KSHV-encoded MiR-K6-3p Promotes Endothelial Cell Migration And Angiogenesis By Regulating SH3BGR/STAT3 Pathway

Background:Kaposi's Sarcoma(KS),is caused by infection of Kaposi's sarcoma(KS)-associated herpesvirus(KSHV),and is a tumor of endothelial cells characterized by angiogenesis and invasiveness.In vitro,KSHV-infected endothelial cells display an increased invasiveness and angiogenesis feature.KSHV encodes twelve precursor miRNAs(pre-miRINAs),which are processed into at least 25 mature miRNAs.However,the roles of these miRNAs in KSHV-induced tumor dissemination and angiogenesis remain unknown.Objective:To explore the involvement of miR-K6-3p in endothelial cell motility and angiogenesis,and and the molecular mechanisms involved in the process.Methods:We first constructed the recombinant plasmid mpCDH-miR-K6-3p carrying miR-K6-3p,and packed recombination lentivirus Lentivirus-K6-3p and control Lentivirus-mpCDH through three plasmids expression system and then infected HUVECs,respectively.Then,we conducted Transwell migration,microtubule formation and Matrigel plug assays to determine whether miR-K6-3p had effect on cell motility and angiogenesis.To identify the targets of miR-K6-3p,we examined proteins differential expressed between miR-K6-3p-and mpCDH-transduced HUVECs by mass spectrometry analysis.Next,bioinformatics analysis with several programs including TargetScan,RNAhybrid,Findtar,and Pictar,was performed to predict the putative miR-K6-3p targets and the luciferase reporter assay,point mutation analyses and Western blot were then performed to validate the putative target of miR-K6-3p.Subsequently,overexpressing target gene in miR-K6-3p-expression cells and further analyzing for migration and angiogenesis activities were performed to determined whether the target gene mediated miR-K6-3p-induced cell migration and angiogenesis.Meanwhile,Western blot,co-immunoprecipitation,and GST pull-down assays were performed to examine the downstream pathway involved in miR-K6-3p induction of endothelial cell migration and angiogenesis.Finally,we deleted miR-K6 from the KSHV genome to further confirm that miR-K6-3p induced endothelial cell migration and angiogenesis by regulating its downstream signaling pathway.Results:The recombinant plasmid carrying miR-K6-3p was successfully constructed.Phenotypic experiments showed that ectopic expression of miR-K6-3p promoted endothelial cell migration and angiogenesis.Mass spectrometry showed that there are 47 cellular proteins were downregulated>2.0 folds by miR-K6-3p.We predicted 3 proteins that might have miR-K6-3p putative binding sites in their 3'UTR using bioinformatics analysis.Luciferase reporter analysis and Western blot revealed that miR-K6-3p directly targeted sequence in the 3' untranslated region(UTR)of SH3 domain binding glutamate-rich protein(SH3BGR).Overexpression of SH3BGR reversed miR-K6-3p induction of cell migration and angiogenesis.Mechanistically,miR-K6-3p downregulated SH3BGR,hence relieved STAT3 from SH3BGR direct binding and inhibition,which was required for miR-K6-3p maximum activation of STAT3 and induction of cell migration and angiogenesis.Finally,deletion of miR-K6 from the KSHV genome abrogated its effect on the SH3BGR/STAT3 pathway,and KSHV-induced migration and angiogenesis.Conclusion:Our results illustrated that by inhibiting SH3BGR,miR-K6-3p enhances cell migration and angiogenesis by activating the STAT3 pathway,and thus contributes to the dissemination and angiogenesis of KSHV-induced malignancies.