Study On The Effect Of RNA Interference Mediated Stable Knockdown Of HMGB1 To Regulate Epithellial-mesenchymal Transition And Invasion And Metastasis In Hypopharyngeal Carcinoma Cells

Objective To investigate the effect of HMGB1 gene on the epithelial-mesenchymal transition and invasion and metastasis in hypopharyngeal carcinoma cell lines.Furthermore,we also try to discuss the regulatory mechanism of HMGB1 in this process.We hope to find the new target of treatment in hypopharyngeal carcinoma.Method1.TGF-β1 induced Fa Du cells that occurred EMT by exploring literature[1-2] and screening optimal induction concentration of TGF-β1.And Fa Du cells were divided into two groups: Normal group and TGF-β1 group. We used microscope to observe the morphologic change of Fa Du cells after the induction of TGF-β1.To detect the expression status of Vimentin 、 Snail 、 HMGB1 protein in each group by Cell immunofluorescence. RT-PCR and Western blot were used to check the expression status of HMGB1 on m RNA and protein levels before and after TGF-β1 induction in FaDu cells.2.We transfected si RNA-HMGB1 and si RNA-Control to Fa Du cells by liposome transfection method(Fa Du cells were divided into three groups: Normal group 、Control group and si HMGB1 group),and detected the inhibition of si RNA by Cell immunofluorescence、Western Blot and Real Time-PCR. The Vimentin、Snail 、HMGB1 pritein expression in Fa Du cells by using Cell immunofluorescence. RT-PCR technique were used to tset Vimentin、Snail、HMGB1、E-cadherin m RNA expression levels. Western blot were used to check the expression change of HMGB1 and RAGE on protein levels after cell transfection. We observed the ability of Fa Du cells migration after silenced HMGB1 by wound healing assay.We observed the ability of Fa Du cells invasion after silenced HMGB1 by Transwell assay.Results1. Fa Du cells transit to elongated fibroblastic phenotype from epithelial cobblestone phenotype after the induction of TGF-β1.Cell immunofluorescence showed that the expression of Vimentin and Snail were up-regulated after the induction of TGF-β1.2. Cell immunofluorescence showed that the expression of HMGB1 was increased in Fa Du cells after the induction of TGF-β1. And In TGF-β1 group, the expression of HMGB1 was up-regulated obviously both on m RNA(P<0.01) and protein(P<0.01)levels.3. The expression of HMGB1 mRNA was inhibited in FaDu cells by siRNA-HMGB1(P<0.05), and also HMGB1 protein expression was inhibited obviously in Fa Du cells by si RNA-HMGB1(P<0.01).4.Cell immunofluorescence results showed the expression level of Vimentin and Snail were decreased after the transfection with si RNA-HMGB1.5.RT-PCR showed that the expression level of m RNA of Vimentin(P < 0.05) and Snail(P<0.01)were down-regulated,but E-cadherin(P<0.01) was increased after the transfection with si RNA-HMGB1.6.Western Blot showed that the expression level of RAGE was decreased after the transfection with si RNA-HMGB1(P<0.01).7.The wound healing assay proved Fa Du cells migration decrease after the transfection with si RNA-HMGB1.8.The Transwell assay proved Fa Du cells invasion decrease after the transfection with si RNA-HMGB1( P<0.01).Conclusion1.Successfully established epithelial-mesenchymal transition model of hypopharyngeal carcinoma cells after Fa Du cells were inducted 48 h by5ng/ml-TGF-β1.And the expression of HMGB1 was associated with epithelial-mesenchymal transition in hypopharyngeal carcinoma cells.2.Silencing HMGB1 gene can reverse the epithelial-mesenchymal transition of FaDu cells.3.Silencing HMGB1 gene can obviously suppress migration and invasion of hypopharyngeal carcinoma Fa Du cell lines.4.HMGB1-RAGE interaction can regulate invasion and metastasis of hypopharyngeal carcinoma Fa Du cell lines.5. HMGB1 gene can be a molecular target for the clinic treatment of hypopharyngeal carcinoma.