|Year : 2016 | Volume
| Issue : 2 | Page : 663-666
Syk expression in non-small-cell lung cancer and its relation with angiogenesis
Peng Chuanliang, Zhao Yunpeng, Hao Yingtao, Sun Qifeng, Zhao Xiaogang, Cong Bo
Department of Thoracic, Second Hospital of Shandong University, Jinan, China
|Date of Web Publication||25-Jul-2016|
Department of Thoracic, Second Hospital of Shandong University, Jinan-250 033
Source of Support: This research was supported by the Science
and Technology Department of Shandong Province (Y2008C55), Conflict of Interest: None
Objective: To study the expression of spleentyrosine kinase (Syk) gene in non--small--cell lung cancer and the relationship between Syk mRNA and microvessel density (MVD) in the tumor cells.
Materials and Methods: The expression of Syk gene in 70 cases of lung tumor tissues, adjacent tissues, and normal lung tissues were examined with reverse transcription polymerase chain reaction (RT--PCR). The expression of MVD was examined with immunohistochemical streptavidin--biotin complex (SABC). The relation between them was analyzed.
Results: Syk mRNA expression rates were 5.7, 95.7, and 100% in tumor, adjacent lung cells, and normal lung cells, respectively. The expression rate in tumor cells was significantly lower compared with those in normal lung tissue and adjacent lung tissue (P < 0.05), expression rate among different pathologic types, differentiation and clinical stages did not reveal any statistically significant differences (P > 0.05). The positive rate of CD34 in tumor was higher than that in adjacent tissues and normal lung tissues. The expression of Syk mRNA and MVD were negatively correlated.
Conclusions: The lack of Syk mRNA expression in lung cancer play an important role in angiogenesis.
Keywords: Microvessel density, non-small-cell lung cancer, Syk mRNA
|How to cite this article:|
Chuanliang P, Yunpeng Z, Yingtao H, Qifeng S, Xiaogang Z, Bo C. Syk expression in non-small-cell lung cancer and its relation with angiogenesis. J Can Res Ther 2016;12:663-6
|How to cite this URL:|
Chuanliang P, Yunpeng Z, Yingtao H, Qifeng S, Xiaogang Z, Bo C. Syk expression in non-small-cell lung cancer and its relation with angiogenesis. J Can Res Ther [serial online] 2016 [cited 2020 May 31];12:663-6. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/663/154082
| > Introduction|| |
Spleen tyrosine kinase (Syk) plays an important role in the differentiation of immune cell and lymphocyte activation. Recent studies have found that expression of Syk gene was lost in some malignant tumors such as breast cancer and pancreatic cancer, so Syk gene is considered a candidate suppressor one., Lung cancer is a malignant neoplasm of vascular rich entities which have the same mechanism of oncogene activation and inactivation of tumor suppressor genes. In this study, we examined Syk mRNA expression in primary lung cancer by reverse transcription polymerase chain reaction (RT-PCR) and microvessel density (MVD) by immunohistochemistry, and clarified that the Syk gene deletion in lung cancer cells may result in angiogenesis and distant metastasis.
| > Materials and Methods|| |
Seventy patients (54 males and 16 females; median age of 61 years, ranged from 41 to 76 years) with NSCLC were diagnosed in Thoracic Department. In 70 cases, 28 were diagnosed as adenocarcinoma, 36 squamouscell carcinoma, and six largecell carcinoma. According to Primary Lung Cancer Diagnostic and Treatment Practices (2001), 13 cases belonged to stage I, 33 stage II, 19 stage III, and five stage IV. None of the patients received radiation or chemotherapy before surgery. Tumor tissues were taken from primary tumor area, avoiding necrosis and inflammatory area. Adjacent tissues were from 2cm next to the tumor material, and normal lung tissues from 5cm next to tumor margin. All the tissues were stored under 70°C.
Total RNA kit was purchased from Sigma company, and RT-PCR kit from Baisheng Biotechnology Company in Dalian. Syk primers were synthesized by Shanghai Biological Engineering Company according to GenBank Z29630. Upstream primer: 5'-CATGTCAAGGAT AAGAACATCATAGA-3'; downstream primer: 5'-AGTTCACCACGTCATAGTAGTAATT-3'. Length of amplification was 514bp. Streptavidin-biotin complex (SABC) kit, CD34 antibody, and DAB chromogenic reagent were from Wuhan Boster Biotechnology Company.
Total RNA was extracted in accordance with the TRIzol kit's instructions. Absorbance values (optical density (OD)) were measured by ultraviolet (UV) spectrophotometer after formaldehyde denaturing gel electrophoresis. RT and PCR were performed on PCR instrument. Four microliter RNA was digested by RNase-free DNase to get rid of DNA in trace amounts, and reverse transcription was done for 45 min in 42°C and 20 μl system with reverse transcriptase Mu-MLV. cDNA we obtained was ready for amplification including ×10 buffer 5 μl, MgCl2 3 μl, dNTP mi × 1 μl, primers 1 μl, Tag enzyme 0.5 μl, template 5 μl, and nuclease water 33.5μl. The reaction involved 30 cycles of denaturation at 94°C for 45s, annealing at 61°C for 1 min, and extending at 72°C for 45s. PCR products were observed under 1.5% agarose gel electrophoresis and analyzed with UV analyzer.
CD34 is a marker of capillaries, which shows vascular endothelial cells with the highest specificity. So we examined the expression of CD34 by SABC method with working concentration of 1:50 according to kit's instruction. Firstly, we looked for high vascular density areas (hot spot) under 40 times optical microscope, then counted the stained microvascular in hot spots under 200 times. Vascular endothelial cell were stained brown, it was regarded as a capillary if separated with the adjacent capillaries, tumor cells, and connective tissue. The maximum value of the number of vessels was thought of MVD under one vision.
The results were analyzed with Statistical Package for Social Sciences (SPSS) software, difference of Syk-positive rate were tested with the Chi-square test (α = 0.05). MVD were expressed as mean ± standard, t-test was used between two groups, and variance analysis and Spearman's correlation analysis between three groups.
| > Results|| |
Syk mRNA expresses rate were 5.7, 95.7, and 100% in tumor, adjacent lung tissues, and normal lung tissues, respectively. Syk expression rate in tumor cells was significantly lower compared with those in adjacent tissue and normal lung tissue (P < 0.05) [Figure 1], while there was no difference between adjacent lung tissue and normal lung tissue (P > 0.05). The positive rate were 5.6% in squamouscell carcinoma, 3.6% in adenocarcinoma, and 16.7%in large cell carcinoma; there is no significant difference among them (P = 0.394). Syk mRNA expression has no correlation with pathologic type, differentiation, and clinical stages (P > 0.05) [Table 1].
|Figure 1: Spleen tyrosine kinase (Syk) mRNA expression in tumor tissue, adjacent lung tissue, and normal lung tissue. 1,2,3 = Adjacent lung tissue, 4,5,6 = normal lung tissue, 7,8,9 = tumor tissue|
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|Table 1: Spleen tyrosine kinase expression in different types and differentiation of lung cancer|
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CD34 immunostaining localized in microvascular endothelial cells. The shape of microvascular in tumor was irregular with uneven distribution; it was most densely tufted in foci edge. Microvessel lumen was relative rule near tumor area, but unevenly distributed. Less microvessel were at cutting edge [Figure 2], [Figure 3], [Figure 4]. CD34 expression was statistically different between tumor tissue and normal lung tissue, and MVD was related to the degree of differentiation. With Spearman's correlation analysis, there was a significant negative correlation between Syk mRNA expression and MVD expression (r = −1.000) [Table 2] and [Figure 5].
|Figure 2: CD34 immunostaining locates in microvascular endothelial cells with brown granular in tumor tissue SABC × 200|
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| > Discussion|| |
Syk, a nonreceptor tyrosine protein kinase, is considered as an inhibition of tumor growth recently. It is characterized by two homology ribbon Src (SH2) in multiple autophosohorylation site, and Syk can be activated by binding nonreceptor tyrosine special sequence (ITAM) in SH2 domain. Lack of Syk gene can result in disorder of the immune cell development, maturation, and even severe immunodeficiency disease. Mutation and abnormal proliferation cells are prone to immune escape and lead to tumorigenesis. Coopman et al., thought that Syk can effectively regulate the growth of epithelial cells and is a potential inhibitory factor in growth of malignant breast cancer and distant metastasis. Recent studies also showed that Syk gene promoter methylation is the cause of Syk gene inactivation, and Syk plays an important role in incidence and metastasis of breast cancer., In our study, we found very low Syk mRNA expression in primary lung cancer cells. These results are similar to previous reports about Syk expression in other types of malignant tumors. However, unlike those reported in previous studies, the Syk expression rates did not reveal any statistically significant differences among patients of different pathologic types, differentiation, and clinical stages. We also noticed that the positive rate of Syk expression in our study was much lower than those reported in previous literatures. This may be explained by the following factors: Small sample size, experimental techniques, and very low expression in lung cancer cell; and large sample volume and other experimental methods are needed for further study.
On the other hand, vascular endothelial precursor cells are from hematopoietic stem cells. Endothelial progenitor cells circulate in the blood, implant in the vessel to generate the active region, and then differentiate into endothelial cell. Carter  thought that Syk and HER2/neu gene have opposite function, over expression of HER2/neu can induce contraction of vascular endothelial cells which help tumor cells through the blood vessel barrier and metastasis. Syk can inhibit cell contraction action of vascular endothelium, and thereby inhibiting tumor metastasis. MVD expression was higher in tumor tissue than that in adjacent tissue and normal lung tissue. We counted MVD based on the positive rate of CD34. CD34 is mainly expressed in endothelial cells, which is most sensitive and specific among many vascular endothelial cell marker and can be observed easily. There was a significant negative correlation between Syk mRNA expression and MVD. Therefore, Syk may inhibit tumor growth by influencing angiogenesis as a tumor suppressor gene from the experimental results.
Distant metastasis can occur in early stage of lung cancer by blood transfer, angiogenesis plays a crucial role, so it is important to find a new way to block angiogenesis. Lung cancer is a malignant entity with rich vascular; upregulating Syk expression in lung cancer cells may block angiogenesis and distant metastasis. However, Syk's role in maintaining the integrity of the blood vessels is only one of many mechanisms. We will have a comprehensive understanding on tumor angiogenesis with time and deep research, and the following issues should be addressed to reveal the molecular mechanism of Syk's tumor suppressive function: Regulation mechanisms in the tumor, Syk subtypers detection and functional studies, relationship between transduction network and tumor, Syk-specific positioning, and functional differences in tumor.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]