Knockdown Cyclin T1 Expression By RNA Interference And Its Effect On Target Cells And HIV-1

RNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), can be used not only as a research tool but also as a therapeutic strategy for viral infection. RNAi with short hairpin RNA (shRNA) has been successfully utilized in a number of recent studies in cultured mammalian cells to reduce the expression of specific cellular and viral genes.Here, intracellularly expressed shRNA based on plasmid vector were utilized in 293T, HeLa and U373-MAGI-CCR5e cells. Our results indicated that intracellularly generated shRNAs could specifically down-regulate cyclin T1 expression. Similar phenomena were demonstrated in 293T and U373-MAGI-CCR5e cells. Additionally, our results indicated that the inhibition of cyclin Tl expression by shRNAs was both dose- and time-dependent.CDK9/cyclin T directs its activity in a cell cycle independent manner and was involved in transcription during the elongation steps. This means that cyclin Tl is important for cell survival. Surprisingly, knockdown of cyclin Tl protein neither influenced the cell cycle nor induced apoptosis in cultured cell lines, and this was consistent with a previous report that knockdown of P-TEFb led to inhibition of HIV-1 replication but not resulted in cell death. Our data indicated that down regulation ofcyclin Tl did not induce significant apoptosis of transfected cells and cyclin Tl could be used as a potential target for inhibiting HIV-1 replication.We have also shown that knockdown of cyclin Tl expression by RNA interference inhibited HIV-1 replication specifically and effectively. Furthermore, we observed the correlation between the levels of down-regulation of cyclin Tl expression and the inhibition of HIV-1 replication, indicating that the replication level of HIV-1 may be dependent on the amount of cyclin Tl in cultured cells.In previous reports about RNAi with HIV-1 replication and infection, chemically synthesized siRNAs were most commonly used, however, synthetic siRNAs do not persist for long period in cells and are not feasible to be delivered into the vast number of target cells like T lymphocytes and macrophages. Our result renders a possibility to deliver the anti-HIV shRNA-encoding genes by viral vectors into target cells and stably express the shRNAs.Taken together, this study demonstrated the cyclin Tl could be a promising cellular target for suppression of HIV-1 replication by DNA-based shRNAs. Now, We are focusing on the delivery of shRNA-encoding genes into primary cells, such as T lymphocytes, monocytes and macrophages by lentiviral vectors and examine the feasibility to use cyclin Tl as target in genetic therapy of AIDS.