| Stromal cell-derived factor-1(SDF-1) also known as CXCL12,belongs to CXC chemokine subfamily,it is a chemotactic protein secreted by bone marrow cells and other epithelial and stromal cells.With the binding of the receptor CXCR4,it plays a critical role in many physiological and pathological processes, such as embryonic development, angiogenesis, hematopoiesis, stem cell migration, inflammation and tumor metastasis. This experiment detects the expression of CXCR4 on small cell lung cancer cell line NCI-H446 and focuses on the effects of SDF-1 on proliferation, invasion, migration and secretion of VEGF and MMP-9,as well as to explore the mechanisms and signaling pathway of the SDF-1/CXCR4 axis in small cell lung cancer invasion and migration by the application of specific CXCR4 inhibitor, PI3K inhibitor and siRNA technology, which may contribute to the immunotherapy of small cell lung cancer.Part one Research the ability of SDF-1/CXCR4 axis to proliferation, adhesion and invasion of small lung cancer cellObjective: To investigate the effect on tumor invasion and the role of PI3K signaling pathway after the binding of SDF-1 and CXCR4 receptor, which is fundamental to the molecular-targeted therapy of small cell lung cancer.Methods: Flow cytometry (FCM) and RT-PCR were utilized to detect the expression of CXCR4 in NCI-H446. CCK-8 assay was utilized to detect the proliferation of tumor cells treated by SDF-1,CXCR4 antagonist AMD3100 and the PI3K inhibitor LY294002. In order to observe the ability of adhesion and invasion, there were five groups in this study: control group; SDF-1 50ng/ml group;SDF-1 100ng/ml group;SDF-1(100ng/ml)+AMD 3100 group;SDF-1(100ng/ml)+LY294002 group. Chemotaxis and transwell invasion experiments were performed in different groups to observe the changes of adhesion and invasion ability.Results: FCM analysis showed that the NCI-H446 express high level of CXCR4(91.6±2.1%).Compared with the control group,SDF-1,AMD3100 did not affect the proliferation of NCI-H446,LY294002(20μmol/L) could inhibit the proliferation of NCI-H446, 24h, 48h, 72h inhibition rates were 19.4%, 31.3%, 18.4%. Compared with the normal control group,100ng/ml of SDF-1 increased the adhesion ability of NCI-H446,OD values were 1.253±0.107 VS 0.783±0.071(P<0.05); it also increased invasion capacity, the penetrating number was 83.2±15.7 VS 30.8±6.5(P <0.001). Compared with 100ng/ml of SDF-1 treatment group, AMD3100 and LY294002 could inhibit the adhesion and invasion, OD values were 0.759±0.088 VS 1.253±0.107,0.652±0.076 VS 1.253±0.107 (P<0.05 ), the penetrating number were 37.8±5.1 VS 83.2±15.7,37.6±7.2 VS 83.2±15.7 (P <0.001).Conclusion: Small cell lung cancer cell line NCI-H446 expressed high level of the chemokine CXCR4, its ligand SDF-1 could promote the adhesion and invasion of the tumor cells. PI3K signaling pathway involved in the proliferation works in the adhesion and invasion of NCI-H446 by CXCR4 activation.Part two The effect of SDF-1/CXCR4 on expression of VEGF and MMP-9 in small cell lung cancerObjective: To explore the effect of SDF-1, AMD3100, and LY294002 treatment on expression of VEGF and MMP-9 in NCI-H446 cell, and to research the mechanism of small cell lung cancer invasion by SDF-1.Methods: There were 5 groups in the experiment:control group (SDF-1 negative group); SDF-1 50ng/ml group; SDF-1 100ng/ml group; SDF-1 (100ng/ml) + AMD3100 group; SDF-1 (100ng/ml) + LY294002 group.After serum-free medium cultivation for 24 hours,NCI-H446 cells were detected to observe the expression VEGF and MMP-9 by RT-PCR and ELISA. Results: There were expressions of VEGF and MMP-9 gene by RT-PCR, VEGF and MMP-9 expression in supernatants were increased in SDF-1 treatment group and could be inhibited by AMD3100 and LY294002. Compared with the control group, in 100ng/ml SDF-1 treatment group, the concentration of VEGF and MMP-9 were significantly increased, [(826±102) pg / ml VS (360±21) pg / ml], [(105±4) pg / ml VS (30±9) pg / ml] (P <0.05); Compared with the 100ng/ml SDF-1 treatment group, SDF-1 (100ng/ml) + AMD3100 treatment, SDF-1 (100ng/ml) + LY294002 treatment group decreased VEGF concentration, [(224±55) pg / ml VS (826±102) pg / ml], [(379±203) pg / ml VS (826±102) pg / ml] (P <0.05); MMP-9 levels also decreased, [(31±2) pg / ml VS (105±4) pg / ml], [(25±4) pg / ml VS (105±4 ) pg / ml] (P <0.05).Conclusion: After the stimulation With SDF-1, the expression of VEGF and MMP-9 was significantly increased, and could be inhibited by AMD3100 and LY294002, which suggests that SDF-1/CXCR4 participate in tumor invasiveness and metastasis in small lung cancer by promoting the secretion of VEGF and MMP-9.Objective: To study the inhibitory effect of CXCR4-targeted small interference RNA on invasion capability of NCI-H446 in vitro.Methods: To design chemical synthesis of CXCR4-specific siRNA based on the target sequence for CXCR4cDNA ,then NCI-H446 cells were transfected with siRNA.To detect the expression of CXCR4 by using the method of RT-PCR and"Western Blotting". Invasion capability of NCI-H446 cells in vitro was evaluated by using transwell chamber model, and the proliferation capability was determined by CCK-8 assay. Results: After being transfected with CXCR4-siRNA, the expression of CXCR4 mRNA and protein was down-regulated significantly. The capability of cells to invade in vitro decreased, compared with the empty liposome group, the penetrating number in CXCR4-siRNA transfected group was 32.3±3.8 VS 62.1±8.2(P<0.001).There was no effect on cell proliferation after transfection of CXCR4 siRNA on NCI-H446 cells.Conclusion: CXCR4 siRNA effectively down-regulates the expression of CXCR4 gene and decreases invasion capability of NCI-H446 cells in vitro. |
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