The Effect Of HIV-1 Tat Protein On Angiogenesis And Tumorigenesis Induced By KSHV K1 And The Molecular Mechanism

Background:Kaposi’s sarcoma-associated herpesvirus (KSHV) infection is associated with KS (Kaposi’s sarcoma), PEL (primary effusion lymphoma) and MCD (multicentric Castleman’s disease). Although KSHV can encode some of oncogeneic proteins such as vGPCR, kaposin A and Kl et al, to promote cell transformation and tumorigenesis, other factors are necessary for KS development. Epidemiological study shows that human immunodeficiency virus type 1 (HIV-1) infection increased the risk of KS occurrence in KSHV carrier significantly. Our previous study has showed that HIV-1 regulatory protein Tat could promote the tumorigenesis induced by Kaposin A and vIL-6. However, whether HIV-1 Tat could promote the tumorigenesis of K1 was not clear.Objective:To explore whether HIV-1 Tat could modulate angiogenesis and tumorigenesis induced by KSHV K1 and the related signaling pathway.Methods:First, sorting endothelial cell line (EA.hy 926) which can strain expressed Kl by Flow cytometry. The level of Tat expression was measured by western blot in K1 cells following lentivirus-Tat infection. To detect the effect of Tat on proliferation mediated by KSHV Kl, CCK-8, Clony formation assay and Flow Cytometry assay (FCM) were used in vitro. Microtube formation analysis used to detect the angiogenesis. In vivo, inoculate the cells with the chick embryo chorioallantoic membrane (CAM) and nude mice models to anlysis the angiogenesis and tumorigenesis of Kl mediated by Tat. Meanwhile, key proteins involoved by signaling pathway were measured by western blot, and tumor tissue structure was observed by HE stain. Immunohistochemisty (IHC) was carried out to detect the molecular mark proteins CD31 and smooth muscle actin (SMA) of vascular endothelial cell, vascular endothelial growth factor (VEGF) expression. Finally, to further explore the signaling pathway involved in processes mentioned above, dominant-negative (DN) constructs and inhibitors of signal pathway-related kinases were used.Results:According to cell proliferation assay and microtube formation analysis, Tat protein could promote the cell proliferation, transformation, shorten Cell cycle and angiogenesis of K1 cells in vitro. And Tat protein could also enhance the angiogenesis and tumorigenesis of Kl cells on the CAM and the nude mice models in vivo. At last, IHC and western blot results reveal that PI3K/Akt and NF-κB signaling pathways participated in Tat protein facilitating angiogenesis and tumorigenesis induced by K1 from the obtained tumor tissues. Mechanistic studies demonstrated that Kl-induced angiogenesis and tumorigenesis promoted by Tat were greatly suppressed through inhibition of PI3K phosphorylation with PI3K-DN, Akt phosphorylation with Akt-DN and IκB phosphorylation with IκB-DN. Addition of Ly294002, PI3K inhibitor and Bay 11-7082, NF-κB inhibitor into CAM and nude mice could significantly decreased the angiogenesis and tumorigenesis.Conclusions:HIV-1 Tat protein could promote KSHV Kl-induced cell proliferation, angiogenesis and tumorigenesis via activating PI3K/Akt and NF-κB signaling pathways. PI3K/Akt and NF-κB pathways may be potential of clinical therapeutic targets in KS patients.