Author(s): Huang M, Wang Q, Zhang W, Li DR, Li L
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Abstract OBJECTIVE: To investigate the efficiency of biology function of ITIH4 gene silenced by small interfering RNA (siRNA) on ovarian cancer. METHODS: The four pairs ITIH4 gene siRNA interference fragments (ITIH4-546, ITIH4-795, ITIH4-917 and ITIH4-1568) were designed respectively, and transfected into HO8910pm cells with ITIH4 mRNA high expression by liposomal method transiently. Quantitative PCR method was used to detect the ITIH4 mRNA expression in HO8910pm cells transfected with interference fragment. The ITIH4 917 was selected as the best silencing effect of siRNA interference fragment and then the recombinant plasmid expression vector pGPU6/GFP/Neo-shRNA-ITIH4-917 was constructed and transfected into HO8910pm cells. The stably transfected cells- pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm cells was obtained by screening of aminoglycoside antibiotics (G418). The experiment was divided into three groups, namely ITIH4-917 transfection group, the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA plasmid (empty vector group), and the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA-ITIH4-NC the plasmid (negative control group). Fluorescence quantitative reverse transcription (RT) PCR and western blot were used to detect the ITIH4 mRNA and protein expression. The cell proliferation, the cell cycle, colony formation of cells, cells migration and invasion in vitro were determined by using methyl thiazolyl tetrazolium (MTT), flow cytometry, colony formation assay and transmembrane (transwell) small chamber method [value represented by absorbance (A)], respectively. RESULTS: The fluorescent quantitative PCR results showed that the ITIH4 mRNA expression levels in ITIH4-917 HO8910pm cells was significantly lower than that in the control cells, the relative copy number was only 0.26 ± 0.15. Also the relative copy number of ITIH4 mRNA in ITIH4-917 transfection group cells was 0.34 ± 0.10, it significantly lower than that in empty vector group (1.87 ± 0.12, P = 0.008) and negative control group (1.58 ± 0.21, P = 0.032); Western blot results showed that the ITIH4 relative expression levels of the protein in ITIH4-917 HO8910pm group cells, empty vector group and negative control group were 0.51, 1.64 and 1.74, respectively, there were statistically significant differences (0.51 vs. 1.64, P = 0.012; 0.51 vs. 1.74, P = 0.014). MTT colorimetric assay showed that the proliferation of ITIH4-917 HO8910pm group cells was significantly faster than that in the empty vector group and negative control group, and there were statistically significant differences among them (P = 0.001). The S + G2/M phase cell ratio in ITIH4-917 HO8910pm group cells was 54.2\%, which was significantly higher than that in the empty vector group or negative control group (26.3\% and 31.3\%, respectively, all P < 0.05). The colony formation rate (55.7 ± 0.7)\% in ITIH4-917 HO8910pm group cells was also significantly higher than that in empty vector group (29.7 ± 0.9)\% (P = 0.037) and negative control group (31.4 ± 0.3)\% (P = 0.043). Migration and invasion experiments showed that cell migration in ITIH4-917 HO8910pm group cells was 0.40 ± 0.18, whicht was significantly higher than that in the negative control group or empty vector (0.30 ± 0.03, P = 0.031;0.25 ± 0.03, P = 0.028, respectively). Although the invasive ability of ITIH4-917 HO8910pm group cells (1.31 ± 0.34) was higher than that in the control cells (1.05 ± 0.68) and empty vector group (1.14 ± 0.08), while there were not significant difference (P > 0.05). CONCLUSION: It would be to promote the cell doubling time and increase the migration capability in HO8910pm cells that ITIH4 expression was down-regulating by ITIH4 mRNA interference.
This article was published in Zhonghua Fu Chan Ke Za Zhi
and referenced in Journal of Clinical & Cellular Immunology