of data and materials
The data and materials are obtained from the corresponding author.