| Background: Epstein-Barr virus(EBV)is the first DNA virus connected with human tumors to be discovered.The infection rate of EBV in adults is up to 90%,and EBV infection is a risk factor for many human tumors.EBV-associated gastric carcinoma(EBVaGC)is one of the common EBV-associated tumors and a unique subtype of gastric carcinoma(GC),accounting for about 10% ofGC.The pathogenesis of EBVaGC is different from that of EBV-negative gastric carcinoma(EBVnGC).Although increasing studies have focused on the pathogenesis of EBVaGC,the specific mechanism remains unclear.Krüppel-like factor 5(KLF5),one of zinc-finger transcription factors,is involved in a variety of intracellular biological processes.A growing number of studies has shown that KLF5 plays a crucial role in tumor formation and progression.However,but the biological significance of KLF5 in EBV-associated tumors remains unclear,and the role of EBV infection on KLF5 is worth studying.Objective: This study sought to investigate the expression and biological role of KLF5 in EBVaGC and EBVnGC and to clarify the relationship between EBV infection and KLF5 expression.Materials and Methods:(1)Immunohistochemical method was used to test the expression of KLF5 in 88GC tissues(62 EBVaGC tissues,26 EBVnGC tissues).The expression of latent membrane protein 2A(LMP2A)encoded by EBV was detected in 62 cases of EBVaGC,and the relationship between LMP2 A and KLF5 was analyzed.(2)Western blot and immunofluorescence were used to detect the expression and localization of KLF5 in EBV positive(GT38,GT39,SNU719)and EBV negativeGC cell lines(SGC7901,BGC823,AGS).(3)By transfecting LMP2 A recombinant plasmid in SGC7901 cells and LMP2 A specific small interfering RNA in GT38 cells,the expression of KLF5 was detected to clarify the regulatory effect of LMP2 A on KLF5.(4)GT38 cells and SGC7901 cells were treated with different concentrations of m TOR activator MHY1485 and different concentrations of rapamycin,inhibitor of mTORC1 pathway,respectively.Western blot and immunofluorescence were used to analyse the expression of KLF5.Thus,the regulatory role of mTORC1 pathway on KLF5 was verified.(5)In SGC7901 cells,m TOR,Raptor and 4EBP1 were interfered with specific small interfering RNA(sim TOR,si Raptor,si4EBP1).Cells were collected to extract total protein,and the expression of KLF5 was detected by Western blot and immunofluorescence to further clarify the regulatory role of mTORC1 pathway on KLF5.(6)The expression of p-m TOR,p-p70S6 K and p-4EBP1 was detected in SGC7901-LMP2 A cells,and then the regulation of LMP2 A on mTORC1 pathway was analyzed.(7)MHY1485 was used to treat SGC7901-LMP2 A cells to activate the mTORC1 pathway.Subsequently,the expression of KLF5 was detected by Western blot and immunofluorescence techniques to elucidate the specific mechanism of LMP2 A regulation of KLF5.(8)SNU719 cells were transfected with specific plasmid of KLF5(pc DNA3.1-KLF5),and SGC7901 and BGC823 cells were transfected with si RNA of KLF5(si KLF5)targeting the KLF5 encoding gene.The migration ability was examined by Transwell cell assay to analyze the effect of KLF5 on migration ofGC cells.(9)SNU719 cells were transfected with pc DNA3.1-KLF5 recombinant plasmid to overexpress KLF5,and BGC823 cells were transfected with si KLF5 to interfere with KLF5.Western blot and immunofluorescence were used to detect the expression of autophagy related indicators and then analyze the effect of KLF5 on autophagy inGC cells.Results:(1)The probability of KLF5 overexpression(++)in EBVnGC tissues was61.54% which was significantly higher than that in EBVaGC tissues(33.87%),and the difference was statistically significant(P<0.05).However,there was no significant correlation between LMP2 A expression and KLF5 expression in the 62 EBVaGC tissues(χ2=1.491,P=0.474).(2)The protein expression of KLF5 in EBV negativeGC cell lines was significantly higher than that in EBV positiveGC cell lines,and the fluorescence intensity of KLF5 in EBV negativeGC cell lines was also stronger than that in EBV positiveGC cell lines.The localization of KLF5 was mainly in cell nuclear.(3) Overexpression of exogenous LMP2 A in SGC7901 cells down-regulated the protein expression of KLF5;whereas interference with LMP2 A in EBV positiveGC cell line GT38 cells significantly up-regulated the protein expression of KLF5.(4)MHY1485 could activate mTORC1 signaling pathway and up-regulate KLF5 expression in GT38 cells. Rapamycin treatment could inhibit mTORC1 signaling pathway and down-regulate KLF5 expression in SGC7901 cells.(5)Interference with m TOR,Raptor and 4EBP1 expression in SGC7901 cells resulted in inhibition of KLF5 expression.(6)Compared with SGC7901-NC,exogenous LMP2 A expression in SGC79001-LMP2 A cells could inhibit the expression of p-m TOR,p-p70S6 K and p-4EBP1,which meant that overexpression of LMP2 A could lead to the inactivation of mTORC1 pathway.(7)Treatment with MHY1485 activated the mTORC1 pathway in SGC7901-LMP2 A cells and partially reversed the down-regulation of KLF5 expression induced by LMP2 A.(8)KLF5 was overexpressed in SNU719 cells,and interference with KLF5 in SGC7901 and BGC823 cells enhanced or inhibited the migration ofGC cells,respectively.(9)Overexpression of KLF5 in SNU719 could enhance the expression levels of autophagy related indicators-LC3 B,Beclin1,ATG7,ATG12-5,and ATG16L1;Interference with KLF5 expression in BGC823 cell lines down-regulated the expression of autophagy related markers LC3 B,Beclin1,ATG7,ATG12-5,and ATG16L1.Conclusions:(1).EBV inhibited the expression of KLF5 in EBVaGC tissues and positive cell lines,and LMP2 A down-regulated KLF5 expression by inactivating the mTORC1 pathway in EBV positiveGC cell lines.(2)Overexpression of KLF5 could enhance the migration ability ofGC cells and induce autophagy. |
PDF Full Text Request