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

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.


Introduction
Bone morphogenetic proteins (BMPs), a member of transforming growth factor beta (TGF-β) family [1], are involved in cell proliferation, migration, differentiation and apoptosis [2]. Neovascularization is important for tumor's invasion and metastasis. Vascular endothelial growth factor (VEGF) plays an important role in solid tumor's growth, progression, metastasis, proliferation and differentiation [3]. VEGF-C is present not only in endothelial cells, but also in tumor cells, and plays regulatory roles in tumor angiogenesis and lymphogenesis [4]. BMPs also play an important role in embryonic angiogenesis [5]. However, BMPs perform their biological functions through its receptor, bone morphogenetic protein receptor II (BMPR-II). BMPR-II plays an important role in tumor's invasion and proliferation [6,7]. Many physiologic functions of BMP-II are achieved through activating mitogen-activated protein kinase (MAPK) and PI3K pathways [8,9]. MAPK is an important signal transduction system in cells and a converging point of various signal pathways. ERK1/2 pathway is mainly involved in cell growth and differentiation, while JNK and p38 pathways participate in stress reactions such as inflammation and apoptosis [10]. Little research has been done about the effects of BMPR-II on invasion and proliferation of human liver cancer cells and its mechanism. Therefore, we observed the effects of small interfering RNA (siRNA) targeting BMPR-II on the invasion, proliferation, apoptosis and cell cycle of liver cancer cells and explored its mechanism. This study provides a theoretical and experimental basis for exploring the occurrence and progression of human liver cancer.

Results
Screening the cell line with higher expression of BMPR-II from liver cancer cell lines HepG2, SMMC7721 and Hep3B RT-PCR showed that the ratios of BMPR-II to β-actin in Hep3B, SMMC7721 and HepG2 were 0.58 ± 0.00, 0.76 ± 0.05 and 1.00 ± 0.04, respectively, and Western blot showed that the ratio of BMPR-II to β-actin were 0.48 ± 0.07, 0.65 ± 0.44 and 1.01 ± 0.06, respectively. Therefore, the expression of BMPR-II in HepG2 cells was the highest among the three liver cancer cell lines (P < 0.01) (Figure 1).

Transfection efficiency
Green fluorescence could be seen under a fluorescence microscope when cells had been successfully transfected with siRNA, because siRNAs carried fluorescence mark. The siRNA transfection efficiency was the highest at 80% when cells were transfected with 50 nmol/L of siRNA for 12 h.

Effects of BMPR-II silence on the growth and morphology of HepG2 cells
Cell growth and morphology were observed under an invert microscope 48 h after cells were transfected with siRNA targeting BMPR-II. Cells grew well with good refractivity in normal control and negative control groups. In siRNA-BMPR-II-a group, cells were shrunken with poor refractivity and cell debris, and adherent cells were significantly reduced ( Figure 3).

MTT assay
MTT assay indicated that there were no statistical differences in survival rate of HepG2 cells between the three groups when cells were transfected for 24 h (P > 0.05), but the survival rate of HepG2 cells were lower in BMPR-II-siRNA-a group (48.27% ± 0.76% and 46.03% ± 0.62%) than in negative control group (81.21% ± 0.80% and 79.18% ± 0.68%) and normal control group (82.64% ± 0.67% and 81.55% ± 0.71%) when cells were transfected for 48 h and 72 h, respectively (P < 0.05). There was also no statistical difference in survival rate between HepG2 cells treated for 48 h and 72 h in BMPR-II-siRNA-a group.

Transwell assay
The number of cells to penetrate matrigel from Transwell upper chamber to Transwell lower chamber reflects the ability of cell invasion. Five visual fields in each group were randomly selected to count the number of invading cells. The number of cells to penetrate the membrane was significantly lower in BMPR-II-siRNA-a group (25.20 ± 1.60) than in negative control group (59.50 ± 1.85) and normal control group (60.40 ± 1.39) (P < 0.05). The results indicated that the invasion ability of liver cancer HepG2 cells was significantly decreased after the cells were treated with BMPR-II-siRNA-a ( Figure 4).

Effects of BMPR-II silence on cell cycle of liver cancer HepG2
Flow cytometer indicated that cells were significantly blocked in S phase 48 h after cells were transfected in BMPR-II-siRNA-a group; but in normal control and negative control groups, cell cycle was not markedly changed (Table 1, Figure 6).

Effects of BMPR-II silence on the expressions of MAPK signal pathway-related proteins and VEGF-C protein
Western blot indicated that the protein expressions of BMPR-II, VEGF-C, p-P38 and p-ERK1/2 were significantly lower in BMPR-II-siRNA-a group than in normal control and negative control groups (P < 0.01, Table 2), but there were no significant differences in p-JNK protein expression between the three groups (P > 0.05, Table 2).

Discussion
BMPs, a group of functional proteins, are widely involved in proliferation, differentiation and apoptosis of many cells, and play an important role in tumor's proliferation, invasion and metastasis. BMPs perform their biological functions through their receptors, BMPR-II. BMPR-II gene mutation allows BMPR-II-mediated BMP signal transduction to inactivate, leading to carcinogenesis [11]. Park et al. [11] have reported that the activation of BMPR-II-mediated BMP signal pathway is one of mechanisms of stomach or colon cancer development. Ye et al. [12] have described that hepatocyte growth factor can up-regulate BMPR-IB; and in prostate cancer, BMPR-II promotes bone metastasis of prostate cancer.
Angiogenesis is strongly associated with tumor's growth, invasion and metastasis [13], and VEGF-C plays an important role in angiogenesis and lymphangiogenesis [14].
MAPKs, a kind of serine/threonine kinase, are an important signal transduction system in cells and a converging point of various signal pathways. ERK1/2 pathway is mainly involved in cell growth and differentiation. JNK and p38 pathways are called stress-activated protein kinase (SAPK) because they participate in stress reactions such as inflammation and apoptosis. JNK and p38 pathways are also involved in cell proliferation and differentiation, and respond to extracellular stimuli [15]. Surgical injury can cause inflammatory reaction to stimulate the production of P38MAPK, in the same, solid tumor also can release cytokine to stimulate the production of P38MAPK [16].
It is reported that Ras can up-regulate VEGF expression through activating Raf-mek-ERK1/2-MAPK pathway [17]. Compared with p-ERK, BMP receptor activation more readily mediates p-P38 activation, and BMP-2 is strongly associated with VEGF in angiogenesis [18]. However, the specific subtype and signal pathway of BMPR-II are not clear yet. Therefore, we observed the changes in liver cancer cell's invasion, proliferation, apoptosis and cell cycle;  and the changes in MAPK signal pathway-related proteins (p-P38, p-ERK1/2, p-JNK and VEGF-C) after BMPR-II silence to explore the biomechanism that BMPs affect liver cancer's invasion, proliferation and metastasis.
In previous study, we found that BMP-2 could promote liver cancer cell's proliferation and migration, and played an important role in liver cancer invasion through downregulation of MMP2 and MMP9 [19]. However, BMPR-II mechanisms about vascular invasion and lymphatic metastasis are not clear in liver cancer. Therefore, in this study, BMPR-II gene was interfered with siRNA. We first selected the liver cancer HepG2 cells with the highest expression of BMPR-II from three liver cancer cell lines, then observed the changes in liver cancer cell's invasion, proliferation, apoptosis and cell cycle after BMPR-II silence; and the changes in MAPK signal pathway-related proteins (p-P38, p-ERK1/2, p-JNK and VEGF-C) after BMPR-II silence, and BMPR-II silence combined with inhibiting MAPK signal pathway (with SB203580: P38 inhibitor, PD98059: ERK1/2 inhibitor and SP600125: JNK inhibitor, respectively). Our results indicated that BMPR-II expression was the highest in HepG2; after HepG2 was transfected with BMPR-II-siRNA-a, invasion and proliferation of HepG2 was significantly decreased, but HepG2 apoptosis was significantly increased, and HepG2 cells were significantly blocked in S phase. At the same time, we also found that after BMPR-II silence, MAPKs signal pathway-related proteins p-P38 and p-ERK were significantly down-regulated and VEGF-C protein was also down-regulated, but p-JNK was unchanged. Subsequently, after MAPKs-related pathways were inhibited, we found that P38 and ERK signal pathways were inhibited with down-regulation of VEGF-C expression, but JNK signal pathway was inhibited with unchanged VEGF-C expression. In order to explore the relationship between BMPR-II-ERK/P38 and VEGF-C, VEGF-C expression after BMPR-II silence combined with inhibiting MAPK sub-signal pathways was observed, results indicated that VEGF-C expression was significantly down-regulated in P38 group (si-BMPR-II + SB203580) and ERK group (si-BMPR-II + PD98059), especially in p-P38 group, but was unchanged in JNK group (si-BMPR-II + SP600125). Based on the results above, we conclude that for human liver cancer HepG2, specific siRNA targeting BMPR-IIcan markedly inhibit cell proliferation and invasion, promote cell apoptosis and block cells 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.   The clinical treatment for liver cancer is difficult due to its malignant biological characteristics such as invasion and metastasis. In this study, we explored the mechanisms of liver cancer's proliferation, invasion and metastasis, providing a new targeted therapy for liver cancer.
BMPs promote VEGF expression, and VEGF finally affects vascular endothelial cells. Whether BMPs directly promote vascular endothelial cell proliferation will be investigated in our further studies.

Reagents
Human liver cancer cell lines (HepG2, SMMC7721 and Hep3B) were provided by Jiangxi Province Key laboratory of molecular medicine (Nanchang, China). Fetal calf serum DMEM, RPMI1640 and MEM medium were purchased from Hyclone (Logan, USA). Trypsin was from Solarbio (Beijing, China). Trizol reagent was provided by Tiangen (Beijing, China). RT kit was purchased from TAKALA (Kyoto, Japan). The primers of BMPR-II and control βactin were synthesized by Invitrogen (Carlsbad, USA). Annexin V-EGFP Apoptosis Detection Kit was purchased from Multiscience (Hangzhou, China). Cell cycle kit was purchased from KEY GEN (Nanjing, China). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, USA). Mouse anti-human monoclonal antibodies of p-JNK and β-actin; and rabbit anti-human polyclonal antibodies of BMPR-II, p-ERK1/2, p-p38 and VEGF-C were purchased from CST (Boston, USA). Secondary antibodies of goat anti-mouse and goat anti-rabbit were purchased from Beijing Zhongshan Golden Bridge Bio (Beijing, China).

Cell recovery and culture
Liver cancer HepG2, SMMC7721 and Hep3B cells were taken from liquid nitrogen, and then thawed in 37°C water bath followed by quickly placing in DMEM medium containing 10% fetal calf serum at 37°C in an atmosphere of 5% CO2 until they grew covering 90% of the culture bottle. These cells were seeded in new culture bottles after they were digested with 0.25% trypsin.
RT-PCR to select the cell line with highest level of BMPR-II mRNA from HepG-2, SMMC7721 and Hep3B cell lines Total RNA was extracted from cells using one-step method. The primers of BMPR-II and control β-actin were synthesized by Invitrogen (Carlsbad, USA). The upstream of BMPR-II primer was 5′-CAAGAACGGCT ATGTGCG-3′ and downstream 5′-CTCGGTTAAATG AATGAGG-TG-3′ with a length of 355 bp of PCR product. The upstream of control β-actin primer was 5′-TAAG AAGCTGCTGTGCTACG-3′ and downstream, 5′-GAC TCGTCATACTCCTGCTT-3′with a length of 459 bp of PCR product. PCR conditions were as follows: predenaturing at 94°C for 5 min, denaturing at 94°C for 40 s, reannealing at 63°C for 30 s, elongation at 72°C for 40 s, 30 cycles; finally elongation at 72°C for 8 min. PCR products underwent 1% agarose gel electrophoresis.

Western blot to select the cell line with highest level of BMPR-II protein from HepG-2, SMMC7721 and Hep3B cell lines
Pre-cooled 4°C lysis buffer (volume of five times) was added in differently cultured cells to extract protein. The protein was stored at −20°C for future use. Protein concentration was determined using BCA method. Protein and buffer were mixed at a ratio of 5:1, and then placed in boiling water for 5-10 min.
A total of 80 μg of sample per well underwent 12% SDS-PAGE, and then was transferred onto nitrocellulose membrane followed by sealing using 10 ml of TBST containing 0.5% dried skim milk at 4°C overnight. Following washing two times using TBST with each time for several seconds, rabbit anti-human antibody of BMPR-II (1:1000) was added at 4°C overnight. Next day, following washing three times using TBST with each time for 10 min, HRP-labeled goat anti-rabbit and goat anti-mouse IgG (1:5000) were added for 2 h. The mixture was washed three times using TBST with each time for 10 min followed by visualization using substrate electrochemiluminescence.

Design and synthesis of sequences of siRNAs targeting BMPR-II
The three pairs of sequences of siRNAs targeting BMPR-II were designed according to hBMPR-II mRNA (NM_001200) published by Genbank. These siRNAs were called BMPR-II-siRNA-a, BMPR-II-siRNA-b and BMPR-II-siRNA-c, and a negative control sequences was also designed. The transcription templates of siRNAs were synthesized by Invitrogen (Carlsbad, USA) and their sequences are shown in Table 4.

Grouping and transfection
In this study, there were 6 groups including group I (normal control, non-transfected cells), group II (blank Table 3 Expressions of MAPK signal pathway-related proteins and VEGF-C protein after BMPR-II silence combined with inhibiting MAPK signal pathway in HepG2 cells (RGS, x ± s, n = 3) control, only liposome-transfected cells), group III (negative control, non-specific siRNA-transfected cells) and group IV-VI (BMPR-II-siRNA-a, siRNA-b and siRNAc-transfected cells, respectively). Cells were adjusted to 1 × 10 5 /ml, and then seeded in 6-well plate for 24 h. Cells covering 70-80% of the hole wall were incubated in 3 ml of serum-free DMEM for 2 h followed by transfection according to the instructions of kit. Five hours later, cells were incubated in DMEM containing 10% fetal calf serum for 24 h or 48 h followed by extraction of RNA or protein.
Western blot and RT-PCR to select the best sequence of siRNA targeting BMPR-II from the three sequences The cells transfected for 24 h or 48 h were used for extraction of RNA or protein, and then the levels of mRNA and protein of were determined with RT-PCR and Western blot. RT-PCR and Western blot were performed as previously described.
MTT assay to assess the proliferation of liver cancer cells There were normal control group, negative group and BMPR-II-siRNA-a group. Cells were seeded in a 96well plate at a density of 1 × 10 5 cells/well with a final volume of 100 μl. After cells covering 70-80% of the hole wall were transfected for 24, 48 and 72 h, respectively; 20 μl of MTT was added at 37°C for 4 h. After the supernatant fluid was removed, 150 ml of DMSO was added for 10 min with shaking. Cell viability was assessed by measuring the absorbance at 490 nm using an Enzymelabeling instrument (EX-800 type). All measurements were performed in triplicate. The results were expressed as the average of three independent experiments.

Transwell assay to evaluate the invasion of liver cancer cells
Cell invasion was assessed using 8 μm pore size Borden chamber (24-well plate). Borden chamber was washed using serum-free DMEM, and then 50 μl of Matrigel (1:8) was used to coat the upper surface of the filter. After cells in each group (normal control group, negative control group and BMPR-II-siRNA-a group) were transfected for 48 h, 200 μl of cell suspension (1 × 10 5 /ml) was added in the upper compartment with serum-free medium containing 10 g/L of BSA, and then 500 μl of 10% fetal bovine serum was added in the lower compartment. Borden chamber was placed in an atmosphere of 5% CO2 at 37°C for 24 h followed by washing with PBS. The cells not to penetrate the membrane of the upper chamber were wiped out using cotton swabs. The cells to penetrate the membrane were fixed with 95% of ethanol for 5 min, and then stained with 4 g/L of crystal violet. The cell-invading numbers in five visual fields were counted under a light microscope and the mean was calculated. The invasive ability of tumor cells was expressed as the cell-invading number. Testing was performed in triplicate in each group.

Apoptosis and cell cycle to detect with flow cytometer
The samples were washed twice and adjusted to a concentration of 1 × 10 6 cells/ml with 4°C PBS. The Falcon tubes (12 mm × 75 mm, polystyrene round bottom) were used in this experiment. For some samples, 100 μl of suspensions, 10 μl of annexin V-FITC and 10 μl of propidiom iodide (PI, 20 μg/ml) were respectively added into the labeled tube to detect apoptosis at room temperature in the dark for 10 min. Other samples were fixed with 70% of ice ethanol at 4°C for 12 h, and then treated with PI for 30 min to observe cell cycle.
Effects of BMPR-II silence on the expressions of MAPK signal pathway-related proteins and VEGF-C protein Total protein was extracted from the cells transfected with siRNA targeting BMPR-II for 48 h. The protein expressions of p-P38/P38, p-ERK1/2/ERK1/2, p-JNK/ JNK and VEGF-C were detected with Western blot.
Expression of VEGF-C protein after inhibiting MAPK signal pathway with SB203580, PD98059 and SP600125, respectively Cells were adjusted to 1 × 10 5 /ml, seeded in 6-well plate, and then treated with serum-free DMEM for 2 h followed by addition of SB203580 (P38 inhibitor, 10 uM), PD98059 (ERK1/2 inhibitor, 40 uM) and SP600125 (JNK inhibitor, 50 uM), respectively. Two hours later, protein was extracted, and then the level of VEGF-C protein in each group was determined with Western blot respectively. Table 4 Sequences of Specific BMPR-II-siRNAs and Negative siRNA Targeting BMPR-II   Name  Sequence (5′-3′ Expression of VEGF-C protein after BMPR-II silence combined with inhibiting MAPK signal pathway Cells were adjusted to 1 × 10 5 /ml, seeded in 6-well plate, and then transfected with siRNA targeting BMPR-II for 48 hours. These transfected cells were treated with DMEM for 1-2 followed by addition 50 μm of SB203580 (P38 inhibitor, 10 uM), PD98059 (ERK1/2 inhibitor, 40 uM) and SP600125 (JNK inhibitor, 50 uM), respectively. Two hours later, protein was extracted, and then the expression of VEGF-C protein in each group was determined with Western blot.

Statistical analysis
Statistical treatment was performed with SPSS 19.0 software. All data were expressed as x ± s (mean ± s.d.).
Single-factor analysis of variance was used for comparison among multiple groups. t-test was used for comparison between two groups. Statistical significance was established at P < 0.05.

Competing interests
There was no any competing financial interest in relation to the work.
Author's contributions PZ: had made acquisition of data and involved in drafting the manuscript. SC: had made acquisition of data. JZ: had made acquisition of data. FY: had made analysis and interpretation of data. WJ: had made acquisition of data. JW: had made substantial contributions and given final approval of the version to be published. All authors read and approved the final manuscript.