| Purpose: To investigate the function of KIAA0008 gene, one of the leading genes in the signature associated with metastasis of hepatocellular carcinoma (HCC) from the cDNA microarray analysis, especially its possible roles in the invasion and metastasis of HCC as well as the possible mechanisms by which it works. Methods: Expression levels of KIAA0008 in 27 HCCs specimens were detected by semi-quantitative RT-PCR, then followed by more sensitively quantitative Real-time RT-PCR; of those 27 HCCs, 23 cases of matched non-tumor liver tissues and 5 cases of matched HCC tissues were also brought into detection. According to the clinicopathological features, 27 HCCs were divided into high-invasiveness, median-invasiveness or low-invasiveness groups; the difference of the KIAA0008 expression levels between these different groups of HCCs was also evaluated. Then, the expression levels of KIAA0008 in three serial HCC cell lines: MHCC97L, MHCC97H and HCCLM3, which have the identical genetic background but stepwise higher potentials of invasion and metastasis, were also detected by Real-time RT-PCR. To further explore the effect of KIAA0008 overexpression on HCC cell proliferation and invasiveness in vitro, recombinant expression plasmid vector of pIRES2-EGFP-KIAA0008 was constructed and transfected into MHCC97L, then the cell proliferation, colony formation and in vitro Matrigel invasion were assayed. In order to study the possible mechanisms by which KIAA0008 influences the invasiveness potential, a second recombinant plasmid vector of pEGFP-C3-KIAA0008 was constructed, by which a fusion protein of KIAA0008 gene product and EGFP would be produced to show the subcellular localization of KIAA0008 gene product in HCCLM3 cell line; then the synchronizations of HCCLM3 cells were achieved to study the expression of KIAA0008 in different phases in cell cycle. Results: Expression levels of KIAA0008 in 23 HCC specimens were significantly higher than that of the paired non-tumorous liver tissues (1.05E-3 vs. 0.397E-3, medians, P<0.001); the expression levels of KIAA0008 in HCCs with high invasiveness were significantly higher than those of low invasiveness (1.29E-3 vs. 0.162E-3, medians, P=0.002). For the results in HCC cell lines, from MHCC97L to HCCLM3, with the increase of invasive and metastatic potentials, the expression levels of KIAA0008 increased correspondingly, its level in MHCC97H was higher than that in MHCC97L, but no statistically significant difference was proved (P=0.324), and its expression level in HCCLM3 was significantly higher than those in MHCC97L and MHCC97H (P<0.001). After the transfection of recombinant plasmid vector of pIRES2-EGFP-KIAA0008, MHCC97L cells showed a significant overexpression of KIAA0008 gene, and overexpression of KIAA0008 in MHCC97L cell line resulted in the enhanced ability of cell proliferation (1.79±0.06 vs. 0.96±0.10; mean±SD; P<0.001), colony formation (14.4±3.97 vs. 8.6±2.40; mean±SD; P=0.024) and invasion (18.8±5.31 vs. 11.8±3.34, mean±SD, P=0.037) compared with the control groups. In HCCLM3 cells transfected by pEGFP-C3-KIAA0008, confocal microscopic analyses showed that the fluorescent signals, which standed for the fusion protein of KIAA0008 gene product and EGFP, was concentrated on the nucleus and cell membrane, especially in the nucleus; while in the control group which transfected with pEGFP-C3, fluorescent signals was found to distribute evenly in the whole cell. Expression level of KIAA0008 remained relatively low from G0 phase till S phase of the cell cycle, and then a notably high level expression was observed at G2/M-phase. Furthermore, with the Pearson Correlation analysis, a significant correlation was found between the expression level of KIAA0008 and percentage of G2/M-phase (r=0.993, P<0.01). Conclusions: KIAA0008 expression is associated with invasiveness of HCC, overexpression of KIAA0008 leads to a more invasive phenotype of HCC cells, which might be through its possible roles in the cell cycle regulation. Further investigation in this aspect is pro...
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