Effects Of Lentivirus-mediated HIV-1 Tat On Proliferation And Migration In Human Prostate Cancer Cell Line PC-3

Objective: To construct the lentiviral vector expressing HIV-1 Tat and explore the effects of lentivirus-mediated HIV-1 Tat on proliferation and migration in human prostate cancer cell line PC-3.Methods: (1) A pair of PCR primers with restriction enzyme cut sites Nhe I and Bam HI was designed to amplify HIV-1 Tat gene according to the nucleotide sequence registered in GenBank. The Flag gene sequence was also added at the 5'end of downstream primer. Then the genome of plasmid pEV was taken as template DNA and Tat gene was amplified using PCR. Subsequently, the PCR product were digested with the two above-mentioned enzymes, and then subcloned to lentiviral transfer vector pHAGE-CMV-MCSIZsGreen, which was designated as pHAGE-Tat., which was introduced with Flag sequence to facilitate the detection of Tat protein. Then the constructed pHAGE-Tat was used to co-transfect the packing 293T cells, together with the plasmids pMD2.G and psPAX2. The packaged viral particles designated as LV-Tat were used to infect the 293T cells and the viral titer was calculated. The expression of HIV-1 Tat in 293T and PC-3 cells was confirmed using RT-PCR and western blot. (2) PC-3 cells were cultured by routine method. The proliferation and cell cycle in PC-3 cells was respectively measured by MTT and FCS at different time points. The wound healing assay was used to detect the cell migration.Results: Lentiviral vector expressing HIV-1 Tat gene was successfully constructed, and the virus titer was 5.73×106 ifu/ml. At 24 and 48 hours after PC-3 and 293T cells were respectively infected, HIV-1 Tat protein could be detected by western blot. Compared with that in Non-treatment group, the average proliferation rate in LV-Tat-infected and LV-pHAGE-infected group was 126.86±10.41% and 99.35±5.05% at 24, 48, 72, 96 and 120 hours after treatment, as proved with MTT. No significant difference was found between Non-treatment and LV-pHAGE-infected groups (P>0.05). However, proliferation rate in LV-Tat-infected group was significantly different from that in LV-pHAGE-infected and Non-treatment groups (P<0.05). At 48 hours after treatment, the S phase cell in LV-Tat-infected, Non-treatment and LV-PHAGE-infected group was 28.71±1.16%, 17.19±0.99% and 18.50±1.07%, respectively. The S phase cell in LV-Tat-infected group was significantly higher than that of other two groups (P<0.05), as manifested by FCS. HIV-1 Tat significantly caused a cell accumulation in S phase. The wound healing assay showed cell migration rate in LV-Tat-infected, Non-treatment and LV-PHAGE-infected grous was 34.70±5.62%, 37.40±4.90% and 34.27±3.40% at 24 hours after PC-3 cells with MMC treatment were infected with lentivirus. There was no significant difference between any two groups of them (P>0.05). Tat insignificantly affected the migration in PC-3 cells, when cell cycle was inhibited by MMC.Conclusion: The constructed lentivirus could express Tat protein in human prostate cancer cell line PC-3. Tat protein, in PC-3 cells, had a positive effect on proliferation. However, no significant effect was detected on migration.