Study On The Role Of RTA In Promoting Endothelial Cell Growth And Invasion Through MiR-155 Regulatory Network

Object:Kaposi’s sarcoma(KS)is a malignant tumor with endothelial cells as the main source,and the lesion mostly occurs in soft tissues,which is the most common malignant tumor in AIDS patients and has a certain aggressiveness.Kaposi’s sarcoma-associated herpesvirus(KSHV)can also affect the growth and proliferation of host cells as the pathogen of KS.KSHV infects cells and exists in both a latent state and a lytic state.The main protein expressed by the virus in the latent infection state is Latent associated nuclear antigen(LANA),and the main protein expressed in the lytic infection state is Replication and transcription activator(RTA).RTA is one of the important virus-encoding proteins during the KSHV lytic stage and is essential for the lytic reactivation of KSHV.At present,the effect of RTA on cell proliferation and invasion is not clear.This study aims to clarify the function of RTA in endothelial cell growth and invasion.Previous studies have confirmed that micro RNA 155(mi R-155)could target and inhibit the expression of GATA-binding protein 3(GATA3),and the latter could regulate the expression of Signal transducers and activators of transcription 3(STAT3),a crucial transcription factor in the JAK-STAT signaling pathway.Therefore,this study continues to explore the regulation and specific regulatory mechanism of RTA on the expression of mi R-155/GATA3/STAT3 to clarify whether RTA promotes endothelial cell growth and invasion through the mi R-155 regulatory network,in order to open up a new path for the treatment of KSHV-related diseases.Method:PCR and WB were employed to detect the overexpression or interference efficacy of RTA following transfection of either the overexpression or interference plasmid of RTA into endothelial cells EAhy926 and i SLK-RTA,respectively.CCK-8,scratch healing,Transwell migration and invasion experiments were conducted to detect the changes of endothelial cell proliferation and invasion after RTA overexpression or interference.In addition,q RT-PCR and WB were used to detect the expression of molecules related to epithelial-mesenchymal transition(EMT).A double luciferase reporter gene experiment was conducted to identify the specific region where RTA transcription activated the mi R-155 promoter.The overexpressing and interfering RTA plasmids were transfected into EAhy926 and i SLK-RTA cells,respectively,and the expression levels of mi R-155,GATA3 and STAT3 were detected by q RT-PCR,the expression levels of GATA3,STAT3 and p-STAT3 were detected by WB.The KSHV virus was prepared by i SLK-219 cell for infecting endothelial cells EAhy926 and HOMEC.The expression levels of green fluorescent protein(GFP)at different time points were observed under fluorescence microscope,and the expression levels of RTA and LANA were detected by q RT-PCR and WB.Subsequently,the expression changes of mi R-155,GATA3 and STAT3 were detected by q RT-PCR,and the expression changes of GATA3,STAT3 and p-STAT3 were detected by WB.The RTA overexpression plasmid and mi R-155 mimic were co-transfected into EAhy926 cell,and the expression changes of mi R-155,GATA3 and STAT3 in each group were detected by q RT-PCR;and the expression changes of GATA3,STAT3 and p-STAT3 in each group were detected by WB.The RTA interference plasmid and mi R-155 mimic were co-transfected into i SLK-RTA cell,and the expression changes of mi R-155,GATA3 and STAT3 in each group were detected by q RT-PCR;and the expression changes of GATA3,STAT3 and p-STAT3 in each group were detected by WB;CCK-8,scratch healing,Transwell migration and invasion experiments were conducted to detect the changes of cell proliferation and invasion in each group;q RT-PCR and WB were conducted to detect the expression of EMT-related molecules in each group.Finally,the website of JASPAR was used to predict whether GATA3 binds to the STAT3 promoter region and it was verified by the double luciferase reporter gene experiment.Results:1.The abilities of cell proliferation,scratch healing,migration and invasion were all enhanced after overexpressing RTA,and these abilities were all inhibited after interfering RTA.2.The expression of epithelial markers ZO-1 and E-cadherin were weakened,whereas mesenchymal markers Vimentin and N-cadherin were increased after overexpressing RTA,and it would produce the opposite results after interfering RTA.3.RTA could bind to the-1500～-1000 bp region of the mi R-155 promoter region and to transcriptionally activate mi R-155.4.The expression levels of mi R-155、STAT3 and p-STAT3 were increasing,and the expression level of GATA3 was decreasing after overexpressing RTA,and which would lead to the opposite results after interfering RTA.5.KSHV virus could infect endothelial cells EAhy926 and HOMEC successfully.6.The expression level of RTA was gradually decreased and the expression level of LANA tended to be stable when KSHV infected the endothelial cells EAhy926 and HOMEC.7.The expression levels of mi R-155、STAT3 and p-STAT3 were up-regulated,and the expression level of GATA3 was down-regulated when KSHV infected endothelial cells EAhy926 and HOMEC.8.The expression level of GATA3 was declining more significantly and the expression levels of STAT3 and p-STAT3 were rising more significantly when RTA overexpression plasmid and mi R-155 mimic were co-transfected into EAhy926 cell compared with transfected RTA overexpression plasmid alone.9.The mi R-155 mimic partially reversed the promotion of sh-RTA on GATA3 expression and the inhibition of sh-RTA on STAT3 and p-STAT3 expression when RTA interference plasmid and mi R-155 mimic were co-transfected in i SLK-RTA cell.10.The inhibition of sh-RTA on cell proliferation,scratch healing,migration and invasion was partially reversed after co-transfection of sh-RTA and mi R-155 mimic in i SLK-RTA cell.11.The mi R-155 mimic partially reversed the promotion of sh-RTA on epithelial markers ZO-1 and E-cadherin expression and the inhibition of sh-RTA on Vimentin and N-cadherin expression when the shRTA and mi R-155 mimic were co-transfected in i SLK-RTA cell.12.GATA3 bound to the STAT3 promoter and inhibited STAT3 transcriptional activity.Conclusion:1.RTA promotes the proliferation、migration、invasion and EMT of endothelial cells..2.RTA transcriptionally activates mi R-155 by binding to the-1500～-1000 bp region of the mi R-155 promoter region,thereby strengthening the expression of mi R-155、STAT3 and p-STAT3 and weakening the expression of GATA3.3.RTA promotes the proliferation、migration、invasion and EMT of endothelial cells through the mi R-155 regulatory network.