The Clinical Significane And Biological Mechanism Of B7-H3 Expression On Human Prostate Cancer PC-3 Cells

Prostate cancer (PCa) is one of the most common cancers in man world. Radical prostatectomy, radiotherapy and hormonal therapy are considered as major therapeutic approaches for prostate cancer. Patients with clinical localized prostate cancer is highly curable by surgery and radiation therapy, while the others with in moderate and advanced stage usually choose hormonal therapy that is effective in the initial period. But almost all prostate cancer would progress into androgen-independent or hormone-refractory and with a bad prognosis. Therefore, revealing the internal molecular mechanism in the progression of prostate cancer has been a research hotspot. It was reported that B7-H3, a new member of the B7 co-stimulatory molecule family, was aberrantly expressed in prostate cancer, and correlated negatively with the clinicopathological features of the disease. Although, the biological functions of B7-H3 in the progression of prostate cancer is still unclear. In the present study, we first detected and compared the B7-H3 expression in prostate cancer and benign prostatic hyperplasia (BPH) tissues. After chemical synthesis and efficiency screening of 7 siRNA, we further investigated the effects of B7-H3 expression on biological behavior of PC-3 cells and its putative mechanism.This study consists of three parts.PartⅠThe expression of B7-H3 in prostate cancer and benign prostatic hyperplasiaObjective:To detect and compare the expression of B7-H3 in the tissue samples of prostate cancer and benign prostatic hyperplasia.Methods:Tissue samples from 13 patients with prostate cancer and 19 benign prostatic hyperplasia were collected and separated into two groups. After samples were weighed, immunohistochemistry (IHC) and enzyme-linked immunosorbent assays (ELISA) were performed to detect the expression of B7-H3 in each sample and its clinical significance was also analyzed.Results:Immunohistochemical staining demonstrated that B7-H3 was significantly over-expressed in the tumor tissue (p=0.0002). B7-H3 expression was detected in more than 50% cells in 7 of the PCa specimens, while none in the BPH specimens. In 4 of the PCa specimens and 5 of the BPH specimens, their 25%~50% of cells were stained positively. In 2 of the PCa specimens and 14 of the BPH specimens, the positive staining was less than 25%. And the ELISA assays showed that B7-H3 levels in PCa group were significantly higher than that of BPH group (174.73±56.80 vs 82.69±46.19 ng/g, p<0.001).Conclusions:B7-H3 expression in PCa group was significantly higher than that of BPH group. And we, for the first time, precisely quantifed the B7-H3 content in prostate tissue with a reference value.PartⅡScreening of the effective siRNA targeting B7-H3 geneObjective:To design and synthesize siRNA targeting B7-H3 gene and select out the most effective siRNA for subsequent research.Methods:Seven chemical synthesis Oligos of siRNA targeting human B7-H3 mRNA and two control siRNAs were designed. The transfection efficiency was evaluated by fluorescence microscope and flow cytometry after transfection for 6 hours. Disturb efficiency of each group was determined by RT-PCR after transfection for 24 hours, and the most efficient siRNA was selected. Real-time PCR and flow cytometry were used to detect the mRNA expression and protein secretion of the selected siRNA group after transfection for 24, 48, 72hours, respectively.Results:The transfection efficiency was over than 80% after transfection for 6 hours. And the 4th siRNA (B7-H3-4 siRNA) had the highest disturb efficiency with an inhibition of mRNA and protein expression up to 60% after transfection for 72 hours.Conclusions:One of the B7-H3 siRNAs which had the best disturb efficiency, was selected and laid a foundation for next studing of biological functions. The B7-H3-4 siRNA had the best disturb efficiency, and was selected for next partition of our study.PartⅢThe effect of B7-H3 expression on biological behavior of human prostate cancer PC-3 cellsObjective:To investigate the effect of B7-H3 low-expression on the biological features of human prostate cancer PC-3 cells.Methods:PC-3 cells were divided into three groups: blank control group B (untransfected group); negative control group NC (NC siRNA group); experimental group (B7-H3-4 siRNA group). MTT assays were used to measure the proliferation of the PC-3 cells; flow cytometry was used to analyze the cell cycle distribution; Annexin V/PI test was used to measure cell apoptosis; cell adhesion assays were used to measure the adherence ability; the wound scrape assays were used to measure the cell motility; transwell invasion assays were used to analyze the invasiveness in vitro. And all data were analyzed statistically.Results:(1) MTT assays showed that the OD values of blank control group and negative control group were slightly higher than that of experimental group on 24h, 48h, 72h, but the differences were not significant (p>0.05). (2) The differences in cell cycle distribution of the three groups were also not significant (p>0.05). (3) The apoptosis ratios of blank control group, negative control group and experimental group were 2.8±0.5%、2.6±0.4%、2.6±0.3%, respectively. And the difference was not statistical (p>0.05). (4) The adherence ability of experimental group was significantly lower than that of blank control and negative control groups (p<0.01). (5) The relative width of wound of experimental group on 12h, 18h, 24h were significantly larger than that of blank control and negative control groups (p<0.05). (6) The number of invaded PC-3 cells of experimental group was smaller than that of blank control and negative control groups (p<0.01).Conclusions:(1) The influence of B7-H3 low-expression on cell proliferation, cell cycle distribution and apoptosis of PC-3 cells were not significant. (2) But the downregulated B7-H3 expression reduced cell adhesion and migration as well as matrigel-invasion ability.