|Year : 2018 | Volume
| Issue : 8 | Page : 100-104
Effect of spleen tyrosine kinase on nonsmall cell lung cancer
Ying Tao Hao, Chuan Liang Peng, Yun Peng Zhao, Qi Feng Sun, Xiao Gang Zhao, Bo Cong
Department of Thoracic, The Second Hospital of Shandong University, Jinan, Shandong, China
|Date of Web Publication||26-Mar-2018|
Department of Thoracic, The Second Hospital of Shandong University, 247, Beiyuan Street, Jinan 250033, Shandong
Source of Support: None, Conflict of Interest: None
Aim of Study: To investigate the anti.tumor effect of spleen tyrosine kinase. (Syk) on the human nonsmall cell lung cancer cells in vitro and its mechanism.
Materials and Methods: In this study, we constructed a eukaryotic expression vector pcDNA3.1D/V5-His-TOPO/Syk and transfected it into human nonsmall cell lung cancer cells A549. Then, we not only analyzed the expression of Syk in transfected cells and its invasion but also the expression of matrix metalloproteinase-9 (MMP-9).
Results: The results showed that overexpressed Syk significantly inhibited A549 cell invasive ability by decreasing the expression of MMP-9.
Conclusion: The overexpressed Syk plays an important role in tumor invasion and metastasis, and a negative role in human nonsmall cell lung cancer cells.
Keywords: Invasion, matrix metalloproteinase, nonsmall cell lung cancer, spleen tyrosine kinase
|How to cite this article:|
Hao YT, Peng CL, Zhao YP, Sun QF, Zhao XG, Cong B. Effect of spleen tyrosine kinase on nonsmall cell lung cancer. J Can Res Ther 2018;14, Suppl S1:100-4
| > Introduction|| |
Spleen tyrosine kinase (Syk) is the most important kinase in B-cell signal transduction pathway, belongs to protein-tyrosine kinases (PTKs) family and plays a key role in the maturation and activation of T-cells and B-cells., Recently, some studies have proved a correlation between Syk and tumor occurrence and development. Syk can inhibit the growth of malignant tumors, which leads to the research of its effect on tumor development and metastasis recently. The development and metastasis of tumor are a multi-stage and multi-factor process, and evading from immune surveillance is one of the most important aspects. Therefore, to analysis the anti-tumor immune activity and tumor immune escape mechanism resulted from Syk expression are valuable. To study the role and mechanism of Syk in the regulation of lung cancer cell growth and metastasis, we constructed eukaryotic expression vector pcDNA3.1D/V5-His-TOPO ®/Syk, transfected them into A549 cells by lipofectamine protocols and detect changes in cell function induced by Syk gene.
| > Materials and Methods|| |
Reverse transcriptase avian myeloblastosis virus was purchased from Promega corporation. Fidelity enzyme KOD plus was from Toyobo company. Trizol reagent, eukaryotic expression vector pcDNA3.1D/V5-His-TPOP and transfection kit plus™ reagent were from American Invitrogen Company of USA. Human lung cancer cell line A549 (Institute of Biochemistry and cell biology of Chinese Academy of Sciences) were maintained in RPMI 1640 (Gibco BRL) with 10% fetal bovine serum. Transwell ® invasion chambers and matrigel gum were from Coaster Company and BD Company. Apa I Restriction endonuclease was presented by Molecular Biology Laboratory of Shandong University.
Amplification of spleen tyrosine kinase by reverse transcription-polymerase chain reaction
Total RNA was extracted from human spleen tissue in accordance with the TRIzol ® kit's instructions. Primers were designed based on Syk cDNA sequence in GenBank ® and pcDNA3.1D/V5-His-TPOP vector. Upstream primer: 5'-CACCATGGCCAGCAGCGGCATGGC T-3, Downstream primer: 5'-GTTCACCACGTCATAGTAGTAATTGCC-3'. Syk cDNA was amplified with 0.1 μg RNA according to reverse transcription-polymerase chain reaction (RT-PCR) kit's instructions. The amplification products were analyzed by 5 L agarose gel and purified by PCR product gel extraction kit from Omega Company.
Construction and identification of recombinant plasmid
The reaction system included fresh Syk PCR product 2 μl, salt solution 1 μl, sterile water 2 μl, and TOPO vector 1 μl. The reaction involved 30 cycles of initial denaturation at 94°C for 1 min, denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The ligation product was transformed into TOP 10 competent cells directly after water bath at 22°C for 30 min and the following ice bath. Amp positive colonies were collected; plasmid was obtained after multiplication, amplified by PCR, and identified by Apa I restriction endonuclease. DNA sequencing was done to ensure correctness of insert Syk gene.
Twenty-four well plates, in which were added A549 cells, were coated in a humidified atmosphere of 50 ml/L CO2 at 37°C. Transfection was done according to Plus ™ Reagent instruction when the cell density reached 70–80%. Plasmid-free Syk gene fragment was regarded as empty vector transfection and control group. Expressing Syk gene clones stably were obtained by G418 selection.
Western blot analysis
A549 cells were collected after 24 h transfection. Cytoplasmic protein was extracted, and the protein concentration was determined using the Bradford method. The protein (100 μg) was added to each well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) film and the position of the target protein. The PVDF membrane was followed by incubation with mouse anti-human Syk monoclonal antibody (1:500) and then with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G antibody at 37°C for 1 h, respectively. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal reference. The signals were detected with an enhanced chemiluminescence kit. The experiment in each group was repeated 3 times.
Expression of matrix metalloproteinase-9 mRNA
Primers were designed based on the sequence in GenBank as follows: Upstream 5'-TTCATCTT CCAAGGCCAATC-3', downstream 5'-CTTGTCGCTGTCAAAGTTCG-3'. We could get 287 bp fragment. The primers of β-actin were as follows: Upstream 5'-ATCTGGCACCACACCTCCTAC AATGAGCTGCG-3', downstream 5'-CGTCATACTCCTGCTTGCTGA TCCACATCTGC-3'. 838 bp fragment could be amplified. The amplification products were analyzed by 10 g/L agarose gel, absorbance value of each zone was measured with gel analysis for semi-quantitative analysis. The experiment was repeated 3 times.
Invasive analysis: 24-well matrix gel invasion chambers (from BD Company) were used. The size of membrane pore between upper and lower chamber is 8 μm, extracellular matrix gel was covered onto the upper chamber membrane surface, which can simulate the body extracellular matrix and basement membrane environment. The cells were mixed with serum-free RPMI 1640 to the concentration of 2 × 105/ml, 200 μl of the cell suspension was added to the upper chamber and 500 μl RPMI 1640 with 10% fetal calf serum to the lower chamber. After 48 h in an incubator filled with 5% CO2 at 37°C, the cells into the lower chamber were fixed with formaldehyde (hematoxylin and eosin staining) and observed under the light microscope (400 times). We counted the cells in six fields randomly; the mean was the number of the invasive cells.
The experimental data were analyzed with Statistical Package for the Social Sciences 11.0 (SPSS. INC., Chicago, IL) for statistical analysis, all the results were expressed as mean±standard deviation (SD), the means among more groups were compared with analysis of variance. The value P < 0.05 was considered statistically significant.
| > Results|| |
Amplification of the target gene Syk using specific primers of Syk gene, total RNA of splenocytes was amplified by RT-PCR and DNA fragment was obtained. Its size is 1909 bp which is consistent with expected [Figure 1]. Identification of Syk recombinant plasmid was digested by Apa I restriction endonuclease, two different size of fragments could be obtained after agarose gel electrophoresis (5809 bp and 1610 bp) [Figure 2]. DNA sequencing results showed that the full length of inserted Syk gene fragment is 1909 bp (including 5'-CACC). The coding sequence was consistent with the sequence in Genbank, and the reading frame remained the same. Syk expression analysis Syk expression was detected by RT-PCR and Western blot techniques. The results showed that the transfected cells (pcDNA3.1D/V5-His-TOPO/Syk) had a higher expression than empty plasmid cells (pcDNA3.1D/V5-His-TOPO) and untransfected cells at mRNA level and protein level (P < 0.05) [Figure 3]. And the empty plasmid pcDNA3.1D/V5-His-TOPO transfected cells had no significant difference when compared with the untransfected cells in the expression of Syk (P > 0.05).
|Figure 1: Reverse transcription-polymerase chain reaction amplification products of spleen tyrosine kinase gene by agarose gel electrophoresis|
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|Figure 2: Electrophoresis result of pcDNA3.1D/V5-His-TPOP/ spleen tyrosine kinase by Apa I restriction endonuclease. M.DL10000 DNA marker. (1) Recombinant plasmid pcDNA3.1D/V5-His-TPOP/ spleen tyrosine kinase. (2) Recombinant plasmid digested by Apa I|
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|Figure 3: Expression of spleen tyrosine kinase gene in different cells detected by reverse transcription-polymerase chain reaction and spleen tyrosine kinase protein by western blot. (1) Empty plasmid cells (pcDNA3.1D/V5-His-TOPO). (2) Untransfected cells; (3) Transfected cells (pcDNA3.1D/V5-His-TOPO/spleen tyrosine kinase)|
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Expression of matrix metalloproteinase-9 (MMP-9) mRNA in different cells. RT-PCR and flow cytometry results showed that MMP-9 mRNA and protein expressed lower in the transfected cells than that in empty plasmid cells and untransfected cells (P < 0.05) [Table 1].
|Table 1: Expression of matrix metalloproteinase-9 mRNA and protein in different cells|
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There is no significant difference between empty plasmid transfected cells and untransfected cells (P > 0.05).
Syk reduced A549 cell invasion. To investigate the invasive ability of the cells, we performed the invasion assay. The results showed that the number of transmembrane cell was significantly reduced in the transfected cells than in other two groups (P < 0.05). The cell structure was not clear under a light microscope, and the invasion on the reconstituted basement membrane was weakened [Table 2] and [Figure 4].
|Table 2: Effect of spleen tyrosine kinase gene transfection on invasion ability of different cells|
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|Figure 4: Cell invasion in different cells (×400). (a) pcDNA3.1D/V5-His-TOPO cells; (b) Untransfected cells. (c) pcDNA3.1D/V5-His-TOPO/spleen tyrosine kinase cells|
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| > Discussion|| |
Invasion and metastasis are not only important feature of malignant tumors but also the main reason for treatment failure and even death in cancer patients. Tumor invasion and metastasis are a complicated progress involving tumor cell proliferation and transformation, angiogenesis, degradation of the matrix by MMPs and tumor cells migration, multi-factor, and multi-link interactions. Degradation of the matrix by MMPs is essential for the primary tumor. MMP-9 is a kind of gelatin enzymes belonging to the family of MMP, which can degrade and destruct collagen IV in extracellular matrix near the surface of the tumor after being activated into collagenase IV. The tumor cells are able to infiltrate into the surrounding tissue along the damaged basement membrane, eventually leading to tumor invasion and metastasis. Many studies results showed that MMP-9 plays an important role in migration, invasion, and metastasis of tumor cells can effectively degrade extracellular matrix and basement membrane and participate in the invasion and metastasis of the tumor., In our study, the results showed that MMP-9's expression is lower in Syk cDNA transfected cells than in empty plasmid cells and untransfected cells (P < 0.05). It suggested that Syk gene can decrease the expression of MMP-9 in lung cancer cells, reduce the degradation of the matrix, and the basement membrane and inhibit tumor invasion and metastasis.
The PTKs is a group of enzyme protein which can catalyze substrate protein tyrosine residues to phosphorylation and is involved in many signal transduction pathways. It also plays an important role in the control of cell differentiation, proliferation, and spread. Recent studies have found that Syk, as a tumor suppressor of PTKs, is closely related to tumor invasion and metastasis., It was long thought to be a hematopoietic cell-specific signaling molecule by many nonhematopoietic cell types , and that it plays a negative role in cancer. Many studies showed the potential inhibitory effect of the gene Syk in breast, gastric, and pancreatic cancer. A few literatures have been reported about Syk expression in lung cancer.,,
In this study, the gene of Syk was obtained from human spleen tissue and clone into the expression plasmid pcDNA3.1D/V5-His-TOPO. The recombinant expression plasmid pcDNA3.1D/V5-His-TOPO/Syk was transfected into human cancer cells A549 using lipofectamine. We found that Syk expression was higher in transfected cells. We also established invasion model in vitro with transwell chamber and matrigel to evaluate the relation between Syk gene expression and cells invasion. Matrigel is extracted from the extracellular matrix of rat Esh sarcoma, its ingredients include collagen IV, laminin, heparan sulfate proteoglycans and integrins, and other components. Matrigel can simulate tissue basement membrane and is used to observe the interaction between the cells and extracellular matrix. Matrigel is laid on the upper polycarbonate membrane of transwell chamber, the number of transmembrane cells can reflect the invasion of tumor cells. The study showed that the number of transmembrane cells was significantly reduced in Syk gene transfected cells than empty plasmid cells and untransfected cells (P < 0.05). It is further confirmed that high expression of Syk gene reduces invasive ability of lung cancer cell. These results are similar to previous reports about Syk expression in other types of malignant tumors.,
| > Conclusion|| |
Syk involved in lung cancer cell invasion as a candidate suppressor gene. Maybe it inhibits migration and invasion of lung cancer cell by reducing the expression level of MMP-9. These observations led us to hypothesize that upregulation of Syk expression could inhibit tumor cell invasion, and these may serve as part of the basis for gene therapy. However, further study should be done and demonstrate on Syk subtypes function, the relationship between transduction network and tumor and further regulation mechanisms in the tumor.
Financial support and sponsorship
This research was supported by the Natural Science Foundation of Shandong Province (Y2008C55).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]