The Effect Of HIV-1 Tat On HHV-8 K12-Induced Tumorigenesis And The Preliminary Studies Of Its Molecular Mechanisms

This study was focused on the effect of HIV-1 transactivativetranscription protein (Tat) on tumorigenesis in nude mice induced byoncogene K12 of human herpesvirus 8 (HHV-8) and the molecularmechanisms lied behind. First, eukaryotic expression plasmid pK12Fcontaining HHV-8 K12 gene was constructed. Four cell lines namedN/K12,N/Tat,N/K12/Tat and N/pcDNA3.1 expressing K12-Flag,Tat-Flag,K12-Flag plus Tat-flag and pcDNA3.1(+), respectively, wereobtained by stable transfection of NIH3T3 cells with the correspondingplasmids. Second, ~3HTdR assay and flow. cytometric analysis wereperformanced to measure the proliferation ability and cell cycle of fourstable cell lines metioned above. The results of ~3HTdR assay indicatedthat the proliferation ability of four stable cell lines took a descendingorder as follows: N/K12/Tat, N/K12, N/pcDNA3.1, N/Tat one by one(P＜0.05). The result from flow cytometric analysis showed that theS-phase fraction (SPF) of four stable cell lines followed the same order asthe results of ~3HTdR assay. Third, cell colony formation assay onsoft-agar demonstrated that all of these four stable cell lines had theability of transformation. Subsequently, nude mice tumorigenesis testsindicated that both autocrine and short-distance paracrine Tat couldaccelerate the formation of tumor induced by HHV-8 K12 (Log-rank test, P＜0.05). However, long-distance paracrine Tat did not exhibit suchfunction (P＞0.05). To further determine the mechanism by which the Tatinteracts with K12, the cDNA array technique was carried out on thesefour stable cell lines and corresponding tumor tissue. This array contains96 genes involved in 15 signal pathways related to tumor formation.Although there was a little of differences between the result of cells andturmer tissue, all of the results still displayed that HIV-1 Tat facilitatedK12 gene to activate more tumor signal genes. Eventually, Western blotanalysis demonstrated that HIV-1 Tat promoted K12-inducedphosphorylation of signal transducers and activator of transcription 3(STAT3) in vitro. Immunohistochemistry staining manifested that theexpression of Cyclin D1, vascular endothelial growth factor (VEGF) andSurvivin in K12 induced tumor could be reduced by HIV-1 Tat.These data suggested that HIV-1 Tat could promote the proliferationability of K12 cells in vitro; both autocrine and short-distance paracrineof Tat could facilitate the HHV-8 K12-induced tumorigenesis in vivo;STAT3 signaling was one of tumor signal pathways, which played animportant role in the K12-induced tumorigenesis accelerated by Tat;Meanwhile, Tat lowered the expression of the VEGF, cyclin D1, andsurvivin in K12-induced tumors.