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Hepatitis B VIRUS REGULATORY PROTEIN X-CAUSED
DEGRADATION OF SMC5/6 IMPAIRS HOST DNA DAMAGE
REPAIR AND PROMOTES TUMORIGENESIS
Kazuma Sekiba1, Motoyuki Otsuka1 and Kazuhiko Koike2, (1)
Department of Gastroenterology, The University of Tokyo,
(2) Department of Gastroenterology, Graduate School of
Medicine, the University of Tokyo
Background: Chronic hepatitis B virus (HBV) infection is
a major global cause of hepatocellular carcinoma (HCC)
Although HBV regulatory protein X (HBx) is a well-known
Direct driver of HCC, its precise oncogenic targets remain
Unclear. Recently, Smc5/6 complex was identified as a host
Restriction factor of HBV replication and is degraded by
HBx protein Because one of the principal roles of Smc5/6
Is promoting homologous recombination (HR) repair at the
DNA double-strand breaks (DSBs), we hypothesized that the
HBx-mediated Smc5/6 degradation leads to the accumulation
Of DNA damages by the impairment of HR repair function,
Resulting in the driving of HCC Methods: 1) We analyzed the
Liver tissues from HBV-infected human liver-chimeric mice and
HBx transgenic mice to determine whether HBV infection and
HBx expression induce DNA damages in vivo 2) To investigate
Whether the degradation of Smc5/6 protein is involved in
The accumulation of DNA damages, we evaluated the DNA
Damage repair efficiency in a stable cell line which expresses
Short hairpin (sh)-RNA targeting Smc5 (HepG2shSmc5) and in
That expresses FLAG-tagged HBx (HepG2Flag-HBx) The DNA
Damage was assessed by the comet assay and quantification
Of γH2Ax expressing levels. 3) For further confirmation
About the involvement of HBx protein in the HR pathway, we
Revised the HR at the double-stranded DNA break which
Was induced by CRISPR-Cas9 system. 4) To further confirm
The dependency of Smc5/6 proteins in the mechanism of the
Impairment of DNA damage repair function by HBx protein,
We analyzed the DNA damage repair with the transient
Expression of Smc5/6 protein in HepG2Flag-HBx cells. 5) We
Applied another Smc5/6 restoration method using nitazoxanide
(NTZ), a compound which has an inhibitory effect on HBx–
DDB1 binding, resulting in the restoration of Smc5/6 protein
Levels, as we previously reported 6) Finally, we performed a
Clonogenic assay to assess whether degradation of Smc5/6
Protein by HBx leads to the promotion of tumorigenesis
Results: 1) The DNA damage was accumulated both in HBV
Infected human liver-chimeric mice and in HBx transgenic
Mice 2) Smc5 knockdown and HBx expression impaired
The repair of DSBs 3) HBx protein suppressed HR function
At the DSBs induced by CRISPR-Cas9 system 4) HBx
Suppressed DNA damage repair in a Smc5/6-dependent
Mode. 5) NTZ restored the DNA damage repair function
Through the restoration of Smc5/6 under HBx expression
6) Both HepG2shSmc5 and HepG2Flag-HBx cells increased the
Cluster formation, which suggested that the degradation of
Smc5/6 protein by HBx has a promoting effect on cellular
Transformation Conclusion: HBx-triggered degradation of
The Smc5/6 complex contributes to the impairment of the
Repair of DSBs, which may lead to the accumulation of DNA
Damages and promotion of tumorigenesis
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发表于 2019-10-23 15:20