Figure 1.
Expression of VASH2 in tissues.
IHC analysis of VASH2 in human normal liver tissue (A) and human liver cancer tissue (B–D) at 20× magnification. In (A), we did not observed VASH2 protein in the cytoplasm. (B) The level of VASH2 protein localized in the cytoplasm was weakly stained. (C) The cytoplasm was moderately stained. (D) Strong staining was observed.
Table 1.
Patient Demographic Features.
Table 2.
Relationship between VASH2 Expression, Pathological grade and Clinical TNM classification (n = 97).
Figure 2.
Generation and identification of stably transfected HepG2 cells.
(A) Measurement of VASH2 expression using qRT-PCR. HepG2-VASH2 means VASH2-overexpressing HepG2 cells; HepG2-EGFP means HepG2 cells transfected with vector-expressing EGFP. HepG2-wt means wild-type HepG2 cells. HepG2-shcont means HepG2 cells transfected with a vector-expressing control shRNA of shVASH2. HepG2-shVASH2 means VASH2-knockdown HepG2 cells. VASH2-overexpressing HepG2 cells showed almost 1200-fold higher VASH2 expression than wild-type HepG2 cells, whereas VASH2-knockdown HepG2 cells had 80% lower expression compared with wild-type HepG2 cells (*P<0.05, compared with the control group). (B) Western blot analyses were used to confirm the knockdown efficiency. The results were similar to those of qRT-PCR analyses.
Figure 3.
Effects of VASH2 on the sensitivity of HepG2 cells to CDDP.
(A) Cell proliferation−toxicity test was conducted using Cell Counting Kit-8 (CCK-8) assay for 48 h. The overexpression of VASH2 decreased the sensitivity of CDDP (*P<0.05). By contrast, the knockdown of VASH2 increased the sensitivity of CDDP (#P<0.05). (B) After treatment with 0, 10 or 20 µg/ml CDDP for 48 h, the apoptosis rate was analyzed with flow cytometry. UR + LR indicated apoptosis. (C, D, and E) Quantification of the data from Figure 3B (*P<0.05).
Figure 4.
VASH2 downregulated the p53-Bax-caspase-3 pathway in HepG2 cells in vitro.
Western blot analyses were performed to detect the protein level of p53, Bax, and CC-3. GAPDH served as a loading control.
Figure 5.
VASH2 influenced the sensitivity of tumor cells to CDDP in vivo.
HepG2-VASH2, HepG2-EGFP, HepG2-shVASH2, or HepG2-shcont cells were suspended at a density of 107 cells/ml, and 100 µl was injected into the flank of nude mice (n = 9). On day 9, mice in CDDP(+) group were given CDDP (10 mg/kg) intraperitoneally every 3 days. After six 3-day cycles of treatment, all nude mice were sacrificed, and tumors were excised from nude mice. (A) The HepG2-shVASH2 tumors were smaller than those of the HepG2-shcont groups, whereas the size of the HepG2-VASH2 tumors did not significantly differ from that of HepG2-EGFP tumors. (B) Tumor growth curves. Tumor volume was calculated 3 days after the first treatment with CDDP using the formula (W2×L)/2 every 3 days. The data are presented as the mean ± SD of nine tumors per group. *A significant difference was found between the HepG2-shVASH2 and HepG2-shcont groups (P<0.05). (C) The comparison of tumor volume between CDDP(+) group and CDDP(−) group(*P<0.05). (D and E) Total RNA and protein were extracted from CDDP(+) group randomly, and VASH2 expression was measured by qRT-PCR and Western blot analyses.
Figure 6.
Knockdown of VASH2 enhanced the apoptosis of tumor cells by CDDP treatment.
(A and B) TUNEL analysis showing enhanced apoptosis rate in tumor cells when the HepG2-shVASH2 group was compared with the HepG2-shcont group (P<0.05). However, no statistical difference was found in HepG2-VASH2 vs. HepG2-EGFP (P>0.05) at 40× magnification. (C) Western blot analysis showed that p53, Bax, and CC-3 were highly expressed in the HepG2-shVSAH2 group.