TLR4-MD-2 complex plays a key role in LPS recognition and its signal transduction. These steps are the vital elements of the host's defensive reaction. Studying the functional domain of TLR4 and MD-2 is very important to further understand the mechanism of LPS signal transduction. It was studied the interaction domain of TLR4 and MD-2 in living cells based on gene mutation, gene transfection and fluorescence resonance energy tramsfer(FRET) which is considered as one of the best methods used for intracellular protein-protein interaction study. CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control, while co-expressed CFP and YFP proteins were used as negative control. The results showed that the ability of TLR4 binding to MD-2 decreased dramatically after the deletion of Glu~(24) ～ Met~(41) in N terminal of TLR4. Aggregation of TLR4 to LPS stimulation was observed, however, TLR4 without the Glu~(24)～ Met~(41) mutation did not aggregate. All these results indicated that TLR4 Glu~(24)～ Met~(41) might be the interaction domain of TLR4 binding to MD-2 and participate in the aggregation effect of TLR4 upon LPS stimulation.

OBJECTIVETo investigate the effect and mechanism of B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine on pulmonary metastasis in mouse model of melanoma.

METHODSTwelve 8-week old female C57BL/6 mice were used in this study. The mice were injected with wild-type B16F10 cells through tail vein after immunization with B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine, and the pulmonary metastasis was observed. The CD4(+) and CD8(+) T cells were isolated by magnetic activated cell sorting, and then used for the detection of CFSE/7-AAD cytotoxicity by flow cytometry. Serum from the mice immunized with tumor-cell vaccine was used to detect IFN-γ expression by ELISA. The expression of TGF-β2, ZEB1, E-cadherin, and N-cadherin of tumor tissues was detected by RT-PCR and immunofluorescence, respectively.

RESULTSThe mice vaccinated with B16F10-ESAT-6-gpi/IL-21 had significantly fewer nodules in the lung and lower lung weight [(285.8 ± 19.01) mg vs. (406.3 ± 27.12) mg], with lower levels of TGF-β2, ZEB1 and N-cadherin proteins but higher level of E-cadherin protein within the tumor tissue, as compared with the control mice. Meanwhile, the immunized mice had significantly increased CD8(+) T cell killing activity [(42.62 ± 3.465)% vs. (22.29 ± 1.804)%] and IFN-γ expression level [(55.200 ± 7.173) pg/ml vs. (6.435 ± 1.339) pg/ml] over the control mice.

CONCLUSIONSThe B16F10-ESAT-6-gpi/IL-21 vaccine can inhibit the metastasis of melanoma in the lung in vaccinated melanoma-bearing mice. This inhibitory effect is associated with CD8(+) T cell immune response and a higher level of IFN-γ, which may influence on the mesenchymal-epithelial transition of tumor cells.

Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cadherins ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Female ; Homeodomain Proteins ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukins ; immunology ; Lung ; pathology ; Lung Neoplasms ; metabolism ; secondary ; Melanoma ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Organ Size ; Transcription Factors ; metabolism ; Transforming Growth Factor beta2 ; metabolism ; Zinc Finger E-box-Binding Homeobox 1

Objective To explore the expression of serine proteinase inhibitor family E member 1(SERPINE1)in colon cancer and its effect on the malignant biological behaviors of colon cancer cells.Methods starBase and TISIDB databases were used to analyze the expression of SERPINE1 in 471 patients with colon cancer and 41 paracancerous tissues,and the relationships of its differential expression with clinical stage and prognostic survival were analyzed.The cancer specimens and paracancerous tissues from 5 patients with colon cancer were collected in No.940 Hospital of Joint Logistic Support Force,and RT-qPCR and Western blotting were employed to detect the expression of SERPINE1 at mRNA and protein levels.After lentiviral vectors of SERPINE1 overexpression and interference were constructed and transfected into colon cancer RKO and LoVo cells.The experimental cells were assessed by RT-qPCR and Western blotting to verify the efficiency of overexpression and interference.Then cell proliferation,migration,invasion and apoptosis were evaluated by CCK-8 assay,clone formation test,Transwell test and Hoechst apoptotic nuclear staining,respectviely.Finally,Western blotting was applied to determine the effect of SERPINE1 on phosphatidyl-inositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway.Results Database analysis showed that SERPINE1 was highly expressed,positively correlated with the clinical stage and negatively correlated with the overall survival in colon cancer patients(P＜0.05).Transfection of overexpression lentiviral vector resulted in enhanced cell proliferation,invasion and migration abilities while weakened apoptosis in RKO and LoVo cells.On the contrary,SERPINE1 interference reduced the abilities of cell proliferation,invasion and migration but improved cell apoptosis(P＜0.05).Western blotting showed that overexpression of SERPINE1 had no effect on the expression of PI3K and AKT,but increased the expression of p-PI3K and p-AKT,and SERPINE1 interference also had no change in PI3K and AKT expression and reduced the levels of their phosphorylation levels.Statistical differences were observed in relative quantitative analysis on the ratios of p-PI3K/PI3K and p-AKT/AKT(P＜0.05).Conclusion SERPINE1 is highly expressed,and correlated with clinical stage and prognosis of patients with colon cancer.Its overexpression promotes the proliferation,migration,invasion and inhibits apoptosis of colon cancer RKO and LoVo cells through PI3K/AKT signaling pathway.