Reduced expression of cenp-e in human hepatocellular carcinoma

Abstract

Background: CENP-E, one of spindle checkpoint proteins, plays a crucial role in the function of spindle checkpoint. Once CENP-E expression was interrupted, the chromosomes can not separate procedurally, and may result in aneuploidy which is a hallmark of most solid cancers, such as hepatocellular carcinoma (HCC). We investigate the expression of CENP-E in human hepatocellular carcinoma,. and analyze the effect of low CENP-E expression on chromosome separation in normal liver cell line (LO2).

Methods: We determined its levels in HCC and para-cancerous tissues, human hepatocellular carcinoma-derived cell line (HepG2) and LO2 cell line using real time quantitative PCR (QPCR) and Western blot. Further to know whether reduction in CENP-E expression impairs chromosomes separation in LO2 cells. we knocked down CENP-E using shRNA expressing vector and then count the aneuploid in LO2 cells using chromosomal counts assay.

Results: We found that both CENP-E mRNA and protein levels were significantly reduced in HCC tissues and HepG2 cells compared with para-cancerous tissues and LO2 cells, respectively. A significantly-increased proportion of aneuploid in these down-knocked LO2 cells compared with those treated with control shRNA vector.

Conclusions: Together with other results, these results reveal that CENP-E expression was reduced in human HCC tissue, and low CENP-E expression result in aneuploidy in LO2 cells.

Figure 1

shows that CENP-E expression in HCC and para-cancerous tissues, LO2 and HepG2 cell lines. (a) Analysis of CENP-E protein levels by Western blot. lysis extracts derived from para-cancerous tissues (lane 5-6), HCC tissues (lane1-4), LO2 (lane 7) and HepG2 cell lines (lane 8), Cyclin B1 was simultaneously immunoprobed for loading control. (b) QPCR and western blot analysis for CENP-E of tissues and cell lines, Cyclin B1 serves as loading control. Data represent the mean ± S.E. of three independent experiments. ^#, P < 0.05 versus HCC tissues; *, P < 0.05 versus HepG2 cells

Figure 2

Analysis interferer efficiency of pGenesil-CENPE. (A)Analysis of CENP-E protein levels by Western blot. Seventy-five micrograms of mitotic extracts derived from LO2 cells treated by nocodazol before detection for 3 h (lane 1-5). (B)shRNA-induced reduction of CENP-E mRNA and protein levels. Reduction of CENP-E mRNA. LO2 cells were transfected with various CENP-E shRNA vectors as indicated, and the mRNA levels were measured 24 h posttransfection by QPCR. Control:negative control (pScramble); mock: transfected Lipofectamine 2000 only alone. (C)Immunofluorescence of CENP-E of LO2 cells 24 h posttransfection with control shRNA vector or CENP-E siRNA. Cells were double stained with DAPI (4,6-diamidino-2-phenylindole) and CENP-E antibodies. Identical exposure times were used for imaging both control and CENP-E shRNA-transfected cells (white arrow point to misaligned chromosome). Bar, 5 μm.

Figure 3

proliferation and apoptosis analysis by MTT assay and flow cytomerty. (A) shows that proliferation of LO2 cells expression shRNA. Proliferation of shRNA-transfected LO2 cells (clone 3), shRNA scramble control and un-transfected LO2 cells were analyzed by MTT assay as described earlier. The mean ± SE of three independent experiments are shown. LO2 cells transfeced with pGenesil-CENPE vector proliferation slowed. (B) the result of flow cytometry showed that the percent of apoptosis cells of LO2 cells transfected with pGenesil-CENPE vector is higher than cells transfected with scrambler control shRNA vector or mock.

Figure 4

Effect of pGenesil-CENP-E on chromosome sepration in LO₂ cells. (A) shows that karyotype of LO2 cells, tetraploid (middle) and subdiploid karyotype (right) present in pGenesil-CENPE mediated LO₂ cells. (fig 4A). (B) aneuploid cells in the LO2 cells treated with shRNA vector is largely high, compare with cells transfected with scrambler control shRNA vector or mock. Data represent the mean ± S.E. of three independent experiments. *, P < 0.05 versus mock; ^#, P > 0.05 versus mock; (fig 4B)