The Role Of Fascin-1 In The Migration, Invasion And Proliferation Of Non Small Cell Lung Cancer

AimsLung cancer is the leading cancer-related cause of morbidity and mortalityworldwide. Cancer metastasis and recurrence account for the lower survival ofpatients with lung cancer. Although the actin binding protein, Fascin-1, is upregulatedin several types of tumors and associated with tumor metastasis, therole of Fascin-1 in lung cancer is still unclear. The aim of this study was toexplore the role of Fascin-1 in the migration, invasion and proliferation of nonsmall cell lung cancer (NSCLC), which may provide a new target and open up anew way for the prevention and treatment of lung cancer metastasis.Methods(1) Smaples of 98 patients with NSCLC including cancer tissues and nornallung tissues, and clinical pathological data were collected. Immunohistochemicalstaining was used to detect the expressions of Fascin-1 and Ki-67 in NSCLC andnornal lung tissues. The relationship between the experssion of Fascin-1 inNSCLC and clinicopathological characteristics including age, gender, histologicaltype, TNM staging, regional lymph node metastasis and Ki-67 labelling indexwere analyzed. (2) The Fascin-1 gene was coloned and the eukaryotic expression vector,pcDNA3.1(+)-fascin-1, was constructed. The Fascin-1 stable over expressedcolons (A549-fascin-1-2 and A549-fascin-1-5) were established by Fascin-1transfection using liposome. The morphological changes of the stabletransfectants were examined by scanning electron microscopy (SEM), andimmunofluorescence and confocal microscopy.(3) In order to further study the role of Fascin-1 in the migration, invasionand proliferation of A549 lung cancer cells:①the scartch-wound assay and thecell migration assay were performed to test the role of Fascin-1 in the migrationof A549 cells in vitro;②the matrigel cell invasion assay was performed to testthe role of Fascin-1 in the invasion of A549 cells in vitro;③the experimentalmetastasis assay was performed to test the role of Fascin-1 in the metastasis ofA549 cells in vivo;④the MTT assay and cell cycel analysis were performed totest the role of Fascin-1 in the proliferation of A549 cells in vitro;⑤thetumorigenicity assay and immunohistochemical staining were performed to testthe role of Fascin-1 in the proliferation of A549 cells in vivo.Results(1) Fascin-1 protein exhibited and located in the cytoplasm and cellularmembrane of positive cells diffusely or locally by immunostaining with a rate of83.67％(82/98) in NSCLC, which was largely confined to the lung cancer cells,the endothelial cells of the microvessels and dentritic cells, but in the normal lung,the non-neoplastic bronchial and alveolar epithelia cells were non-reactive forFascin-1. There was no difference between gender, age and histological subtypesfor the expression of Fascin-1(P＞0.05). However, the expresssion of Fascin-1was significantly correlated with TNM staging and regional lymph nodemetastasis(P＜0.05). The rates of Fascin-1 expression of NSCLC were 50.00%(7/14)in stage I, 82.05%(32/39)in stage II and 95.56%(43/45)in stageIII; and were 68.97%(20/29)in N0, 87.50%(35/40)in N1 and 93.10%(27/29)inN2. The cases were divided into low proliferation group(Ki-67 labellingindex≤10%) and high proliferation group(Ki-67 labelling index>10%) accordingto the Ki-67 labelling index. There was no statistical difference for Fascin-1expression between groups with different proliferation(85.71% in lowproliferation group and 83.12% in high proliferation group). These resultsindicated that the expression of Fascin-1in NSCLC was associated with TNMstaging and regional lymph node metastasis, but not with gender, age, histologicalsubtypes and proliferation.(2) The Fascin-1 gene was coloned by using mRNA templates of Hela cells.A pcDNA3.1(+)-fascin-1 expressive plasmid was constructed and transfected intoA549 cells by using liposome. A series of stable clones were isolated. RT-PCRand Western blot showed that clone 2 and 5 had middle and high level expressionof Fascin-1. In order to ensure reliability and repeatability, these two clones wereused for further in confirmation of study. The SEM showed that there were muchthinner and longer progections at the edge of A549 cells overexpressiong Fascin-1, which indicated that Fascin-1 could promote the formation of filopodia. Toobserve the influence of Fascin-1 on F-actin in A549 cells, the cells were stainedwith FITC-phalloidin. The immunofluorescent staining showed that there were alot of F-actin protrusions formed in the A549-fascin-1-2 and A549-fascin-1-5cells with polygons, which indicated that Fascin-1 may promote the formation ofprotrusions by modutating the F-actin.(3) To have an insight of the effect of Fascin1 overexpression in A549 lungcanccer cell migration and invasion in vitro, the scratch-wound assay, the cellmigration assay and the matrigel cell invasion assay were performed. Compared with A549 and A549-control cells, the migation and invasion of the A549-fascin-1-2 and A549-fascin-1-5 cells were significantly improved(P＜0.05). The resultsof in vivo exprerimental metastasis assay showed that there were more metastasisnodles formed in lung for A549-fascin-1-5 cells than that of A549-control cells.All these results indicated that Fascin-1 overexpression could promote A549 cellsmigration and invasion both in vitro and in vivo. An MTT assay was performed todraw the cell growth curves, and the cell cycle distribution was analyzed by flowcytometry. The results showed that there was no significant difference amongfour groups. An in vivo tumorigenicity assay was further performed to evalute theeffect of Fascin-1 on lung cells proliferation. The results showed that Fascin-1overexpression in A549 cells had little effect on tumor growth(P＞0.05).Conclusions(1) In NSCLC tissures, the expression of Fascin-1 was upregulated. Theexpression of Fascin-1 was significantly associated with TNM staging andregional lymoh nodes metastasis, but not with age, gender, histological subtyoesand proliferation.(2) The Fascin-1 gene was successfully coloned and the eukaryoticexpression vector, pcDNA3.1(+)-fascin-1, was constructed. A549 colons stablyoverexpression of Fascin-1(A549-fascin-1-2 and A549-fascin-1-5) wereestablished. The results of scanning electron microscopy (SEM), andimmunofluorescence and confocal microscopy showed that overexpression ofFascin-1 could promote the formation of filopodias in A549 cells.(3) Up-regulation of Fascin-1 in A549 lung cancer cells could promote themiration motility and invasiveness, but had little influence on the proliferation ofA549 cells neither in vitro nor in vivo. Fascin-1 may be a specific gene which canpromote lung cancer metastasis. In this study, the expression of Fascin-1 in lung cancer tissures and the role ofFascin-1 in the migration, invasion and proliferation of A549 lung cancer cellswere systematically invastigated. It was proved that Fascin-1 could promote lungcancer cells migration and invasion by promoting the formation of filopodias,which provided a new target and opened up a new way for the prevention andtreatment of lung cancer metastasis.