Bone sialoprotein expression on osteosarcoma cell invasion and metastasis characteristics of
Author: Huang Tao, Min Chen, Guo Jin, Zhang Yi North, Liao Hui
【Abstract】 Objective To construct the human bone
sialoprotein (BSP) sense and antisense eukaryotic expression vector, to study the invasion and metastasis of osteosarcoma cell characteristics. Methods The PCR-amplified open reading
frame of human BSP sequence, with the vector pIRES2 EGFP
linked to constitute a recombinant antisense expression
plasmid. By liposome-mediated transfection into MG 63
cells, by Western blot detection of cell protein. With reconstituted basement membrane invasion assay and scratches Experimental Determination of the invasiveness of osteosarcoma
cell effects. The result successfully constructed a human BSP antisense RNA eukaryotic expression vector, after transfection effectively expressed hBSP, compared with the control group, just after transfection vectors so that MG 63 cells hBSP
elevated levels of protein expression, invasiveness enhancement; antisense vector transfected to MG 63 cells
hBSP reduce the level of protein expression, invasiveness is inhibited. Conclusion BSP expression changes may be the process of invasion and metastasis of osteosarcoma play an
important role, BSP decreased expression can inhibit the invasive ability of osteosarcoma.
Key words bone sialoprotein gene; gene expression; tumor metastasis; Osteosarcoma
Expression of BSP Influence on Behavior of Invasion and
Metastasis of Osteosarcoma
Abstract: Objective To construct sense and antisense human bone sialoprotein (BSP) eukaryotic expression vector and observe influence of its expression in human osteosarcoma cell
line MG 63 cells about invasion and metastasis of
osteosarcoma.Methods An open reading frame of human BSP was amplified by PCR method and was reconstructed into the eukaryotic expressive vector pIRES2 EGFP to construct the
hBSP sense or antisense expressive vector. The sense and antisense hBSP eukaryotic expression vector was transfected into MG 63 cells with lipofec tamine.The expression of BSP on the cells was measured by Western blotting.Transwell
chamber methodand scarification was used to evaluate influence of BSP expression on osteosarcoma cell invasion and metastasis.Results The sense and antisense hBSP eukaryotic vector was successfully constructed and transfected. Compared with control cells, expression of BSP increased in the cells
by sense hBSP eukaryotic vector transfected and invasive power of these cells is enhance, but expression of BSP decreased in the cells by antisense hBSP eukaryotic vector transfected and invasive power of these cells is been inhabited.Conclusion The
change of BSP expression may play an important role in osteosarcoma metastasis.The down regulation of BSP
expression can inhibit abilities of osteosarcoma cell invasion and metastasis.
Key words: Bone sialoprotein gene; Gene expression;
Neoplasm metastasis; Osteosarcoma
Bone sialoprotein (Bone sialoprotein, BSP) is the extracellular matrix in an acidic glycoprotein, its tissue distribution, mainly in mineralized tissues (such as bone, teeth), and calcification of cartilage and bone junction [1,2] . In recent years, the research suggests that it is on the BSP in tumor growth, invasion and metastasis, tumor angiogenesis, tumor immune escape have played an important role. This study attempts to bring antisense nucleic acid
technology to build full-length human BSP sense and antisense eukaryotic expression vector, and transfected human osteosarcoma MG 63 cells to explore the sense and antisense expression of BSP protein in osteosarcoma cell invasion
migration behavior of .
1 Materials and methods
1.1 Main materials and reagents
Human BSP cloning vector pUCm hBSP by the laboratory
building, hBSP vector containing genes from the start codon to the stop codon of the full-length cDNA. MG 63 cell lines
from cell culture of Life Sciences, Wuhan University, are introduced. DH5α bacteria, pIRES2 EGFP plasmid by the room
to save. Gel extraction kit, plasmid extraction purification kit purchased Bio-Engineering Co., Ltd. in Shanghai Waston.
PCR product cloning kit purchased from Shanghai Biological Engineering Technology Health Engineering Services Limited. BamH ?, Hind ? purchased at the Toyobo Corporation.
LipofectAMINETM 2000 transfection kit purchased from Invitrogen Corporation. Sequenced by Shanghai Boya
Biotechnology Co., Ltd. completed. Rabbit anti-human BSP
polyclonal antibody purchased from ALEXIS Biochemicals. Matrigel (basement membrane of artificial rubber), Fn (fibronectin) was purchased from BD Labware, Transwell (cell invasion of the small room) to buy the company in the Coster.
1.2.1 human BSP antisense expression vector
220.127.116.11 Design of primers
According to the full-length human BSP cDNA sequence of design for the person BSP open reading frame antisense
sequence of the upstream and downstream primers, respectively,
the introduction of restriction sites BamH ? and Hind ?,
while the upstream and downstream of the restriction sites introduced with the Building The pUCm hBSP sequence of
multiple cloning restriction sites in reverse order. Antisense primer: upstream 5 ' GCAAGCTT (Hind ?) GCCAGAGGAAGCAATCAC
3', downstream 5 ' GCGGATCC (BamH ?) CTTCACTGGTGGTGGTAG
18.104.22.168 justice and human BSP expression vector
pUCm hBSP with pIRES2 EGFP were used to BamH ? and
Hind ? restriction enzyme digestion, recycling, and pIRES2
EGFP insert a linear fragment, T4 ligase to form a just containing human BSP eukaryotic expression plasmid.
22.214.171.124 antisense expression vector BSP
With the built pUCm hBSP cloning vector as a template, using the designed antisense primer PCR amplification, the product line of 1% agarose gel electrophoresis, recovery, by T 4 ligase the PCR products were cloned into pUCm T cloning
vector. Then by BamH ?, Hind ? restriction enzyme digestion, will receive the antisense hBSP fragment was subcloned into pIRES2 EGFP expression vector, thereby constituting containing human BSP antisense eukaryotic expression vector.
126.96.36.199 antisense expression vector of human BSP Identification
Competent bacteria were transformed into a product of DH5α, selecting positive clones were amplified, extracted plasmid DNA. The BamH ? and Hind ? restriction enzyme
digestion to identify and DNA sequencing.
1.2.2 Cell culture and transfection
Human osteosarcoma cell MG 63, according to
conventional methods of cultivation. The cell is divided into four groups: Justice transfection group (s hBSP MG),
antisense transfection group (as hBSP MG), positive
control group (Positive control) or empty vector transfected
group, negative control group (Negative control) or untreated group of tumor cells. Transfection reagent for cationic liposome LipofectAMINETM2000, specific methods to carry out according to instructions.
1.2.3 Western blot detection of transfected cells in
each group the expression of bone sialoprotein
After transfection, the four set of cells 50μg of total
protein were extracted by 10% SDS PAGE gel electrophoresis,
transferred to nitrocellulose membrane, 1% skim milk at room temperature overnight closure by adding rabbit anti-BSP
polyclonal antibody , 37 ? under the hybrid 2h, the
corresponding secondary antibody at room temperature
hybridization 1h, the final color reaction and exposed by X-
1.2.4 tumor reconstituted basement membrane invasion
With 50mg / L Matrigel 1:8 dilution-coated Transwell
chamber the bottom of the Transwell chamber will be placed in 24-well plate, in the small outdoor plus chemokines 200μl and
contained 10? S of DMEM200μl; in a small room by adding 100μ
l suspension of tumor cells fluid, cell number 1 × 105, each
repeated six samples. Conventional culture 24h, carefully wiped the microporous membrane with cotton swab the upper cell, 95% ethanol-fixed, Giemsa staining solution. Counted
under the inverted microscope to move the lower microporous membranes of cells, each sample count of 10 vision.
1.2.5 cell scratch test
Cells were inoculated with each of the groups using FN pre-coated 24-well culture plates, each 6 holes, each hole
cell count of 5 × 105, conventional cell culture to form a single layer along the culture plates with the pipette emitter the bottom of the draw "a"-shaped horizontal wounds, continue to use the serum-free DMEM culture. Microscopic observation of
12h, 24h, 36h, 48h, 60h, 72h of cell movement, recording the migration from the migration of the most remote point of origin to the distance between the nuclei, with migration distance reflects the migration speed. Reposted elsewhere in the paper for free download http://
1.2.6 Statistical analysis
SPSS12.0 the experimental data using statistical software for statistical processing, data with ? s said that the
difference between the two groups was significant by t test, statistical methods using analysis of variance. P <0.05
2.1 people BSP antisense expression vector and identification of
Recombinant human BSP eukaryotic sense and antisense plasmid was cloned BamH ? and Hind ? restriction enzyme
digestion, the 1% agarose gel electrophoresis, obtained fragment was about 950bp, and is expected to the same size. Will be derived from pIRES2 EGFP fragment was subcloned
into the corresponding restriction sites, build hBSP antisense expression vector. Vector of the proceeds were once again double-BamH ? and Hind ? restriction enzyme digestion, the
result may be a purpose of the treaty 950bp fragment. BSP on the recombinant plasmid in the open reading frame DNA sequencing fragments, and GenBank hBSP homologous sequence
comparison, sequence composed of the same sense and antisense sequence in the opposite direction, indicating that antisense recombinant vector was successfully constructed, shown in Figure 1.
2.2 Cell Transfection results
After transfection, 24h, 48h and 72h respectively, were observed under a fluorescence microscope, no transfected cells were observed under a fluorescence microscope, no fluorescence, while the transfected cells were observed under a fluorescence microscope, see the green fluorescence, and 48h after transfection to express green fluorescent protein the number of cells than 24h, 72h transfected cells increased the amount of reduced gradually.
2.3 Western blot detection of transfected cells in each
group stability and protein expression of BSP
Western blot test results showed that cells in each group were BSP protein expression, indicating in osteosarcoma cells, there is the expression of BSP, and the same reported in the literature. But the expression intensity of each group is different as compared with the negative control group, justice transfected group cells increased the expression of BSP, and anti-sense transfected group cells was significantly reduced the expression of BSP, positive control group of cells, the
expression of BSP in the amount of non - changes, shown in
2.4 reconstituted basement membrane invasion inhibition
Group 4 cells were able to pass through the membrane
paved with Matrigel. And negative cells compared with the
control group, justice transfection group of tumor cells trans-membrane cell number increased, there is a significant difference (P