Initial Study On IGFBP-2Regulating The Abilities Of Metastasis And Angiogenesis In Non-Small Cell Lung Cancer

Party I The study on clinical significance of the expression of IGFBP-2in non-small cell lung cancerObjective:To investigate the expression of IGFBP-2in non-small cell lung cancer and its clinical significance.Methods:The expression of IGFBP-2mRNA and protein were detected by RT-PCR and Western blot in33fresh cases Non-Small Cell Lung Cancer and pericarcinoma tissue; The expression of IGFBP-2protein and microvessel density (MVD)in110cases laryngeal carcinoma were detected by immunohistochemical methods.To research the expression of IGFBP-2relation to Non-Small Cell Lung Cancer metastasis eand prognosis, the combination of clinical pathology, clinical follow-up data and the expression of IGFBP-2protein and MVD were analyzed by the multi-factor Logistic regression analysis.Results:1. The expression of IGFBP-2mRNA positive rate of fresh Non-Small Cell Lung Cancer tissues (65%,13/20) is significantly higher than that of pericarcinoma tissue(30%,6/20)(x2=4.916,P=0.027).The expression level of IGFBP-2mRNA in fresh Non-Small Cell Lung Cancer tissues was significantly higher than pericarcinoma tissue (3.17±0.50vs0.96±0.10, P<0.01).The positive expression of IGFBP-2protein of fresh Non-Small Cell Lung Cancer tissues is different from peri-carcinoma tissue significantly(55%vs20%)(χ2=5.227, P=0.022). The expression level of IGFBP-2protein in fresh Non-Small Cell Lung Cancer tissues was significantly higher than pericarcinoma tissue (0.57±0.03vs0.23±0.04, P<0.01).2. In total110patients,57were found positive expression of IGFBP-2and53were negative via immunohistochemistry. The overall positive rate was51.8%. In positiveexpression patients, the incidence of metastasis was45.6%, while it was20.8%in patients with negative expression of IGFBP-2. In the metastatic group, the positive ratewas70.3%(26/37), which was higher than the positive rate42.5%(31/73) in the non-metastatic group. The differences between the two groups were significant(x2=7.60,P<0.01).3. The results of Kaplan-Meier model, the mean survival time of patients with IGFBP-2overexpression was62.99±6.10months, while it was75.60±4.57months of those withoutThe difference was statistically significant (χ2=8.64, P<0.01). Based on multi-variate Logistic regression analysis, it was demonstrated that IGFBP-2, MVD and TNM stage are strong predictors of metastasis and poor prognosis (the OR were3.558,3.428and2.137, respectively).Conclusion:1. IGFBP-2is not a good tumor marker for non-small cell lung cancer.2. IGFBP-2is associated with metastasis and poor prognosis in non-small cell lung cancer.3. IGFBP-2might be involved in the metastasis of non-small cell lung cancer by regulating the angiogenesis in non-small cell lung cancer Party II Study on the effect of biological behavior and VEGF expression of human lung cancer95D cell line interfered with siRNA target to IGFBP-2Objective:To investigate the mechanisms of tumor invasion, metastasis and angiogenesis, by observing the changes of biological behavior and VEGF expression of lung cancer95D cell with interfence targeting IGFBP-2silence.Methods:1. The siRNA specific for silencing IGFBP-2was synthesized chemically, using Lipofcctamine reagent lipofectamine2000transfectcd into95D cells. Fluorescent siRNA was transfected into95D cells, then fluoresccnce microscopy and MTT assay were used to detect the transfection efficiency and security. The silencing effects of IGFBP-2was analysed by RT-PCR and Western blot; The most efficient siRNA targeting IGFBP-2was chosen according to the results of the assays above mentioned.2. The expression of VEGF mRNA and protein in95D cells were detected using RT-PCR and Western blot after transfected by the most efficient siRNA targeting IGFBP-2.3. The abilities of95D cells migration, invation and proliferation transfected by the most efficient siRNA targeting IGFBP-2were evaluated with Cell scratch assay, Transwell chamber assay and MTT assay.Results:1.24h after IGFBP-2-siRNA transfected95D cells,the transfection efficiency were detected up to80%observed under the fluorescence microscope; The survival rate of95D cells were up to80%detected by MTT assay after72h. The expression of IGFBP-2mRNA in Group C, Group D and Group E are significantly lower than those of Group A and Group B(relative amount is3.32±1.05,1.18±0.31and4.14±1.27vs9.14±0.35and9.02±0.88). The relative amount expression of IGFBP-2protein in Group D (0.080±0.0070) is significantly lower among the Group A,Group B, Group C, Group D and Group E.So the most efficient siRNA targeting IGFBP-2was IGFBP-2-siRNA-homo-923.2.48h after95D cells were transfected by non-special siRNA (Group B) and IGFBP-2-siRNA-homo-923(Group C), the relative amount expression of VEGF mRNA in95D cells of the Group A(control group),Group B and Group C is4.26±0.95、4.23±1.05and1.09±0.10respectively. The VEGF mRNA expression in Group C is lowest among all groups(all P<0.01). The relative amount expression of VEGF protein in95D cells of the Group A,Group B and Group C is0.414±0.014.0.412±0.012and0.132±0.004respectively. The expression of VEGF protein in Group C is lowest among all groups(all P<0.01).3.24h after95D cells were transfected by non-special siRNA (Group B) and IGFBP-2-siRNA-homo-923(Group C), the migration indexes in95D cells of the Group C ((60.2±5.36)%)is less than G roup A(control group) and Group B ((86±1.58)%and (86.4±4.28)%, respectively)(all P<0.01). Perfield displayed the numbers of passed through the Magtrigel-coated membranes cells of the Group A and Group B were respectively42.00±4.47and42.80±5.47, whereas the Group C was20.20±4.44, and there was statistical significant differences (P<0.01);There was no significant difference of the proliferation in95D cells among all groups(P>0.05).Conclusion:1. The specific siRNA for silencing IGFBP-2synthesized chemically can reduce the expression of IGFBP-2mRNA and protein in95D cell significantly.2. In vitro, inhibition of the expression of IGFBP-2in95D cells might attenuate the expression of VEGF, migration and invasion of95D cells, but has no remarkable affection on the proliferation of95D cells.3. IGFBP-2might promote invasion and metastasis of non-small lung cancer by regulating the expression of VEGF to promote the angiogenesis.Party Ⅲ Study on the effect of human lung cancer95D cell lines to interfere with siRNA targetting to IGFBP-2of angiogenesis in vitroObjective:To investigate the mechanism of the expression of IGFBP-2in non-small lung cancer angiogenesis by interfering with targeting to IGFBP-2silence.Methods:HUVEC cells were co cultured with the conditioned medium of the human lung cancer cell95D transfected by IGFBP-2-siRNA-homo-923for24hours.the abilities of HUVEC migration, proliferation and differentiation into tubes were evaluated by scratch test, MTT method and Matrix assays test.Results:24h after co cultured with the conditioned medium of95D cells were transfected by non-special siRNA(Group B) and IGFBP-2-siRNA-homo-923(Group C), the migration indexes in HUVEC of the Group C((56.8±3.35)%) is less than G roup A(control group)() and Group B ((83±3.16)%and (84.2±8.17)%,respectively)(all P<0.01). Conditioned medium from95D cells with IGFBP-2silenced could significantly reduce the proliferation and capillary like tubules of HUVEC in the Group C among all groups (all P<0.01).Conclusion:1. In vitro, inhibition of the expression of IGFBP-2in95D cells might attenuate migration, proliferation and differentiation into tubes of HUVEC.2. IGFBP-2might become a new target for clinical therapy in the treatment of non-small cell lung cancer...