[Abstract] Objective To construct bone sialoprotein (BSP) and antisense eukaryotic expression vector, and to study the characteristics of osteosarcoma cell invasion and metastasis. Sequence of the open reading frame of the human BSP PCR method to constitute recombinant sense and antisense expression plasmid vector pIRES2  EGFP. Into MG  63 cells, liposome-mediated transfection and protein expression in the cells was detected by Western blot. Reconstituted basement membrane invasion assay and scratches experimental determination of osteosarcoma cell invasion force. BSP positive and negative sense nucleic acid eukaryotic expression vector was successfully constructed and effective expression after transfection hBSP, compared with the control group, after justice vector-transfected MG  63 cells the hBSP protein expression levels of invasion enhanced; antisense vector-transfected MG  63 cells hBSP protein expression level to reduce the invasion force suppressed. Conclusion The change of BSP expression may play an important role in the process of invasion and metastasis of osteosarcoma, BSP downregulation can inhibit the invasive ability of osteosarcoma.

Key words bone sialoprotein gene; gene expression; tumor metastasis; osteosarcoma

    Bone sialoprotein (Bone sialoprotein, BSP) is an acidic glycoprotein in the extracellular matrix, tissue distribution, mainly in the border zone of the mineralized tissue (such as bone, teeth) and calcification of cartilage and bone [1,2] . In recent years prompted the BSP in tumor growth, invasion and metastasis, tumor angiogenesis, and tumor immune escape have played an important role. This experiment attempts to build by antisense technique to carry a full-length human BSP sense and antisense eukaryotic expression vector, and transfected osteosarcoma MG  63 cells to investigate the effects of antisense BSP protein expression in osteosarcoma cell invasion and migration behavior .

    1 Materials and methods

    1.1 major materials and reagents

    The person BSP cloning vector pUCm  hBSP by the lab building, the vector containing hBSP gene full-length cDNA from the start codon to the stop codon. MG  63 cell lines by the introduction of Wuhan University of Life Sciences, cell culture chamber. DH5α bacteria, pIRES2  EGFP plasmid saved by the chamber. Gel extraction kit, plasmid extraction and purification kit was purchased from Shanghai Huashun Sheng Bio-Engineering Co., Ltd.. The PCR product was cloned kit was purchased from Sangon Biological Engineering Technology & Services Co., Ltd. in Shanghai. BamH Ⅰ, Hind Ⅲ purchased from Toyobo Company. LipofectAMINETM 2000 transfection kit was purchased from Invitrogen Corporation. The sequencing was completed by Shanghai Boya Biotechnology Co., Ltd.. Rabbit anti-human BSP polyclonal antibody was purchased from ALEXIS Biochemicals. Matrigel (reconstituted basement membrane glue), Fn (fibronectin) were purchased from BD Labware, the Transwell (cell invasion chamber) was purchased from Coster company.

    1.2 Methods

    1.2.1 antisense human BSP Expression Vector

    1.2.1.1 design primers designed according to the full-length sequence of human BSP cDNA upstream and downstream primers for the antisense sequence of the open reading frame of human BSP, and the introduction of restriction sites BamH Ⅰ and HindIII, while the introduced enzyme in Preparation the polyclonal restriction sites in the locus and the constructed sequence pUCm  hBSP opposite order. Antisense primer: the upstream 5 ‘ GCAAGCTT, (Hind III) GCCAGAGGAAGCAATCAC  3’ downstream 5 ‘the  GCGGATCC (BamH Ⅰ) CTTCACTGGTGGTGGTAG  3’.

    1.2.1.2 righteous BSP BamH Ⅰ and Hind Ⅲ digestion recycling insert and pIRES2  EGFP linear fragments, T4 ligase constitutes containing human BSP to build pUCm  hBSP pIRES2  EGFP expression vector justice really expression plasmid.

    1.2.1.3 antisense human BSP expression vector constructed pUCm  hBSP cloning vector as the template, with the design of an antisense primer for PCR amplification, and the product lines a 1% agarose gel electrophoresis, recovered by T 4 ligase The PCR product was cloned into the cloning vector pUCm  T. By BamH I and Hind Ⅲ digested the the antisense hBSP fragment subcloned into pIRES2  EGFP expression vector, which constitutes the antisense eukaryotic expression vector containing the human BSP.

    1.2.1.4 the identification product of both positive and negative of BSP expression vector of the righteous were transformed into competent bacteria DH5α, positive clones amplified extracted plasmid DNA. With BamH Ⅰ and Hind Ⅲ double enzyme digestion and DNA sequencing.

    1.2.2 Cell culture and transfection

    Human osteosarcoma cell MG  63, according to the conventional method of cultivation. The cells were divided into 4 groups: justice transfected group (s  hBSP  MG) the antisense transfection group (as  hBSP  MG), positive control group (Positive control) that is empty plasmid group, negative control group ( Negative control) untreated tumor cells group. Transfection reagent cationic liposomes LipofectAMINETM2000, and specific instructions.

    1.2.3 Western  blot detection of bone sialoprotein expression in transfected cells in each group

    After transfection, the cells were extracted from the total protein 50μg by 10% SDS  PAGE gel electrophoresis, transferred to nitrocellulose, 1% skim milk was blocked overnight at room temperature, by adding rabbit anti-human BSP polyclonal antibody, 37 ℃ hybrid 2h, corresponding secondary antibodies room temperature hybridization 1h final color reaction and exposure to X-ray film.

    1.2.4 tumor reconstituted basement membrane invasion assay

    50mg / L by Matrigel 1:8 dilution package is the bottom of the Transwell chamber, Transwell chamber placed in a 24-well culture plate, the chemotactic factor 200μl added in small outdoor and containing 10% FBS DMEM200μl; Add 100μl tumor cells suspended in a small chamber solution, the cell number of 1 × 105, each repeated six samples. Routinely cultured for 24 h, with a cotton swab carefully wipe the upper microporous membrane cells, 95% ethanol the Giemsa solution dyed. Moved lower microporous membrane of cells counted under an inverted microscope each sample count 10 vision.

    1.2.5 cells scratches experimental

    The cells in each group were inoculated in the use of the FN pre-coated 24-well culture plates, 6 in each hole, each hole has a cell number of 5 x 105, the conventional culture to form a monolayer of cells with a pipette drippers along the plates the bottom of the program, “a" shaped horizontal line wound, continue with serum-free DMEM culture. Microscope 12h, 24h, 36h, 48h, 60h, 72h cell movement, recorded from the migration point of origin to the migration of the distance between the most distal nucleus to reflect migration speed, migration distance.

    1.2.6 Statistical

    Experimental data using SPSS12.0 statistical software for statistical analysis, the data ± s, significant differences with group t test was used for statistical analysis of variance. P <0.05 was statistically significant.

    2 Results

    2.1 the pros and cons of the righteous BSP expression vector and identification

    The of recombinant antisense human BSP eukaryotic cloning plasmid after BamH Ⅰ and Hind Ⅲ digestion, a 1% agarose gel electrophoresis, the resulting fragment of about 950bp, with the estimated size of the same. The resulting fragment was subcloned into pIRES2  EGFP corresponding restriction sites, build hBSP sense and antisense expression vector. The resulting vector by BamH Ⅰ and Hind Ⅲ restriction enzyme digestion again, the result will be offered to them by a treaty 950bp purpose of fragments. Determination of the DNA sequence of the open reading frame fragment of the recombinant plasmid BSP compare with the GenBank hBSP homologous sequences, the same sequence of sense and antisense sequence in the opposite direction, indicating that antisense recombinant vector was successfully constructed, as shown in Figure 1.

    1: just hBSP expression vector restriction enzyme digestion; 2: antisense hBSP expression the carrier restriction endonuclease Figure 1 antisense recombinant expression vector digested after agarose gel electrophoresis

    2.2 cells transfected into Results

    24 h after transfection, 48h and 72h, respectively, was observed under a fluorescence microscope, non-transfected cells and no fluorescence was observed under a fluorescence microscope, transfected cells were observed under a fluorescence microscope to see the green fluorescent expressing green fluorescent protein, and 48 h after transfection cells more than the number 24h, 72h of transfected cells increased gradually reduced.

    2.3 Western blot to detect the expression of stably transfected cells BSP protein

    Western blot analysis showed that the protein expression of cells in each group are BSP, BSP expression, and consistent with those reported in osteosarcoma cells. Groups express different intensity, compared with the negative control group, justice transfected cells increased amount of BSP expression, antisense BSP expression was significantly reduced in the transfected cells, the expression of the positive control cells in the BSP no changes, see Figure 2.

    1: positive control group; 2: negative control group; 3: Justice transfection group; 4: the antisense transfected Photos 2 Western blot detection cells BSP expression

    2.4 reconstituted basement membrane invasion suppression

    4 cells are able to pass through covered with Matrigel membrane. And compared to the negative control group cells justice tumor cells transfected group transmembrane cell count increased, there is a significant difference (P <0.05). The antisense transfection group penetrating cells was significantly less than the negative control group, there was a significant difference (P <0.01). 0.05),见表1。">The transmembrane cell count of the positive control group and negative control group showed no significant difference (P> 0.05), as shown in Table 1. Table 1 Transwell invasion chamber measured in each group cell invasion (± s)

    Cell membrane cell number grouping wear justice transfection group 107.67 ± 11.33 * △ antisense transfection group 77.67 ± 5.82 *-△ positive control group was 94.83 ± 4.62 negative control group, 95.33 ± 6.92

    * Compared to the positive control group, P <0.05; △ compared with the negative control group, P <0.052.5 scratch test

    Scratches after 12h, cells in each group were observed in the cell is retracted away from the scribe area in each group were recorded scratches zone on both sides of the distance between cells, and as a reference. 24h microscope small set of cells start crawling. 36h microscope, cells crawling to the scratch area, and the antisense transfection group was significantly slower than the other groups, justice transfected group, the positive control group, negative control group crawling speed seems no obvious differences. 48h after antisense transfection group a few cells close to the scratch area, and other groups have a large number of cells near the scratch area. 60h the endoscopic see justice transfection group, positive control group and negative control group scratches region the presence of a small number of cells, antisense transfected cells are still scratches the edge, there is no cell in the scratches. 72h observed justice transfected group, positive control group and negative control group cells scratches district how the amount of cells, the antisense transfection group scratches District cells were scattered. 60h when selected according to the cell migration, cell migration distances were measured, see Table 2. Table 2 scratch wound model to determine the cell migration distance (± s)

    Cells grouped migration distance (μm) justice transfection group 265.38 ± 5.16 antisense transfection group 211.81 ± 12.19 * the △ positive control group 248.79 ± 10.29 negative control group 259.85 ± 4.37 *

    * Compared with the positive control group, P <0.05; △ compared with negative control group, P <0.05

    3 Discussion

    The antisense nucleic acid technology is one of the strategies of the tumor biological treatment, with appropriate design of antisense oligonucleotides specific inhibition of gene expression, is very likely to become the new method of treating tumors. Invasion and metastasis of malignant tumors of the essential characteristics of the transfer law is extremely complex the tumor clinical problem. Therefore, the application of antisense nucleic acid technology control tumor invasion and metastasis is a hot topic in cancer treatment research. Kido et al [3] on the adoption of antisense aminopeptidase N cDNA transfected osteosarcoma cells significantly inhibited osteosarcoma cells in vitro adhesion moving in nude mice effectively reduce the incidence of lung metastasis.

    Recent studies have shown that many tumors are associated with the expression of BSP in the development process, and these tumor expression BSP also more likely to exhibit invasion, a tendency to transfer [4]. Waltregny et al [5] of the breast and prostate cancer research for the first time to clarify BSP in tumor bone metastasis in a significant effect. Zhang [6], Sharp [7] confirmed that human breast cancer cells in a mouse model of BSP overexpression can promote bone metastasis, BSP expression inhibited cells also inhibit bone metastases. Oldberg et al. [8] studied the display the BSP via its RGD motif to promote adhesion between the osteosarcoma cells. BSP molecules, including integrin αvβ3 combination of fine – Gan – days (door) and aspartate (Arg-Gly-Asp, RGD) motif, can participate in integration-mediated signal transduction pathways, thereby mediating cell adhesion and migration [9,10]. As in normal tissues, the expression of BSP only limited to bone cells and trophoblast, so by antisense technique, we build the antisense hBSP expression vector, and osteosarcoma cells transfected as an object, preliminary research BSP expression of the biological behavior of autologous tumor of the bone tissue.

    The experimental artificial simulation of the artificial the basement membrane gum and fibronectin basement membrane and extracellular matrix, determination of transfection sense and antisense hBSP osteosarcoma cell invasion and migration of tumor cells to the basement membrane. In the invasion assay, we found that and antisense transfection the migration ability of tumor cells of the BSP significantly suppressed. Overexpression of the BSP tumor cell migration ability to get a certain degree of strengthening, suggesting that upregulation of BSP level possible invasion and metastasis of osteosarcoma, the BSP level downward to restrain the role of osteosarcoma invasion. Described through antisense hBSP expression vector transfection, direct inhibition of the transcription of DNA and affect the expression of BSP mRNA, so that the reduction of the expression of BSP, thereby reducing the activity of the tumor cells and tendency. Also showed that antisense-transfected osteosarcoma cell migration speed was significantly inhibited in the in vitro the scratches experimental model. It is the principle is the same can be suppressed due to the low expression of BSP FN chemotactic effect of tumor cell motility.

    The study results showed that the BSP in osteosarcoma cell invasion and metastasis process plays an important role, but also shows that the liposomal transfection of antisense BSP cDNA nucleotide chain human osteosarcoma cell invasion and metastasis have some impact, indicate that the use of antisense hBSP blocking the expression of BSP thereby inhibiting the occurrence of osteosarcoma, development as one of the methods of gene therapy for osteosarcoma.