A Experimental Study Of The Relationship Between Expression Of Syk And STAT3 Signaling Pathway In Breast Cancer Cells Line

Objective: Quercetin (Que) can be induced cancer cells oxidatie stress reaction. Detection in oxidatie stress condition, Que can be induced induced the human breast cancer cell lines that MDA-MB-231 and MDA-MB-231/Syk cell apoptosis and the relationship between expression of Syk and STAT3 genes.Design and construct the eukaryotic expression vector of human STAT3 gene short hairpin RNA (shRNA), then transfect to human breast cancer cell MDA-MB-231 and MDA-MB-231/Syk. Meanwhile, combined Que intervention, through in vitro experiments, the relationship between expression of Syk and STAT3 in the breast cancer cell line of MDA-MB-231 and MDA-MB-231/Syk was investigate. The mechanism of STAT3 signaling pathway in the breast cancer line was studied and to provided basis for clinical application.Methods:1 Expression of syk in breast cancer cell line of MDA-MB-231/Syk were detected by using RT-PCR and Western blot methods.2 Expression of STAT3 gene after treatment with quercetin (20μmol/L) for 48h were examined by RT-PCR. Expressions of pSTAT3 were assessed by immunohistochemistry.3 Applying RNAi technique to specifically silence STAT3 gene in MDA-MB-231 and MDA-MB-231/Syk cells, expressions of STAT3 in breast cancer cells were detected by using RT-PCR and Western blot methods. The green fluorescence cells and the transfection efficiency were observed by fluorescence microscope for 48 hours. The morphologic changes of breast cancer cells were observed by Wright and Giemse's staining for 48 hours.4 Effect of RNAi on apoptosis of MDA-MB-231 and MDA-MB-231 /Syk cells was determined by flow cytometry. Expression of apoptosis- associated genes Bax and bcl-2 was determined by semi-quantitative RT-PCR.5 Inhibitory effects of RNAi STAT3 in different concentrations and different time on MDA-MB-231 and MDA-MB-231/Syk proliferation were detected by MTT.6 Effects of RNAi STAT3 for 48 h on the invasion and metastasis of MDA-MB-231 and MDA-MB-231/Syk cells were detected by scratch. The transcriptional levels of matrix metalloproteinases MMP-9 and MMP-2 in MDA-MB-231 and MDA-MB-231/Syk cells treated with RNAi STAT3 for 48 h were detected by RT-PCR.7 Effects of Que and STAT3 RNAi on apoptosis in MDA-MB-231 and MDA-MB-231/Syk cellsFive experimental groups were divided.A group: the blank control group, not taking any intervention, add the RPMI1640 medium without bovine serum in the experiments;B group: Negative plasmid control group, transfected by negative control plasmid into MDA-MB-231 and MDA-MB-231/Syk cells;C group: RNAi group, transfected by STAT3-1and S-STAT3-1 plasmid into MDA-MB-231 and MDA-MB-231/Syk cells;D group: Que group, add Que, its final concentration of 20μmol/L (this concentration is close to IC₅₀ in pre-experiment);E group: RNAi combining Que group, transfected by STAT3-1 and S-STAT3-1 plasmid into MDA-MB-231 and MDA-MB-231/Syk cells, at the same five experimental groups time, add Que, its final concentration of 20μmol/L.After transfection for 48h, five experimental groups, cell proliferation activity was assayed by MTT; tumor cell apoptosis rate were detected by flow cytometry. Results:1 After treatment with quercetin(20μmol/L)for 48h, expression level of STAT3 gene in MDA-MB-231 cell were decreased significantly (P<0.05).But there was no statistical significance in MDA-MB-231/Syk cell after treatment with quercetin(20μmol/L) for 48h (P>0.05).2 The RNAi technology targeted to STAT3 can effectively silence STAT3 expression in MDA-MB-231and MDA-MB-231/Syk cells (P<0.05).3 RNAi targeted to STAT3 can induce apoptosis and down-regulate expression of bcl-2 mRNA and up-regulate expression of Bax mRNA (P<0.05).4 Proliferation of MDA-MB-231and MDA-MB-231/Syk cells were significantly inhibited after eukaryotic expression vectors of STAT3 shRNA for 24, 48 and 72 hour, especially for 48 and 72 hour (P<0.05).5 After transfected STAT3 siRNA 48 hour in MDA-MB-231and MDA-MB-231/Syk cells, the diameters of scratch became broaden than the control group and could down-regulated the expression levels of MMP-9 and MMP-2 mRNAs (P<0.05).There was significant difference between MDA-MB-231 cell group and MDA-MB-231/Syk cell group, the expressions of MMP-9 and MMP-2 mRNA were obviously reduced in MDA-MB-231/Syk cell group (P<0.05).6 Proliferation of MDA-MB-231 cell in RNAi, Que, RNAi combining Que treated groups was inhibited to varying degrees in different time (24h, 48h, 72h). Proliferation inhibitory rates in RNAi, Que, RNAi combining Que groups increased with the extension of time and increased significantly compared to blank control group (P<0.05).There was no statistical significance between blank control group and NC group (P>0.05). Proliferation inhibitory rate in RNAi combining Que group increased significantly compared to RNAi group and Que group (P<0.05).The results suggested that RNAi can significantly enhance the inhibition of Que on cell proliferation, there was certain synergistic effect between the two treatment. MDA-MB-231 cell proliferation inhibition was more obvious if joint using the two methods.Proliferation of MDA-MB-231/Syk cell between RNAi and RNAi combining Que treated groups was inhibited to varying degrees in different time (24h, 48h, 72h). Proliferation inhibitory rates between RNAi and RNAi combining Que groups increased with the extension of time and increased significantly compared to blank control group (P<0.05). There was no statistical significance between blank control group and NC group (P>0.05), Que group significance difference compared to RNAi and RNAi combining Que groups (P<0.05). Proliferation inhibitory rate in RNAi combining Que group increased significantly compared to RNAi group(P<0.05). The results suggested that Que group can not inhibition of cell proliferation.7 The results of flow cytometry showed that the apoptosis rate of MDA-MB-231 cells in RNAi, Que, RNAi combining Que groups increased significantly compared to blank control group(P<0.05). The apoptosis rate was highest in Que combining RNAi group, reached (37.35±3.01)% from (4.10±1.51)%. There was no significant difference between blank control group group and NC group (P<0.05).The results of flow cytometry showed that the apoptosis rate of MDA-MB-231/Syk cells between RNAi and RNAi combining Que treated groups increased significantly compared to blank control group (P<0.05). The apoptosis rate was highest in Que combining RNAi group, reached (38.29±2.85)% from (4.10±1.51)%. There was no statistical significant between blank control group and NC group (P>0.05), Que group significance difference compared to RNAi and RNAi combining Que groups (P<0.05).Conclusions:1 The eukaryotic expression vectors of STAT3 shRNA could effectively inhibit the expression of STAT3 gene.2 The eukaryotic expression vectors of STAT3 shRNA could inhibit proliferation and invasion and metastasis, and induced the apoptosis of breast cancer cells.3 Quercetin can induce breast cancer cells oxidatie stress reaction, Syk plays an important and indispensable role in oxidative stress induced STAT3 activation. The treatment of RNAi combining Que has more significant inhibitory effect on STAT3 signaling pathway.