Expression And Functional Significance Of Semaphorin4D In Non-small Lung Cancer:Its Role In Vasculogenic Mimicry,Migration And Invasion

Part I Expression and clinical significance of Semaphorin4D and vasculogenic mimicry in non-small lung cancerObjective:To detect the expression and clinical significance of Semaphorin4D (Sema4D) and vasculogenic mimicry (VM) in non-small lung cancer.Methods:The expression of Sema4in60non-small lung cancer and10lung benign disease cases were detected by SABC immunohistochemistry using monoclonal antibody of Sema4D. PAS staining and immunohistochemistry of CD31were adopted to detect the expression of VM.Results:Sema4D protein is mainly expressed in the cell cytoplasm. The expression of Sema4D was detected in65%of60NSCLC, while the expression positive rate of lung benign disease was30%, Sema4D expression in NSCLC is more than that in lung benign disease group (P<0.05).VM was absent in lung benign disease tissue, while it was detected in NSCLC (P<0.05). There was a positive relationship between the expression of VM and Sema4D (r=0.322, P<0.05).VM and Sema4D were significantly related to grading, clinical TNM staging and lymphatic metastasis (P<0.05).Conclusion:The level of VM and Sema4D is correlated to poor differentiation, TNM staging and lymphatic metastasis in NSCLC. VM and/or Sema4D can be used as a judgment of prognosis of NSCLC. Part Ⅱ Lentivirus-mediated siRNA targeting Semaphorin4D gene silence its expression in non-small lung cancer cell linesObjective:To silence Semaphorin4D (Sema4D) in lung cancer cell lines (A549, H358, H460and H1975) using small interfering RNA (siRNA) mediated by lentivirus.Methods:Constructed lentivirus vector carrying designed siRNA sequences for Sema4D and identified by genetic testing. Sema4D-siRNA expression vector mediated by lentivirus was transfected into4kinds of lung cancer cells (A549, H358, H460and H1975). The change of expression of Sema4D mRNA and protein were tested respectively by real-time PCR (RT-PCR) and Western blotting.Results:Lentivirus vector carrying designed siRNA sequences for Sema4D has been successfully constructed and transfected in A549, H358, H460and H1975. The expression level of Sema4D mRNA and protein were down-regulated significantly after tansfection of Sema4D-siRNA (P<0.05).Conclusion:Sema4D-siRNA expression vector effective inhibited the expression of STAT1in lung cancer cell H358, A549, H460and H1975, laying the foundation for subsequent experiments. Part III Effects on vasculogenic mimicry, migration and invasion of Lung cancer cell lines by RNA interference targeting Semaphorin4D geneObjective:To explore the effects on vasculogenic mimicry, migration and invasion of Lung cancer cell lines by silencing Sema4D. Methods:3D culture system was used to determine any VM formation in vitro in A549, H358, H460and H1975cell line. Sema4D-siRNA was used to explore the association of Sema4D with VM formation in vitro. Transwell chamber was used to assess the effect of the Sema4D-siRNA on the migration and invasion of lung cancer cell lines.Results:Tunnel-like pattern formation was observed in3D culture system, compared to a scattered pattern in conventional culture system in all4kinds of lung cancer cell lines. The VM area and lenth of RNAi group is decreased compared with the CON group and NCgroup (P<0.05). In migration and invasion assay, the number of penetrating cells in RNAi group is more than control groups (P<0.05). There was no significant difference between CON group and NC group (P>0.05).Conclusion:siRNA targeting Sema4D can inhibit the formation of VM and the ability of migration and invasion in lung cancer cell lines. Sema4D plays a promoting role in lung cancer. So, Sema4D can be served as a potential therapeutic target.