| Purpose: To detect and analyze the expression of B7-H3 in human prostate cancer (PCa). To evaluate the disturb efficiency and duration of validity of the siRNA targeting homo B7-H3. To explore the effects of B7-H3 low-expression on biological features of human prostate cancer PC-3 cells.Methods: This study consists of three parts.1. Tissue samples from 19 patients with prostate cancer and 19 patients with benign prostatic hyperplasia (BPH) as control were collected and separated into two groups. After tissue extracts were prepared, enzyme-linked immunosorbent assays (ELISA) were performed to determine the levels of B7-H3 in each sample and its clinical significance was also analyzed.2. PC-3 cells were transfected with B7-H3 siRNA. Then, Real-time PCR and flow cytometry were utilized to analyze the B7-H3 mRNA and protein expression for 24, 48, 72 hours after transfection, respectively. PC-3 cells were divided into three groups: blank control group (untransfected group), negative control group (NC siRNA group) and experimental group (B7-H3 siRNA group).3. MTT assays were used to measure the proliferation of the PC-3 cells; 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.Results:1. (1) B7-H3 levels in extracts of PCa tissue were statistically higher than that of BPH tissue (4.44±1.70 vs 2.59±1.55 ng/ml, P=0.001). (2) No statistical difference could be found between B7-H3 levels in extracts of patients with PSA≥20ng/ml and that of patients with PSA <20ng/ml (P=0.110). (3) Patients in stageⅢ~Ⅳhad higher B7-H3 levels in tissue extracts than that in stageⅡ(P=0.020). (4) The extracts levels of B7-H3 of patients with Gleason scores 8~10 were higher than that of patients with Gleason scores≤7 (P=0.001).2. (1) The B7-H3 mRNA expression levels of experimental group were 39.53±7.11%, 29.23±6.72% and 33.37±5.47% compared with that of the negative control group on the same time point after transfection for 24, 48, 72 hours (P<0.01). There were no statistical differences between blank control and negative control groups (P>0.05). (2) The B7-H3 expression levels of experimental group were 40.12±5.11%, 31.62±3.15%, 27.06±2.10% compared with that of the negative control group on the same time point after transfection for 24, 48, 72 hours (P<0.01). There were also no statistical differences between blank control and negative control groups (P>0.05).3. (1) The results of MTT assays demonstrated that there were no statistical differences in OD values among the three groups for 0, 24, 48, 72 hours after transfection (F=1.528, P=0.291; F=0.017, P=0.983; F=0.012, P=0.988; F=0.352, P=0.717). (2) Adhesion assays showed that the reduction of adhesive ability of experimental group was so statistically significant (P<0.01), but the difference between blank control and negative control groups was not significant (P>0.05). (3) In the wound scrape assays, time-course analysis of wound closure demonstrated that re-establishment of a monolayer occurred within a significantly longer period in the experimental group comparing to that of the blank control and negative control groups (P<0.01). (4) The number of invaded PC-3 cells was significantly smaller than that of the blank control and negative control groups (P<0.01), while the number of this two control groups were similar (P>0.05).Conclusions: (1) The expression levels of B7-H3 were statistically higher in PCa than that in BPH. And the B7-H3 expression levels in PCa were positively correlated to the clinical stage and postoperative Gleason scores. This indicated that B7-H3 may play an important role in the occurance and progression of PCa, and its expression levels were negatively correlated with the clinicopathologic features of the disease. (2) The B7-H3 siRNA can inhibit the expression of B7-H3 mRNA and protein secretion efficiently after transfection for 72 hours and meet the need of next partition. (3) Low-expression of B7-H3 reduced cell adhesion, migration, and invasion ability of PC-3, while no apparent impact on cell proliferation was observed. This indicated that B7-H3 may regulate the biological functions of PC-3 in a novel way in addition to acting as an immunoregulatory protein. |
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