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Updated: Jul 3, 2026
Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System
Published on: June 23, 2023
[Effect of siRNA targeting centromere protein-A gene on biological behavior of HepG2 cells]
Yong-mei Li1, Zhi Zhu, Ying Chen
1Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Objective:
To study the influence of siRNA inhibition of CENP-A expression on the biological behavior of HepG2 cells.
Methods:
Three pairs of 21 bp reverse repeated motifs of CENP-A target sequence with 9 spacer were synthesized and inserted into vector pSilencer 2.1-U6 neo to generate siRNA eukaryotic expression plasmids. After stable transfection into HepG2 cells, cell growth, apoptosis, cell cycles and plate clone forming efficiency were investigated. Expressions of CENP-A mRNA was monitored by the reverse transcriptase polymerase chain reaction (RT-PCR). The protein expression of CENP-A, bcl-2, Bax, p53, p21waf1 and mdm2 were detected by Western-blotting.
Results:
Two eukaryotic expression plasmids with significant siRNA specific inhibition to the CENP-A gene were created. Compared with control cells, HepG2 cells transfected with the constructs showed G1 phase delay (P < 0.01) and cell number decrease in the S phase (P < 0.001), along with an increased apoptotic rate (P = 0.003), significant increase of Bax expression and decreased bcl-2 expression (P< or =0.001). The protein expressions of p21waf1 was higher and mdm2 was lower than those of the control groups. However, the wild type p53 protein expression was not effected by CENP-A siRNA.
Conclusions:
An altered expression of CENP-A may be related to the proliferation of hepatocellular carcinoma through cell cycle regulation involving an altered bcl-2/Bax expression, that may be p53 independent.
Insights
Silencing CENP-A expression in HepG2 cells induced cell cycle arrest and apoptosis. This suggests CENP-A influences hepatocellular carcinoma proliferation via cell cycle regulation, potentially independent of p53.
Area of Science:
- Cell Biology
- Molecular Oncology
- Epigenetics
Background:
- CENP-A (Centromere Protein A) is crucial for centromere formation and chromosome segregation.
- Dysregulation of CENP-A is implicated in various cancers, including hepatocellular carcinoma (HCC).
- Understanding CENP-A's role in HCC progression is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the functional impact of CENP-A gene silencing on HepG2 cell behavior.
- To elucidate the molecular mechanisms underlying CENP-A's influence on HCC cell proliferation and survival.
Main Methods:
- siRNA-mediated gene silencing of CENP-A in HepG2 cells.
- Assessment of cell proliferation, apoptosis, and cell cycle distribution.
- Analysis of key protein expression (CENP-A, bcl-2, Bax, p53, p21waf1, mdm2) using RT-PCR and Western blotting.
Main Results:
- Successful generation of siRNA constructs targeting CENP-A.
- CENP-A inhibition led to G1 phase cell cycle delay and S phase reduction.
- Increased apoptosis, elevated Bax, and reduced bcl-2 expression were observed.
- p21waf1 expression increased, mdm2 decreased, while p53 expression remained unaffected.
Conclusions:
- CENP-A silencing significantly alters HepG2 cell biological behavior, inducing cell cycle arrest and apoptosis.
- Altered bcl-2/Bax expression ratio contributes to apoptosis induction.
- CENP-A's role in HCC proliferation may involve p53-independent cell cycle regulation.
