College Innovative Experiment

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College Innovative Experiment

Group members

Advisors

Studies about the roles of DLC-1 gene in HBV-induced Hepatocellular Carcinoma

Prof. Wensheng Sun, Liang Xiaohong

Cai JieReporter

Wang Jimeng, Wu Fei

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CONTENTSCONTENTS

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1 Background

DLC-1

New subject

Experimental Procedure

Feasibility analysis

Innovation

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DLC1 mRNA in BEL7402 cells transfected with or without HBc expression vector

BEL-740

2

BEL7402

-HBc

0.0

0.5

1.0

1.5

2.0

DL

C1

rala

tive

exp

ress

ion

Results

The difference of DLC1 expression between HBV-transgenic mice and

their normal littermates

control

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Paper1. A paper has been published on Chin J Curr Adv Gen Surg.

2. Another paper would be submitted to Medical hypothesis soon.

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Interest

Communicating ， consulting

Subject

Designing

Preparing

Experiment

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Tumor-bearing nude mice

Ayw subtype HBV Tg mice

Experimental Procedure

Experiment in vitro

Experiment in clinical samples

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Infection of Hepatitis B Virus (HBV):

the most important reason for Hepatocellular Carcinoma （ HCC） in China

HBV HCC

Background

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cancer

Identification of cancer-related genes

Multiple genetic alterations

Background

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DLC-1 (deleted in liver cancer 1)

DLC-1

First cloned in a human hepatocellular carcinoma.

Located at human chromo-some 8p21.3-22

A potential tumor suppressor

A focal adhesion molecule

preventing cell transformation

DLC-1

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SAM RhoGAP

START

DLC-1

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HBV DLC-1

HBc DLC-1

New subject

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Objectives

To provide responsible theory evidence for prevention and gene therapy of HCC

To explore the role of DLC-1 gene in HBV-induced HCCNO.1

NO.2

New subject

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Tumor-bearing nude mice

Ayw subtype HBV Tg mice

Experimental Procedure

Experiment in vitro

Experiment in clinical samples

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11 Detect the effect of HBV on DLC-1 in thses cells

Plasmids: pcDNA3-HBV1.1 、 pcDNA3

Experiment in vitro

Hepatocyte cell lines: HepG2 、 L02

Method: liposome transfection

Procedure

22 Detect the effect of DLC-1 to HBV-transfected cells

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Total RNA by TRIZOL

qRT- PCR

HepG2 cells or L02 cells

A B

Total protein

Western Blot

Detect the effect of HBV on DLC-1 in thses cells

C

Experiment in vitro

pcDNA3-HBV1.1 pcDNA3 No transfection

Transfection

DLC-1

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Add in TRAIL or FasL

D E F

HepG2-HBV1.1 or L02-HBV1.1

Transfection

Empty plasmid

Gather the cells after 24h

Detect the apoptotic rate with TUNEL

Add in 5mg/L RNaseA to digest

Detect the cell cycle with PI-FCM

Gather cells at different time(0h,24h,48h,72h,5d,7d

)

Detect the cell proliferation with MTT

Detect the effect of DLC-1 to HBV-transfected cells

Experiment in vitro

DLC-1 plasmid No Transfection

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Detect the methylation of DLC-1 promotor

Use LOH to analysis the DLC-1

Mechanisms

HepG2-HBV1.1 L02-HBV1.1

Experiment in vitro

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Use immunohistochemical method to detect the expression of DLC-1 protein

Experiment in clinical

Samples:Samples: Normal liverNormal liver

HBV-related HCC liverHBV-related HCC liver

Use qRT-PCR to detect the expression of DLC-1 mRNA

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HBV Tg mice

Experiment on mice

Neoplasia formation

Nude mice

Tumor suppression

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BALB/c BALB/c HBV-Tg

Total protein

Western Blot

Experiment on ayw subtype HBV Tg mice

6 ， 12 ， 20months

Total RNA by TRIZOL

qRT- PCR

DLC-1

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（ Stable transfected with HBV）

Experiment on tumor-bearing nude mice

HepG2.2.15 cell

DLC-1 plasmid

Neoplasia formation

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Feasible theoretically, and argument reasonable;

Infarctate preliminary work;

Strong technolongical develop-ment and innovation capacity.

Feasibility analysis

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Innovation

DLC-1 gene in HBV with whole expression.

Research both in vitro and vivo ， more clinical significance.