[Frontiers in Bioscience S2, 1127-1144, June 1, 2010]

System biology analysis of cell cycle pathway involved in hepatocellular carcinoma

Meiqian Sun1, Wenjuan Mo1, Xuping Fu1, Gang Wu1, Yan Huang1, Rong Tang1, Yi Guo1, Minyan Qiu2, Feng Zhao3, Lin Li4, Shengdong Huang3, Yumin Mao1, Yao Li1, Yi Xie1

1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, P.R. China, 2 Shanghai BioStar Genechip Inc., Shanghai, P.R. China, 3 Institute of Thoracic Cardiac Surgery, Changhai Hospital, Second Military Medical University, Shanghai, P.R. China, 4 Department of Radiation Therapy, Affiliated Tumor Hospital of Guangxi Medical University, Guangxi, P.R. China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Tissue samples and RNA isolation
3.2. cDNA microarrays
3.3. Reconstruction of cell-cycle genetic network for hepatocellular carcinoma
3.3.1. Gene selection
3.3.2. Modeling genetic network
3.3.3. Identification of genetic network
3.4. Cell culture
3.5. RNA interference
3.6. Quantitative real-time reverse transcription-PCR
3.7. Cell cycle analysis
3.8. Apoptosis detection using flow cytometry
3.9. MTT assay
3.10. Statistical analysis
4. Results
4.1. Identification and functional analysis of differentially expressed genes in HCC
4.2. Results of the cell-cycle network reconstruction
4.3. Reconstructed cell cycle network validation and complement
4.3.1. Rebuilding a published interaction
4.3.2. Unclosing some interesting interactions
4.3.3. Experiment validation and complement of the network
4.3.3.1. Silencing of MCM2 and CCNB1 in HCC cells by siRNA
4.3.3.2. Induction of G2/M-phase arrest, apoptosis and antiproliferation in HCC cells by depletion of MCM2 or/and CCNB1
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

To investigate genetic mechanisms of hepatocarcinogenesis and identify potential anticancer targets in hepatocellular carcinoma (HCC), we analyzed microarray gene expression profiles between 33 HCCs and their corresponding noncancerous liver tissues. Functional analysis of differentially-expressed genes in HCC indicated that cell cycle dysregulation plays an important role in hepatocarcinogenesis. Based on 14 differentially-expressed genes involved in cell cycle in HCC, we applied Structural Equation Modeling (SEM) to establish a potential genetic network which could assist understanding of HCC molecular mechanisms. siRNA-mediated knock-down of two significantly up-regulated genes, minichromosome maintenance protein 2 (MCM2) and cyclin B1 (CCNB1), in HCC cells (SMMC-7721 and QGY-7703) induced G2/M-phase arrest, apoptosis and antiproliferation in HCC. Some up-regulated cell cycle-related genes in HCC were down-regulated following specific depletion of MCM2 or/and CCNB1 in HCC cells, which might well validate and complement the reconstructed cell cycle network. This study may contribute to further disclose hepatocarcinogenesis mechanism through systematically analyzed the HCC-related-cell-cycle pathway. This study also shows that MCM2 and CCNB1 could be promising prognostic and therapeutic targets for HCC.