The apoptotic ratio was increased in NSBP1 knockdown 786-O cells compared to control (Figure 2B). To confirm that NSBP1 knockdown could inhibit proliferation and induce apoptosis in ccRCC cells, we examined the expression of apoptosis and cell cycle related proteins and found that Bax
protein level was significantly increased while CyclinB1 and Bcl-2 protein 17DMAG levels were decreased selleck inhibitor in NSBP1 knockdown cells compared with control (Figure 2C). These data provide evidence that NSBP1 modulates cell cycle and antagonizes apoptosis to promote the oncogenic potential of ccRCC cells. NSBP1 knockdown inhibits the invasion of ccRCC cells Next we assessed the role of NSBP1 in cell invasion, an important aspect of ccRCC metastasis. By transwell assay we found that NSBP1 knockdown cells showed few number of invading cells compared to control group which expressed high level of NSBP1 (Figure 3A). The number of cells crossing the matrigel was 62.3 ± 3.1 in NSBP1 siRNA group versus 110.7 ± 3.1 in scramble siRNA control group (P < 0.05). Moreover, gelatin zymography assay demonstrated that NSBP1 knockdown efficiently decreased MMP-2 and MMP-9 enzymatic activity, especially MMP-9 enzymatic activity (Figure 3B). To address whether decreased
MMP-9 and MMP-2 activity is due to the downreguation of their expression after NSBP1 knockdown, we examined the expression of MMP-9, MMP-2 and their upstream transcription factors c-fos and c-jun. Ruboxistaurin chemical structure Western blot analysis demonstrated that NSBP1 knockdown downregulated the expression of VEGF, VEGFR-2, MMP-2, MMP-9, c-fos and c-jun (Figure 3C). Taken together, these data suggest that NSBP1 upregulates the
expression of MMP-2 and MMP-9 via c-fos and c-jun. The increased MMPs activity and angiogenesis then contributes to the migration and invasion of ccRCC cells. Figure 3 NSBP1 knockdown inhibits the invasion of ccRCC cells. (A), Representative photos showing the invasion of ccRCC cells into the lower chamber of transwell. ×200. (B), Gelatin zymography assay showing that MMP-9 and MMP-2 activities were decreased in NSBP1 knockdown 786-O cells. Data shown Alanine-glyoxylate transaminase were mean ± SEM from three independent experiments. (C), Western blot analysis showing that the expression of VEGF, VEGFR-2, MMP-2, MMP-9, c-fos and c-jun were significantly decreased in NSBP1 knockdown 786-O cells. Data shown were mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, versus the scramble siRNA transfected control group. NSBP1 knockdown inhibits ccRCC growth in xenograft nude mice To further investigate the role of NSBP1 in ccRCC in vivo, we established xenograft ccRCC by subcutaneous injection of 1 × 106 NSBP1 knockdown 786-O cells or the corresponding scramble siRNA transfected control cells into the flanks of BALB/c nude mice (n = 10).