Study Of The Effects Of Fascin1 Gene Induced By TGF-β1 On Invasion And Metastasis Of Gastric Cancer Cells

Gastric carcinoma(GC) is a frequent malignant neoplasm.In China, the mortality of patients with GC is quite high.Invasion and metastasis is the major always responsible for the death of patients with GC. Therefore,further investigations on the mechanism of invasion and metastasis are of great importance.Transforming growth factor beta(TGF-β) has been reported to be expressed in many human tumors and is believed to play an important role in tumor invasion as well as tumor metastasis.Cancer cells overexpressing active TGF-β1 by gene transfection showed increased metastatic ability.Targeting of TGF-βsignaling can prevent metastasis in GC.Our previous study showed that TGF-β1 significantly promoted the invasiveness and metastasis of GC cell lines SGC7901 and BGC823. Similarly,clinical studies showed the positive correlation of TGF-β1 expression with lymph node metastasis and poor prognosis in gastric carcinoma.Although many efforts were made to clarify the role of TGF-βin the invasion and metastasis of neoplasms,the mechanism is not fully elucidated.Study indicated that TGFβ1 actives Smad-dependent and Smadindependent pathways.TGFβ1 combines with TGFβtypeⅡreceptor (TβR-Ⅱ) and then actives TβR-Ⅰ.Smad2 and Smad3 are phosphorylated by TβR-Ⅰand then form the heteromeric complexes by associating with Smad4.These Smad complexes move into the nucleus and regulate expression of their target genes.In Smad-independent pathway,JNK,Erk and P38 have been identified as key signaling processes in response to TGF-β.Fascin1,a 55-kDa globular protein,belongs to a unique family of actin-binding proteins.Fascin1 locates in structures containing actin bundles including filopodia,stress fibers,cell membrane ruffles and microspikes.Studies reported cells expressing high levels of fascin showed an increasing activity of membrane extensions,cell motility and loss of cell-cell adhesion.In human colorectal carcinoma cell SW1222, up-regulation of fascin gene expression contributed to the formation of cell-motility structure and the increased migration ability.In normal gastric mucosa,fascin expression is usually undetectable but is often dramatically up-regulated in gastric carcinoma.Moreover increased expression of fascin was found positively correlated with invasion, metastasis and prognosis in GC.Recently,study showed that TGF-β1 up-regulated fascin1 expression in lung cancer.So,we postulate that TGF-β1 can induce the expression of fascin1 and then promote the invasion and metastasis of GC cells.In recent years,RNA interference(RNAi) has emerged as a powerful method of gene therapy,and has been widely used for silencing malignant cellular and viral genes.In this study,we employed RNAi-mediated suppression of Fascin1 gene to evaluate its effects on invasion and metastasis of GC cells and investigate its preliminary mechanisms.ObjectiveOur current investigation attempts to study the effects and mechanisms of TGF-β1-induced fascin1 expression on invasion and metastasis of GC cells,and to provide theoretical and experimental evidences for the gene therapy of GC through specifically turning off Fascin1 gene.MethodsA single strand DNA was synthesized according to the hair loop RNA sequence,and subcloned into eukaryotic expression vector pGenesil-1 to construct a shRNA-expression pDNAs driven by human U6 promoter of fascin1(pGen-1-FSCN1),then transformed into DH5αcompetent cells.One additional construct of random siRNA (pGen-1-con) not homologous to any human genes was made in the same way as control.Positive clones were identified and verified by using restrictive cleavage and sequence.pGen-1-FSCN1 and pGen-1-con were transfected into gastric carcinoma cell line MKN45 with liposome,respectively.Positive colonies were selected with G418.Expression of fascin1 protein and mRNA in the transfected and non-transfected MKN45 cells(NT) was examined by Western blotting and RT-PCR,respectively.Adherent ability of transfected cells was detected by MTT method; The ability of migration and invasion of transfected cells was examined by Bodern method;Nude mice metastasis models were established by abdominal cavity transfer method.To examine the signaling pathway by which the TGF-βincreases the fascin1 expression and then promotes the ability of invasion and migration,Smad2 siRNA and Smad4 siRNA were transiently transfcted into MKN45 cells,and the the expression of fascin1 were measured. Specific protein kinase inhibitors were used to determine that whether the Smad-independent signaling involved in the changes of fascin1 production and promoting the ability of invasion and migration induced by TGF-β.ResultsFascin1 expression levels were found to markedly decrease in MKN45 cells transfected with pGen-1-FSCN1(siFascin1).Transfection of pGen-1-con plasmid(CON) showed no obvious effect on fascin1 expression in MKN45.Down-regulation of fascin1 was accompanied with decreased adhesiveness,invasiveness and migratory abilities.To investigate whether TGF-β1-mediated fascin1 expression is involved in the invasive and metastatic potential of MKN45 cells,NT, CON and siFascin1 cells with or without TGF-β1 treatment were used in our study.Compared with untreated NT and CON cells,the siFascin1 cells showed lower adherent,invasive and metastatic abilities(p＜0.05). TGF-β1 significantly promoted the adhesiveness,invasiveness and migration of NT and CON cells.However,TGF-β1 showed no effect on the adherent,invasive and metastatic abilities in siFascin1 cells.In vivo, the migration rate of the siFascin1 cells was decreased than NT and CON cells(p＜0.05).In vivo,TGF-β1 pretreatment could increase liver metastasis in NT and CON cells.However,TGF-β1 treatment had no effect on the metastatic potential in siFascin1 cells.To address whether smad2 and smad4 were involved in TGF-β1-mediated fascin1 expression,the levels of fascin1 expression in the cells were detected by western blotting after silencing smad2 and smad4.We found both smad2 and smad4 expressions were suppressed successsfuly(p＜0.05),but fascin1 expression was not altered significantly.The specific inhibitors of P38,JNK and Erk,SB203580,SP600125, and PD98059 were used in our study,PD98059 and SP600125 decreased the TGF-β1-induced fascin1 production(by 78%and 75%,respectively), but SB203580 showed no effect on fascin1 expression.Conclusion1.It is the first time to address that TGF-β1 can induce the fascin1 expression in gastric carcinomacells MKN45.2.siRNA plasmid targeted against fascin1,pGen-1-FSCN1,was successfully constructed and transfected into MKN45 cells,and cell model steadily suppressing fascin1 was obtained.3.We for the first time revealed that RNAi plasmid targeted against fascin1 could obviously suppress the invasion and metastasis of GC cells MKN45 mediated by TGF-β1,in vitro and in vivo.4.TGF-β1 promoted the invasion and metastasis of MKN45 cells by increasing fascin1 expression through JNK and ErK signaling mediated.