Regulation Of KSHV Encoded Protein LANA On The Expression Of MiR-155 And GATA3 And Its Role In Promoting Cell Growth And Invasion

Object:This article studied the effect of latency associated nuclear Antigen(LANA)on the regulation of micro RNA-155(mi R-155)and GATA-binding protein 3(GATA3)and its role in the growth and invasion of KSHV infected cells,and clarified the molecular mechanism of LANA promoting cell growth and invasion by enhancing the interaction of c-Jun/c-Fos to transcriptionally activate mi R-155 and inhibit the expression of GATA3.These findings can provide some experimental and theoretical basis for discoverying therapeutic targets for KS.Method:Doxycycline and sodium butyrate were used to induce i SLK-219 from latency to lytic replication period to synthesize a large amount of KSHV.KSHV was extracted by virus concentration method.The copy number of KSHV virus solution was detected by Taq-man quantitative real-time-PCR(q RT-PCR).The expression of Green fluorescent protein(GFP)in cells was observed at different time points by fluorescence microscope to determine the success of infection.q RT-PCR was used to detect the expression of LANA,mi R-155 and GATA3.Western blot was used to detect the expression of LANA,GATA3 and epithelialmesenchymal transition(EMT)related molecules.EAhy926 cells were infected with KSHV,EAhy926 cells were transfected with p CAGGS-LANA or i SLK-219 cells were transfected with sh-LANA,CCK-8 assay was used to detect the changes of cell proliferation ability in the above groups;Wound healing assay and Transwell migration and invasion assay were used to detect the changes of cell motility in each group.Luciferase reporter assays were used to detect the regulation of LANA,c-Jun and c-Fos on the transcription level of mi R-155 promoter after single transfection or co-transfection.Chromatin immunoprecipitation(Ch IP)and Re-chromatin immunoprecipitation(Re-Ch IP)experiments were used to detect the binding of c-Jun and/or c-Fos in the promoter region of mi R-155.The interaction between c-Jun and c-Fos and the effect of LANA on the interaction between c-Jun and c-Fos were detected by immunoprecipitation assay.Results:1.KSHV virus solution was successfully prepared and infected cells.2.The proliferation,migration and invasion of EAhy926 infected with KSHV were significantly enhanced.3.After overexpression of LANA,mi R-155 was increased,GATA3 was decreased,and the proliferation,migration and invasion of EAhy926 cells were enhanced;On the contrary,after interfering with LANA in i SLK-219 cells,mi R-155 was decreased,GATA3 was increased,and the ability of cell proliferation,migration and invasion reduced significantly.4.Co-transfection experiments showed that LANA enhanced the inhibitory effect of mi R-155 on GATA3 expression and enhanced the promoting effect of mi R-155 on cell growth and invasion.5.Both c-Jun and c-Fos can transcriptionally activate mi R-155.The transcriptional activity of mi R-155 is most significantly enhanced after co-transfected with c-Jun and c-Fos.6.c-Jun and c-Fos interact endogenous in i SLK-219 cells.7.c-Jun,c-fos or c-Jun/c-fos complex can bind to the-95 ～-100 bp site of mi R-155 promoter.8.LANA enhances the expression of c-Jun and c-Fos.9.LANA enhances the interaction between c-Jun and c-Fos.10.LANA enhances the transcriptional activation of mi R-155 by the c-Jun/c-Fos complex Conclusion:1.With the increase of infection time,the abilitity of proliferation,migration and invasion were increased in EAhy926 infected with KSHV.2.LANA inhibits the expression of GATA3 by up regulating mi R-155,thereby promoting cell growth and invasion.3.c-Jun and c-Fos interact to form a complex.LANA up-regulates the expression of c-Jun and c-Fos and enhances the formation of c-Jun/c-Fos complex.The complex binds to the-95 ～-100 bp site of mi R-155 promoter and transcriptionally activates mi R-155.