2016;7:47431\47443. annealed by GenePharma (Shanghai, China). Two focus on sequences of CD44s siRNA, 518 and 688, were selected. These sequences were: 5\CCGCTTTGCAGGTGTATTC\3; and 5\AAATGGTCGCTACAGCATC\3, respectively. An siRNA with a non\targeting sequence (scrambled sequence) was used as a negative?control (shNC) in our ICG-001 experiment. RNA interference was carried out as described previously.11 2.9. In?vitro migration and invasion assays For in?vitro migration and invasion assays, cells were seeded onto the upper chamber of a transwell or ICG-001 on a Matrigel\coated transwell (BD Biosciences, Franklin Lakes, NJ, USA) in serum\free media. The lower chamber contained DMEM with 10% FBS as a chemoattractant. After 12 or 48?hours of incubation, non\migrated cells were gently removed from the upper chamber with a cotton swab. Cells were fixed and stained using Giemsa solution and counted in 5 randomly chosen visual fields. 2.10. Statistical analysis Statistical analyses were carried out using SPSS 16.0 software. All data are presented as the mean??SD. Two\group comparisons were analyzed using the two\tailed Student’s test. Three or more group comparisons were analyzed using one\way ANOVA. P?.05 was considered statistically significant. 3.?RESULTS 3.1. TM suppresses the proliferation of HCC cells by inducing G2/M arrest To ICG-001 determine the effects of TM on the proliferation of cells, HCC cells were treated with different concentrations of TM for 48?hours. Cell proliferation was determined by the MTT assay. As shown in Figure?1A, TM inhibited cell proliferation of HCC cell in a dose\dependent way. To exclude the possibility that the effect of TM on cell proliferation was occurring as a result of drug toxicity, the effect of TM on the immortalized human liver cell line L02 was also investigated. Results showed that L02 cells had a lower sensitivity compared to HCC cells treated with TM (Figure?1A). Open in a separate window Figure 1 Effect of tunicamycin (TM) on hepatocellular carcinoma (HCC) cell growth and cell cycle\related protein expression. A, Growth inhibition rates of Huh7, MHCC\97L, MHCC\97H, MHCC\LM3, HCC\LY5, HepG2 and L02 cells resulting from treatment with TM for 48?h. B, Cell cycle distribution of MHCC\97L cells that were treated with 2.5?g/mL TM for 48?h. C, After the cells were synchronized with thymidine, cell cycle distribution of MHCC\97L cells was determined by flow cytometry. D, After the cells were synchronized with nocadazole, cell cycle distribution of MHCC\97L cells was determined by flow cytometry. E, Expression of proliferating cell nuclear antigen (PCNA), CDC\2 and cyclin B1 was detected by western blotting in MHCC\97L cells treated with 2.5?g/mL TM for the indicated length of time To further investigate the mechanism by which TM affects HCC proliferation, cell cycle distributions of MHCC\97L cells were determined by flow cytometry. Our results showed that TM induced G2/M arrest in MHCC\97L cells (Figure?1B). To further confirm the effects of TM on the cell cycle, MHCC\97L cells were synchronized with thymidine or nocadazole. Results showed that TM induced G2/M arrest in MHCC\97L cells (Figure?1C,D). In addition, we also determined the expression of proteins related to G2/M cell cycle arrest. Our results showed that TM inhibited CDC\2, cyclin B1 and proliferating cell nuclear antigen (PCNA) expression in a dose\dependent way (Figure?1E). Therefore, these results showed that TM suppresses the proliferation of HCC cell by inducing G2/M arrest. 3.2. Tunicamycin induces HCC cell apoptosis by Bcl\2 family proteins Apoptosis is also widely believed to be the major antiproliferative mechanism of anticancer AKAP13 drugs in many tumor cell types. Therefore, we also investigated the effect of TM on HCC cell apoptosis. Increased apoptosis was observed in MHCC\97L cells treated with TM, implying that an increased rate of apoptosis could be one of the mechanisms of TM inhibition of cell proliferation (Figure?2A). To understand the mechanism by which TM induces cell apoptosis, we assessed the expression of Bcl\2 family proteins using western blotting. Results showed that the proapoptotic Bcl\2 family proteins?Bim and Bid were up\regulated, and that the concomitant anti\apoptosis proteins Bcl\xL and Mcl\1 were down\regulated in TM\treated ICG-001 MHCC\97L cells (Figure?2B). However, the expression of Bcl\2 and PDCD4 did not change after TM treatment. Open in a separate window Figure 2 Tunicamycin (TM) ICG-001 induces cell apoptosis and regulates the expression of Bcl\2 family proteins in hepatocellular carcinoma (HCC).