2 kb NDRG2 gene released from plasmid by Sal I—Hind III restriction enzyme digestion

were shown in Fig. 1A. The target segment in AdEasy-GFP-NDRG2 was detected by PCR. Results of electrophoresis on PCR amplification of the target segment in AdEasy-GFP-NDRG2 are shown in Fig. 1B. Five clones were picked. Titers of the adenoviral stocks were 3.1 × 108 cfu/ml. Figure 1 Validation of recombinant adenovirus. (A) The pET44a-NDRG2 Dabrafenib chemical structure plasmid with and without digestion by by Sal I—Hind III restriction enzyme were shown. (B) The PCR product of target segment in AdEasy-GFP-NDRG2. NDRG2 Inhibits CCRCC cell Proliferation To elucidate the functional role of NDRG2 in renal tumorigenesis, we examined the effect of exogenous expression of NDRG2 on the malignant phenotype of CCRCC cells, A-498. Western blotting revealed that A-498 expressed NDRG2 when infected by recombinant adenovirus pAd-GFP-NDRG2 (Fig. 2A). Figure 2 NDRG2 inhibits the proliferation of CCRCC cells. (A) Tthe protein expression was detected by Western blotting. (B) The proliferation of A-498 cells was detected by MTT.* P < 0.05. We then tested the effect of NDRG2 on the Proliferation of A-498 cells. Growth curves were compared in a medium containing 10% fetal calf serum, the curves for cells expressed NDRG2 was significantly lower than those for control cells(P < 0.05;

Fig. 2B). This suggested that NDRG2 had the potential to inhibit selleck chemical the proliferation of CCRCC cells. NDRG2 Induces the Cell Cycle Arrest and apoptosis of CCRCC Cells To further investigate the mechanism by which NDRG2 inhibits CCRCC cell

growth, we studied the effects of NDRG2 expression on the cell cycle by fluorescence activated cell sorter analysis (FASC). The results of the cell cycle showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05, Fig. 3A). In addition, FASC also revealed that there were much more apoptotic cells in NDRG2 -expressing cells than in the controls (P < 0.01, Fig. 3B). We then investigated the mechanism by which NDRG2 induced cell cycle arrest in CCRCC cells. Cell cycle effectors were examined by western Selleckchem MK-3475 blot analysis (Fig. 3C). Our results indicated that upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E proteins, whereas cyclinD2, cyclinD3 and cdk2 were not affected. Figure 3 NDRG2 Induces the Cell Cycle Arrest and apoptosis of CCRCC Cells. (A) and (B) The effects of NDRG2 expression on the cell cycle and apoptosis were detected by FASC. (C) The cell cycle protein were examined by western blot analysis. p53 up-regulates NDRG2 expression in CCRCC cells Bioinformatics analysis suggested that there was a p53 binding site in upstream of NDRG2 promoter. To investigate whether NDRG2 expression was regulated by p53, we first infected A-498 cells with recombinant adenovirus Ad-p53.