Experimental Study On Reversal Of EMT In Laryngeal Squamous Cell Carcinoma By Targeted Regulation Of Snail/VDR Signaling Pathway

Objective:In recent times,Chinese and foreign scholars have used the theory of"epithelia-mesenchymal transition?EMT?"to clarify the mechanism of tumor metastasis.EMT is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity,resulting in the acquisition of migratory and invasive properties.A number of distinct molecular processes are initiated the complex process.Increasing evidence suggests Snail plays a key role in promoting EMT as a transcription factor.Snail has been found to downregulate E?cadherin,and the canonical pathway has been confirmed largely.However,the functional role of the unique non-canonical pathway of repressing the expression of the vitamin D receptor gene remains elusive.Here,we focused on the molecular mechanism of Snail/VDR signaling pathway involved in EMT of laryngeal squamous cell carcinoma and explored its effect on invasion and metastasis of LSCC.Methods:The first part:The content of Snail in LSCC tissues and their conrrespongding adjacent normal laryngeal tissues was assessed by Western blot and RT-PCR.A pair of Snail-specific shRNAs was used to establish the stable Snail-silenced Hep-2cells.After selection,the Hep-2 cells transfected with NC or Snail shRNA were subjected to Western blotting and real-time PCR.The expression levels of MMP-2,MMP-9,VDR,ITG?1,?-catenin,E-cadherin,vimentin,N-cadherin,and FN in Hep-2cells were measured after silencing of Snail by Western blottingand real-time PCR.The adhesive capacity,the migration ability and the invasiveness of Hep-2 cells with or without Snail silencing was assessed by Transwell.The second part:The small interfering RNA of VDR was synthesized and tranfected into Snail-silenced Hep-2 cells.The migration and invasion ability of Hep-2 cells was measured by Transwell after knockdown of Snail with transfection of VDR siRNA,and the expression levels of?-catenin,E-cadherin,and FN were detected.The expression levels of E-cadherin,Vimentin,MMP2 and MMP9 were measured respectively in Hep-2 cells,Snail-silenced Hep-2 cells cultured with 1,25-?OH?₂D₃,Snail-silenced Hep-2 cells and Hep-2 cells cultured with 1,25-?OH?₂D₃.The migration ability of Hep-2 cells from the four groups was evaluated by wound healing assay.Results:The first part:we firstly detected the expression of Snail was higher in cancer tissues compared with adjacent tissues in 14 patients diagnosed LSCC.Then we established Snail-silencd Hep-2 cells.Results revealed that knockdown of Snail suppressed EMT process of Hep-2 cells,as evidenced by downregulation of matrix metallopeptidase?MMP?-2,MMP-9,integrin subunit beta 1?ITG?1?,?-catenin,vimentin,N-cadherin,and fibronectin and upregulation of vitamin D receptor?VDR?and E-cadherin.Further study showed that silencing of Snail significantly reduced the ability of adhesion,migration,and invasion of Hep-2 cells.The second part:The Snail-silenced Hep-2 cells were transfected by small interfering RNA of VDR.Following the process,the expression levels of?-catenin and FN were upregulated reversely and that of E-cadherin was downregulated.Wound healing assay revealed that the migration ability of Snail-shRNA Hep-2cells mixed with 1,25-?OH?₂D₃ was the lowest.Snail-shRNA Hep-2 cells combined with 1,25-?OH?₂D₃ had the highest level of expression of E-cadherin,and the lowest of Vimentin,MMP2 and MMP9 compared with other three groups.Conclusion:1.The content of Snail was higher in LSCC compared with adjacent tissues.2.Snail-silenced Hep-2 cells exhibited significantly higher VDR expression, acquired phenotype convertation and reduced the ability of adhesion,migration, and invasion.It demonstrated that Silencing of Snail effectively inhibited EMT of Hep-2 cells and the findings indirectly revealed Snail induced the EMT of LSCC.3.Knockdown of Snail could inhibit the EMT process of LSCC through the Snail/VDR signaling pathway in vitro.The present study is the first to report that Snail directly induced the LSCC EMT by regulating VDR through Snail-VDR-?–catenin pathway in vitro.4.Downregulation of Snail gene expression combined with 1,25-?OH?₂D₃ can effectively inhibit the invasion and migration of Hep-2 cells synergistically.The present study suggests a novel rationale for inhibiting cancer metastasis using anti-Snail/VDR and 1,25-?OH?₂D₃ therapeutic strategies for LSCC.