A Experimental Study Of The Relationship Between Expression Of Syk And Interleukin-2 Receptor Signaling Pathway In Breast Cancer Cell Line

Objective: To investigate the relationship between expres- stion of syk and interleukin-2 receptor in the breast cancer cell line of MDA-MB-231. In order to study the mechanism of the interleukin-2 receptor signaling pathway in the breast cancer line and advance to reveal the primal anti-cancer effects of syk.Methods:1 The expression of Syk and IL-2 receptor in the breast cancer cell line of MDA-MB-231 were detected by using RT-PCR and Western blot methods. The expression of endoge- nous IL-2 in the breast cancer cell line of MDA-MB-231 was detected by using RT-PCR and ELISA methods.2 Effects for different concentrations of recombinant human interleukin-2 on proliferation of MDA-MB-231 cells were analyzed by MTT assay. Cell cycle and apoptosis rate of MDA-MB-231 cells induced with recombinant human interleu- kin-2 were detected by flowcytometry.The MDA-MB-231 cells were effected by different concentrations of recombinant hum- n interleukin-2 for 48 hours, then we detected the expression of STAT5 by RT-PCR method.3 Eukaryotic expression vector pcDNA3.1D/V5-His-TO PO/syk was constructed. Recombinant plasmid was transfected into human breast cancer cells MDA-MB-231 by using lipofectamin protocols. MDA-MB-231 cells do not express Syk. After G418 selection, MDA-MB-231/syk cells stable express Syk. The expression of Syk on transfected cells was analyzed by Western blot and RT-PCR methods. The expression changes of interleukin-2 receptor on transfected cells were analyzed by Western blot and RT-PCR methods.The expression changes of STAT5 on untransfected and transfected were analyzed by RT-PCR method.4 Effects for different concentrations of recombinant human interleukin-2 on proliferation of untransfected and transfected cells was analyzed by MTT assay.5 The expression of syk was effected by silence the expression of interleukin-2 receptorβin transfected MDA-MB -231 cells.The expression of interleukin-2 receptorβin transfec- ted MDA-MB-231 cells was silenced by RNAi. The most effective plasmid expression vector of inhibiting interleukin-2 receptorβexpression was screened out by RT-PCR and Western blot methods and used for the follow-up experiments. The expression changes of syk on transfected cells of RNAi were analyzed by RT-PCR and Western blot methods.Results:1 Results of RT-PCR and Western blot showed that syk expression is negative in MDA-MB-231 cells, but the expressi- ons of interleukin-2 receptorβandγare positive. The expression level of interleukin-2 receptorβis lower than interleukin-2 receptorγ. Results of RT-PCR and Western blot showed that the expression of interleukin-2 receptorαis negative. The expression of endogenous IL-2 in the breast cancer cell line of MDA-MB-231 detected by using RT-PCR and ELISA is negative.2 By MTT analysis, it is shown that proliferation of MDA-MB-231 cells was significantly inhibited by recombinant human interleukin-2 at different concentrations. The disparatio- n is meanful compared with control group (P<0.05) and showing a dose-and-time dependent manner. Cell cycle and apoptosis rate of MDA-MB-231 cells induced with recombina- nt human interleukin-2 were detected by flowcytometry. After treatment with 100, 500, 2500u/ml human interleukin-2 for 48 h, the percentages of MDA-MB-231 cells at G0/G1 phase were increased(P<0.05), and those at s phase were decreased (P<0.05). Cell apoptosis was induced by a dose dependent manner of recombinant human interleukin-2. Cell apoptosis was increased by the concentration of rhIL-2. The disparation is meanful compared with control group (P<0.05). Results of RT-PCR showed that STAT5 expression is decreased by recombinant human interleukin-2.3 Stable express syk in MDA-MB-231/syk cells was detected by using RT-PCR and Western blot methods.The expression of IL-2Rβin transfected cells detected by using RT-PCR and Western blot methods was upregrated. The expres- sion of STAT5 in transfected cells detected by using RT-PCR methods was downergrated. The disparation is meanful(P<0.05).4 By MTT analysis, it is shown that proliferation of transfected cells was more significantly inhibited by recombina- nt human interleukin-2, compared with untransfected cells. The disparation is meanful(P<0.05).5 The results revealed that the expression of interleukin-2 receptorβwas silenced in transfected cells by RNAi. The expression of syk detected by usying Western blot and RT-PCR methods was downgrated by RNAi. The disparation is meanful(P<0.05).Conclusion:1 Syk expression in MDA-MB-231 cells is negative. The expression of interleukin-2 receptorβandγare positive. The exression level of interleukin-2 receptorβis lower than interle- ukin-2 receptorγ.2 The proliferation of MDA-MB-231 cells was inhibited by recombinant human interleukin-2. The expression of STAT5 was downregrated by recombinant human interleukin-2.3 The expression of interleukin-2 receptorβwas upregra- ted in transfected cells, the expression of STAT5 was downgra- ted.4 The proliferation of transfected cells was more signific- antly inhibited by recombinant human interleukin-2, compared with untransfected cells.5 The expression of syk was downgrated by silence the expression of interleukin-2 receptorβin transfected MDA-MB -231 cells. The results revealed the anti-tumor mechanism of syk maybe relative with the signaling pathway of interleukin-2 receptor.