[Effect of siRNA targeting centromere protein-A gene on biological behavior of HepG2 cells].
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.
Li
Yong-mei
YM
Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Zhu
Zhi
Z
Chen
Ying
Y
Luo
Zhi-gang
ZG
Shi
Min
M
Zhu
Ming-hua
MH
D001324:Autoantigens / Q000187:drug effects / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D045744:Cell Line, Tumor; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000187:drug effects / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D056945:Hep G2 Cells; D006801:Humans; D034622:RNA Interference / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000187:drug effects / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D034741:RNA, Small Interfering