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Differential MicroRNA Expression
Between Hepatitis B and Hepatitis C
Leading Disease Progression to
Hepatocellular Carcinoma
HEPATOLOGY 2009;49:000-000
Yin-Hong Zhu
朱银洪
Overview of Presentation
Backgroud -why
Protocol
-how
Discussion-what
Instruction-learn
1、HBV,HCV,HCC
Genes involvedand
Pro-apoptotic
in inflammation
DNA repair responses
HBV
epidemiologic
Disease
progression
Signal
pathways
Factors
inducing
those
differences
virologic
Different
2006
features
oncogenic
histologic
factors
findings
1998
2001
findings
HCC promotion
inflammatory
and anti-apoptotic
phenotypes
Genes responsible
for Anti-inflammation
HCV
•
Genes expression
Signal pathways
HEPATOLOGY 2009;49:000-000.
?
2、Regulation of gene expression
microRNA
30% of human genes
3、 Gene expression regulated by
microRNAs
.
4、Function of microRNA in
animals and plants
diseases such as
infection and cancer
Development
Proliferation
(Stem cells)
microRNA
Differentiation
Apoptosis
5、microRNA and viral infection
疱疹病毒家族
禽流感病毒
EBV,KSHV,MHV-68,
HCMV,HSV-1
多瘤病毒家族
Avian influenza virus
microRNA
HBV,HCV
host or virus
SV40(Simian virus 40)
腺病毒家族
逆转录病毒家族
hAV(adenovir)
HIV-1
HEPATOLOGY 2009;49:000-000.
6、 microRNAs ,HBV,HCV,HCC
HBV or HCV
mirRNA-122
HBV or HCV ,HCC
HBV between HCV ,HCC
Normal
SPC3649
LNA-antimiRTM-122
Clinic trial phase I on
health person
Blank
7、 Scientific supposition of this
research
Dignosis ,Therapy
HBV
≠
Genes
expression
≠
HCV
≠
≠
microRNAs
Signal
Disease progression
pathways HCC promotion
Overview of Presentation
Backgroud-why
Protocol
-how
Discussion -what
Instruction-learn
1、Basic problems for miRNA
researches
When and where the
miRNAs are expressed？
Diagnosis
miRNA profiles analysis
What miRNAs
are expressed
Isolation,clone,sequence
Which gene is the
target of the miRNAs
Functional analysis
Which important miRNA influence
Some certain bio-process
Therapy
miRNA library selection
2、Researches of microRNA
and diseases
miRNA功能研究
miRNA表达的分析
Compare miRNA profiles of diseases
and normal samples
Identify miRNAs that affect cell
division,apoptosis,angiogenesis,cell
proliferation,telomerrase activity
在疾病样本中差异性表达的
miRNAs
诱导或消除了疾病表型的
miRNAs
Target genes
3、Protocol for this research
RTD-PCR
Microarray Analysis.
miRNAs expressing profiles analysis
Functional analysis
Identification of
microRNA Target genes
Verification in HBV/
Verification of Regulation HCV Infection Model
of genes by microRNA
3、Protocol for this research
RTD-PCR
Hierarchical cluster analaysis
Class Prediction
Binary tree calssification
188 known miRNAs
Control 9 normal liver tissue from patients with metastatic
liver cancer
HBV
12 patients
HCV
14 patients
Normal
13significantly differential microRNAs
3、Protocol for this research
Microarray Analysis.
Global analysis of
mRNA expression
in the liver tissues
3、Protocol for this research
Identification of microRNA Target genes
Step 1
Step 2
Identification of
microRNA Target genes
Predicted by
MIRANDA Pro3.0
3、Protocol for this research
1、Gain of function：transfect
Huh7.5 cells with Pre-miRNA
2、Lost of funtcion：transfect
with Small interfering RNAs
specific to 13 candidate miRNAs
Verification of Regulation
of genes by microRNA
miRNAs and Target genes’
mRNA were detected by
RTD-PCR
3、Protocol for this research
pHBV
Huh 7.5 cells
JFH1-RNA
Virus proteins and DNA/RNA
were deteced
miRNA expression
Verification in HBV/
HCV Infection Model
Overview of Presentation
Backgroud-why
Protocol -how
Discussion -What
Instruction -learn
1、CH and HCC miRNAs in the
same patient
HBV
No dependence
HCV
Disease
＞
Normal
HEPATOLOGY 2009;49:000-000.
Paired condition
2、 HBV and HCV infectionspecific miRNAs
HBV
HCV
6 miRNAs
13 miRNAs
Cell death,DNA damage and recombination,
trancription signals
！
Immune response,antigen presentation,
Cell cycle,proteasome,and lipid metabolism
Endogennous miRNA involved in defense mechanisms,mainly RNA viruses
Normal
HEPATOLOGY 2009;49:000-000.
3、Tumor-associated
miRNAs
has-let-7a
ras and c-myc genes
has-miR-34
p53 tumor supressor pathway
has-miR-191
Worse survival in acute myeloid leukemia
HBV
HCV
has-miR-133b
Sequamous cell carcinoma
has-miR-142-5p
Hematopoietic specific
Normal
HEPATOLOGY 2009;49:000-000.
4、HCC-specific miRNAs
Cytoskelton
miRNAs Cell cycle
HBV Ubiqutin proteasome
Transiation
Immune
response
HBV
HCV
Pathway
Cancer-associated
Inflammation-mediated
Normal
HEPATOLOGY 2009;49:000-000.
5、miRNAs in HBV/HCV disease
progression and HCC promotion
HEPATOLOGY 2009;49:000-000.
Overview of Presentation
Backgroud-why
Protocol -how
Discussion - What
Instruction -learn
1、The same problem in different aspects
Researches are still in need
HBV
2009
microRNAs
HCV
2001
Genes
expression
2006
Disease progression
Signal
pathways HCC promotion
2、The same aspect with diffeCellular
rent protocols
apoptosis
Cellular
qRT-PCR
表达谱系分析
功能鉴定
invasion
Cellular
miRNarray
model
Cellular
Northern Blot
proliferationCellular
MicroRNA
Bioinformatics
cycle
prediction microarray
5’RACE
Luciferase
Western Blot
reporter
qRT-PCR
靶基因筛查
Sensor
reporter
EMSA
CHIP
表达调控研究
3、The same protocol for the
similar researchs
miRNA功能研究
miRNA表达的分析
Compare miRNA profiles of cancer
and normal samples
Identify miRNAs that affect cell
division,apoptosis,angiogenesis,cell
proliferation,telomerrase activity
在疾病样本中差异性表达的
miRNAs
诱导或消除了疾病表型的
miRNAs
Target genes