| Objective: SPC24 is an important component of the nuclear division cycle 80(Ndc80) kinetochore complexes, which participated in and regulated a great of major molecular events during mitosis, such as the precision coupling of kinetochore to spindle microtubules(MTs), the correct chromosome alignment, and the proper chromosome segregation. However, the relationship between SPC24 and hepatocellular carcinoma(HCC) development, progress, invasion and metastasis remain unknown. Here, we detected the expression of SPC24 in HCC and analyzed its association with clinicopathologic features for providing a new diagnostic index and a potential therapeutic target of HCC patients.Methods:1. A total of 212 patients who were diagnosed with HCC and underwent routine curative surgery between November 2001 and April 2007 at the Affiliated Hospital of Guilin Medical University(Guilin, Guangxi, China) were recruited and retrospectively analyzed. We collected all 212 pairs of HCC samples and matched adjacent noncancerous liver tissues(ANLT) samples, 9 specimens of normal liver tissues surrounding the hepatic hemangioma tissues were collected as normal liver tissues.2. The relative m RNA level of SPC24 was evaluated in 20 HCC specimens and corresponding ANLT, as well as 9 normal liver tissues by semi-quantitative RT-PCR. The m RNA levels of SPC24 in 212 paired HCC specimens and ANLT were further determined by quantitative real-time PCR.3. Tissue microarrays containing 69 pairs of HCC and 9 normal liver tissues were examined by means of IHC staining with a specific antibody against SPC24, and SPC24 expression was also analyzed in 40 HCC specimens and corresponding ANLT, as well as 9 normal liver tissues by using the western blotting.4. We measured the m RNA expression levels of SPC24 in 12 HCC cell lines(Hep3B, SK-hep1, Huh7, SMMC7721, MHCC97 L, MHCC97 H, PLC, Hep G2, QGY7703, BEL7402, BEL7404, BEL7405)and 1 available normal liver cell(LO2)in order to screen the target HCC cell lines that used for investigating the cell biology.5. There small interfering RNAs(si RNAs) against SPC24 and the control scrambled si RNA were designed on the Whitehead Institute Web Server(http://jura.wi.mit.edu/bioc/si RNAext/) and chemically synthesized(Shanghai Gene Pharma Co). SMMC7721 and Hep G2 cells were seeded in 6-well plates and transiently transfected with specific si RNAs against SPC24 until they became 60-80% confluent, analysis of genes and proteins expression was performed 48 hours and 72 hours post transfection, the transfection efficiency of SPC24 si RNA was evaluated by RT-PCR and western blotting.6. We choose SPC24 si RNA-2 as the optimal silenced efficiency of endogenous SPC24 on HCC cell lines, after si RNA-2 was transiently transfected into SMMC7721 and Hep G2 cells, cell proliferation and adhesion ability was determined by Cell Counting Kit-8(CCK-8) kits.7. After si RNA-2 was transiently transfected into SMMC7721 and Hep G2 cells, tumor cell invasion was measured in vitro using a transwell insert.8. western blotting was used to detect the activated cleaved caspase-3 in SMMC7721 and Hep G2 cells transiently transfected with si RNA2 for analyzing the tumor cells apoptosis.Results:1. As illustrated in the results of semi-quantitative RT-PCR, SPC24 m RNA levels were aberrantly elevated in 18 of the 20 analyzed HCC tissues(90%) in comparison to non-tumor controls, while the SPC24 m RNA was faintly expressed in 9 cases of normal liver tissues from hepatic hemangioma’ surrounding liver tissues. The expression level of SPC24 was further detected in 212 paired HCC tissue specimens by quantitative real-time RT-PCR showed that SPC24 m RNA were increased in 73.1% HCC cases(155 in 212 cases), and decreased in 26.9% HCC cases(57 in 212 cases). The relative expression of SPC24 m RNA in HCC specimens was significantly higher than that in adjacent noncancerous livers(mean ± SD, 1.123 ± 0.072 VS. 0.350 ± 0.036, p < 0.0001).2. The IHC data showed that none of the 9 normal liver tissues, 31.90%(22 out of 69) of ANLT showed positive for SPC24 staining, however, 73.90%(51 out of 69) of HCC tissues was stained positively by using SPC24 antibody. Consistent with above result, SPC24 protein expression was highly expressed in 87.50%(35 out of 40) of HCC tissues, but faintly expressed in ANLT and normal liver tissues.3. RT-PCR showed that the expression level of SPC24 was abundant in SMMC7721, PLC, Hep3 B, BEL7404, BEL7405, MHCC97 H, Hep G2, Huh7, and QGY7703 HCC cells, but weak in LO2, MHCC97 L, SK-hep1, BEL7402 cell lines.4. Three specific si RNAs against SPC24 were employed to inhibit endogenous SPC24, RT-PCR and Western blotting show that the expression of SPC24 in SMMC7721 and Hep G2 cells transfected with SPC24 si RNAs was significantly decreased. The si RNA-2 knocked down SPC24 most effectively among the three SPC24 si RNAs.5. Compared with the cells transfected with scrambled si RNA control, we observed cell proliferation, adhesion and invasion ability was significantly inhibited in SMMC7721 and Hep G2 cells transfected by si RNA2. And, western blotting showed an increase of cleaved caspase 3 protein expressions in SMMC7721 and Hep G2 human HCC cells transfected with si RNA2 than that of vector control or scrambled si RNA control, suggesting down-regulation of SPC24 may induce HCC tumor cells apoptosis.6. The correlation analysis demonstrated that the High expression of SPC24 was significantly correlated with alpha-fetoprotein(AFP)(p = 0.044), median size(p = 0.030), tumor number(p = 0.019), and Barcelona-Clinic Liver Cancer(BCLC) stage(p = 0.015).7. Kaplan-Meier analysis showed that the disease-free survival(DFS) and overall survival(OS) of high SPC24 expression group was significantly shorter than that of low SPC24 expression group(p < 0.001; p = 0.001; respectively). Multivariate analysis identified SPC24 upregualtion as independent risk factor of DFS and OS for HCC patients(p = 0.001; p < 0.001; respectively).Conclusions: These results showed that SPC24 expression was significantly up-regulated in HCC, which may act as a novel prognostic biomarker for patients suffering from this deadly disease. Additionally, silence of SPC24 inhibiting HCC cell growth indicated that SPC24 may be a promising molecular target for HCC therapy. |
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