The present results indicated that the high expression levels of CCNB1 were associated with the invasion degree of pituitary adenoma. and non-invasive pituitary adenomas. Knockdown Ispronicline (TC-1734, AZD-3480) of CCNB1 resulted in significant decreases in cell viability and proliferation, arrested cell cycle at the G2/M phase and increased apoptosis. In addition, knockdown of CCNB1 significantly decreased the expression levels of the mesothelial cell marker N-cadherin (P<0.001), but significantly increased the expression levels of the epithelial cell markers E-cadherin (P<0.01) and p120-catenin (P<0.001). Further analyses identified that RES inhibited the expression level of CCNB1, and RES treatment exhibited a similar effect as CCNB1 shRNA infection. The present study suggested that suppressing the expression level of CCNB1 could regulate the proliferation and apoptosis of pituitary tumor cells and alter the expression level of various EMT markers. In addition, RES treatment could be used as an inhibitor of CCNB1. The present study also identified the molecular mechanisms underlying CCNB1 role in EMT. CCNB1 inhibitor experiments, RES (Beijing Solarbio Science & Technology, Co., Ltd.) was dissolved in dimethyl sulfoxide (DMSO; Beijing Solarbio Science & Technology, Co., Ltd.) and added to the F-12 culture medium, based on the research by Joe (30). Briefly, cells were treated with 0.2% DMSO (negative control) and RES inhibitor (100 and 300 M) at 37C in a humidified Ispronicline (TC-1734, AZD-3480) incubator with 5% CO2. After Ispronicline (TC-1734, AZD-3480) 48 h of treatment, both adherent and floating cells were harvested for Ispronicline (TC-1734, AZD-3480) further examinations. Statistical analysis Statistical analyses were performed using GraphPad Prism (version 7.0; GraphPad Software, Inc.). All quantitative data are presented as the mean standard deviation. Differences between groups were determined using one-way ANOVA test with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated three times. Results CCNB1 expression is upregulated in pituitary adenomas and is higher in the invasive group The present results revealed that the expression of CCNB1 was upregulated in tumor samples compared with the normal control (Fig. 1A). In addition, the experimental results of another group of tumor specimens proved that the expression of CCNB1 was markedly higher in the invasive group compared with the non-invasive group (Fig. 1B). Open in a separate window Figure 1. Protein expression levels of CCNB1 in pituitary tissues. The samples were divided into two experimental groups. (A) In the first group, the expression level of CCNB1 was analyzed in 14 pituitary adenoma tissue specimens and a normal pituitary gland. Expression levels of CCNB1 in normal (n=1) and pituitary adenomas (n=14) were detected by western blotting. (B) In the second group, four non-invasive, four invasive pituitary adenoma tissue specimens and a normal pituitary were investigated. Expression levels of CCNB1 in normal (n=1) and non-invasive (n=4) and invasive pituitary adenomas (n=4) were detected by western blotting. Blots were applied to the same exposure condition. Band intensities were quantified and standardized to -actin for active CCNB1. *P<0.05. CCNB1, cyclin B1; Con, pituitary tissue sample; patients, pituitary adenoma tissue samples. sh-CCNB1 downregulates the expression of CCNB1 Furthermore, it was demonstrated that CCNB1 expression was affected by the lentiviral-mediated shRNA infection. The infection effect was observed using fluorescent imaging of GFP-positive GH3 and MMQ cells following lentivirus transfection (Fig. 2A-F). The cells were transfected with the shRNA and after 72 h, the transfection rates of shRNA-CCNB1 group and shRNA-NC group were both ~80%. The interference Thbd effect was validated via RT-qPCR and western blotting. These results revealed that CCNB1 was significantly decreased both at the mRNA and protein levels compared with the control and NC groups (Fig. 2G and H). Open in a separate window Open in a separate window Figure 2. Effect of shRNA on CCNB1 expression, proliferation,.
