The Role Of SNAIL In Gastric Cancer And Its Relationship With EBV

Background: Epstein-Barr virus(EBV),a ubiquitous oncogenic herpesvirus,approximately infects more than 90% of the worldwide adult population.Micro RNAs(mi RNAs)are 19-25 nucleotides non-coding RNA molecules that repress gene expression by inhibiting translation or degrading target m RNA.EBV was the first virus found to encode mi RNAs.To date,there are 25 mi RNA precursors with 44 mature mi RNAs produced by EBV.mi R-BARTs are highly expressed in NPC and EBVa GC regulating the expression of various genes at the post-transcriptional level.SNAIL is a member of the group of conservative zinc finger transcription factors.There are three members in SNAIL family protein,including SNAI1(SNAIL),SNAI2(SLUG)and SNAI3(SMUC).SNAIL is a hallmark of Epithelial-mesenchymal transition(EMT).EMT is a process that induces cell motility during morphogenesis at the time of embryonic development.Overexpression of SNAIL increases tumor recurrence and promotes tumor metastasis by repressing host immune surveillance.Objective: To explore the expression of the SNAIL in EBV-associated gastric cancer(EBVa GC)and EBV-negative gastric cancer(EBVn GC),and to clarify the regulatory role of EBV-mi R-BART12 on the SNAIL,so as to provide a theoretical basis for the development of EBVa GC.Materials and methods: 1 The expression levels of SNAIL in EBVa GC cell lines(GT38,GT39,SNU719)and EBVn GC cell lines(SGC7901,AGS,and BGC823)were detected by quantitative real-time PCR(q RT-PCR)and Western Blot.2 Immunofluorescence assay was used to detect SNAIL’s intracellular localization.3 Western Blot was applied to detect the expression of SNAIL after transfecting EBV-mi R-BART12 inhibitor and mimics into EBVa GC and EBVn GC cell lines.4 The dual luciferase reporter assays detected whether SNAIL was the target gene of EBV-mi R-BART12.5 Transwell and CCK8 assay detected the effects of EBV-mi R-BART12 and SANIL small interfering RNA(si SNAIL)on the migration and proliferation of gastric cancer cells.The effects of EBV-mi R-BART12 and si SNAIL on cell cycle or apoptosis were analyzed by flow cytometry.6 The degradation of protein was detected by Cycloheximide(CHX)chase assay.Result: 1 The transcription levels of SNAIL in the EBVa GC cell lines were significantly lower than that of the EBVa GC cell lines.Western Blot results showed a significant decrease of SNAIL protein in EBVa GC cell line.2 The SNAIL protein in the EBVa GC cell lines was mainly located in the cytoplasm and its fluorescence intensity was significantly weaker than that of the EBVn GC cell lines.3 The expression of SNAIL protein was up-regulated by EBV-mi R-BART12 inhibitor in GT39 and SNU719 cell lines,while the expression of SNAIL protein was significantly inhibited by EBV-mi R-BART12 mimics in SGC7901 and BGC823.4 The dual luciferase reporter assays results showed that EBV-mi R-BART12 could directly target and bind to the 3’-UTR region of the SNAIL.5 CCK8 and Transwell experiments showed that EBV-mi R-BART12 mimics inhibited the proliferation and migration of gastric cancer cells.6 Downregulation of SNAIL expression by si RNA could significantly inhibit the migration and proliferation of gastric cancer cells.7 CHX chase assay results showed that the EBV-mi R-BART12 mimics could accelerate SNAIL protein degradation.8 Western Blot results showed that EBV-mi R-BART12 inhibits NF-κB pathway.Conclusion: In the EBVa GC cell lines,SNAIL protein was significantly down-regulated,and EBV-mi R-BART12 could inhibit expression of SNAIL protein by targeting its 3’-UTR region and accelerated SNAIL protein degradation.EBV-mi R-BART12 inhibited the proliferation,migration and EMT process of gastric cancer cells by targeting the SNAIL.