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. 2014 Jun 19:14:55.
doi: 10.1186/1475-2867-14-55. eCollection 2014.

Effects of siRNA targeting BMPR-II on the biological activities of human liver cancer cells and its mechanism

Peng Zeng  1 , Sheng Cai  1 , Jia-Na Zhang  2 , Feng-Ming Yi  3 , Wei-Min Jiang  3 , Jian-Bing Wu  4
Affiliations

Effects of siRNA targeting BMPR-II on the biological activities of human liver cancer cells and its mechanism

Peng Zeng et al. Cancer Cell Int. .

Abstract

Background: Bone morphogenetic protein receptor II (BMPR-II) plays an important role in tumor's invasion and proliferation. In this study, we observed the effects of small interfering RNA (siRNA) targeting bone morphogenetic protein receptor II (BMPR-II) on the biological activities of human liver cells and explore its mechanism.

Methods: The molecular sequences of three siRNA targeting BMPR-IIwere designed and synthesized. In this study, there were 6 groups including group I (normal control), group II (blank control), group III (negative control) and group IV-VI (BMPR-II-siRNA-a, siRNA-b and siRNA-c-transfected cells, respectively). The levels of mRNA and protein of BMPR-II were determined to select the best sequence for BMPR-II silence. After liver cancer cells were transfected with the best sequence, proliferation and invasion of transfected cells were assessed, and apoptosis and cell cycle were detected. The expressions of mitogen-activated protein kinases (MAPKs) signal pathway-related VEGF-C protein were observed after BMPR-II silence and BMPR-II silence combined with inhibiting MAPKs signal pathway, respectively.

Results: RT-PCR and Western blot indicated that BMPR-II expression was the highest in HepG2 among the three liver cancer lines (P < 0.01) and the lowest in group IV among the six groups (P < 0.01). MTT assay and transwell assay revealed that the numbers of cell growth and cell transmembrane were significantly lower in group IV than in control groups 48 h after cells were transfected (P < 0.05). Flow cytometer showed that apoptosis was the highest and cells were significantly blocked in S phase 48 h after cells were transfected in group IV (P < 0.01). Western blot indicated that the protein levels of p-P38 (P < 0.01) and vascular endothelial growth factor-C (VEGF-C) (P < 0.01) were significantly decreased after BMPR-II silence. The protein level of VEGF-C was significantly decreased in PD98059 + siRNA-BMPR-II-a and SB203580 + siRNA-BMPR-II-a groups (P < 0.01), especially in SB203580 + siRNA-BMPR-II-a group (P < 0.01).

Conclusions: siRNA targeting BMPR-IIcan markedly inhibit HepG2 proliferation and invasion, promote apoptosis and block HepG2 in S phase. Its mechanism may be that BMPR-II silence down-regulates VEGF-C expression through MAPK/P38 and MAPK/ERK1/2 pathways, especially MAPK/P38. This study provides a new targeted therapy for liver cancer.

Keywords: Bone morphogenetic protein receptor II; Liver cancer; Mitogen-activated protein kinases; Small interfering RNA; Vascular endothelial growth factor-C.

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Figures

Figure 1
Figure 1
Expressions of BMPR-II mRNA and protein in each cell line. A: Expressions of BMPR-II mRNA in each cell line. B: Expressions of BMPR-II protein in each cell line. A: Hep3B cells; B: SMMC7721 cells; C: HepG2 cells.
Figure 2
Figure 2
Expressions of BMPR-II mRNA and protein in each group. A: Expressions of BMPR-II mRNA in each group. B: Expressions of BMPR-II protein in each group. A: Normal group; B: Blank control group; C: Negative control group; D: BMPR-II-siRNA-c group; E: BMPR-II-siRNA-b group; F: BMPR-II-siRNA-a group.
Figure 3
Figure 3
Effects of BMPR-II silence on the growth and morphology of HepG2 cells (×200). A: BMPR-II-siRNA-a group; B: Negative control group; C: Normal control group.
Figure 4
Figure 4
Effects of BMPR-II silence on the invasion of HepG2 cells (×200). A: BMPR-II-siRNA-a group; B: Negative control group; C: Normal control group.
Figure 5
Figure 5
Effects of BMPR-II silence on liver cancer HepG2 apoptosis. A: Normal control group; B: Negative control group; C: BMPR-II-siRNA-a group.
Figure 6
Figure 6
Effects of BMPR-II silence on cell cycle of liver cancer HepG2. A: Normal control group; B: Negative control group; C: BMPR-II-siRNA-a group.
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