The Effect Of Syk On Metastasis Of Breast Cancer

ObjectiveIn breast cancer ,lymphatic metastasis is a frequent early event. Lymphangiogenesis plays a considerable role in lymphatic metastasis of tumor,so we could study the mechanism of lymphangiogenesis to suppress lymphatic metastasis of tumor.Recently,it was found that Syk closely correlated with lymphangiogenesis in mouse embryos.But the problem that if Syk also correlated with lymphangiogenesis and lymph node metastasis of tumor still remains unknow.First,we want to initially analysis the relationship between Syk and lymph node metastasis of breast cancer,as well as the dependablity between Syk,VEGF-C and NFκB ( p65 ) by immunohistochemistry method; then make Syk express in MDA-MB-231 cells to observe the effect of Syk on activity of NFκB and expression of VEGF-C.We intend to investigate the effect of Syk on metastasis of breast cancer and its mechanism.Methods1. Immunohistochemistry method was used to detect the expression of Syk , NFκB(p65) and VEGF-C in 55 cases of breast cancer tissue. The relationships between protein expression of Syk , NFκB(p65),VEGF-C and patient age,tumor histological type , lymph node metastasis,as well as dependablity between Syk , NFκB(p65)and VEGF-C were analysesed respectively.2. Total RNA was isolated from human breast cancer cells MCF-7, Syk gene encoding fragment was amplified by RT-PCR and T clone, and then was cloned into the eukaryotic expression vector pcDNA3.1(-) to obtain recombinant plasmid pcDNA3.1(-)-Syk. XholⅠand BamHⅠdigestion , DNA sequencing were adopted to identify this recombinant plasmid.3. Recombinant plasmid was transiently transfected into human breast cancer cells MDA-MB-231 lacking Syk by using lipoplast protocols. 48h later, the expression of Syk in MDA-MB-231 cells was detected by RT-PCR and Western blot methods.Then,expression of VEGF-C were detected by real-time quantitation PCR and Western blot methods.And the activity of NFκB was detected by ELISA method.Results1. The positive rates of Syk,VEGF-C and NFκB(p65)were 50.9%,56.4% and 81.8% ,respectively, in 55 cases of breast cancer tissue. The positive rate of Syk in lymph-node metastasis group ( 30.8% ) was significantly lower than that in non-lymph-node metastasis group(69.0%)(P<0.05); the positive rate of VEGF-C in lymph-node metastasis group ( 84.6% ) was significantly higher than that in non-lymph-node metastasis group(31.0%)(P<0.05);but no significant differences was found in p65(P >0.05). The positive rate of p65 nuclear expression in lymph-node metastasis group(57.7%)was significantly higher than that in non-lymph-node metastasis group(27.6%) (P<0.05).The expression of Syk was negatively correlated with VEGF-C (r=-0.620,P=0.000);The nuclear expression of p65 was associated with the low expression of Syk(r=-0.448,P=0.002)and the high expression of VEGF-C (r =0.310,P =0.036).2. The double digestion demonstrated that the length and direction of inserted sequence were exactly correct. DNA sequencing demonstrated that the accuracy of inserted sequence was 99.7%,and the changes in which have no influence on Syk protein.3. Syk expressed in the cells transfected by pcDNA3.1(-)-Syk,but did not express in negative and control group.4. The expression of VEGF-C mRNA and protein were decreased significantly in the cells transfected by pcDNA3.1(-)-Syk compared with the control group(P<0.05),while the negative and control groups showed no significant differences(P>0.05). 5. The activity of NFκB p65 was decreased significantly in the cells transfected by pcDNA3.1(-)-Syk compared with the control group(P<0.05),while the negative and control groups showed no significant differences(P>0.05).Conclusions1. By immunohistochemistry method we initially conjectured that Syk may down regulate the expression of VEGF-C by inhibiting activity of NFκB in breast cancer to suppress lymph node metastasis .2. The recombinant plasmid pcDNA3.1(-)-Syk expressing Syk has been constructed successfully,and been proved to effectively express Syk mRNA and protein in transfected MDA-MB-231 cells.3. Our study demonstrates that after Syk expressing in MDA-MB-231 cells ,the activity of NFκB and the expression of VEGF-C decrease.It suggests that Syk down regulate by inhibiting the activity of NFκB, to suppress lymph node metastasis of breast cancer.