Effect of siRNA targeting centromere protein-A gene on biological behavior of HepG2 cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the influence of siRNA inhibition of CENP-A expression on the biological behavior of HepG2 cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Expression of centromere protein A in hepatocellular carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression of centromere protein A (CENP-A) and its significance in hepatocellular carcinoma (HCC) and adjacent non-neoplastic liver tissue.</p><p><b>METHODS</b>The expression levels of CENP-A mRNA in 20 samples of HCC and adjacent non-neoplastic liver tissue were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (qRT-PCR). Immunohistochemical study for CENP-A and p53 proteins was also performed on tissue microarrays containing 80 samples of HCC and adjacent liver tissue.</p><p><b>RESULTS</b>The expression level of CENP-A mRNA in HCC (0.64 +/- 0.18) was higher than that in adjacent non-neoplastic liver tissue (0.09 +/- 0.09) (t = 12.78, P < 0.01). Of the 80 samples of HCC, 57 cases (71.25%) and 60 cases (75%) expressed CENP-A and p53 proteins respectively. The positivity rates of CENP-A and p53 proteins in non-neoplastic liver tissue were 43.75% (35/80) and 16.25% (13/80) respectively. There was a statistically significant difference in CENP-A and p53 protein expression between HCC and non-neoplastic liver tissue (P < 0.01). The coincident rate between CENP-A and p53 expression was 88.75% (71/80). Expression of CENP-A protein showed a positive correlation with that of p53 protein (r = 0.57, P < 0.01).</p><p><b>CONCLUSION</b>The over-expression of CENP-A occurs at transcriptional level and may be related to malignant proliferation of HCC via possible interaction with p53 gene.</p>