Study On The Effect And Mechanism Of Cell Growth Induced By Bcl-3in HTLV-1-infected Cells

Background Human T cell leukemia virus type1(HTLV-1) is a kind of retrovirus which infects theCD4+, CD8+T cells and dendritic cells. It is the pathogene of adult T cell leukemia (ATL). Tax, the virus protein of HTLV-1, can regulate the cellular transformation and viral replication through a variety of cellular mechanisms and signal pathway. Tax induced NF-κB signal pathway persistent activation is considered to be the main cause of ATL. Bcl-3is a member of IκB family, it can promote or inhibit the NF-κB dependent gene transcription. An elevated Bcl-3expression was found in many HTLV-1infected cells and ATL cells. However, the mechanism of Bcl-3in the pathogenesis of HTLV-1is still not clear.Objective Study the influence on the growth, proliferation, DNA damage and NF-κB activity after knockdown of Bcl-3in HTLV-1infected cells.Methods1. RT-PCR and Western blot:Measure the level of Bcl-3mRNA and protein in MT2, Jurkat, IM9, THP-1and MT4transfected Bcl-3shRNA.2. Immunofluorescence:Observe the nuclear translocation of Bcl-3and p65in Tax infected Jurkat cells and TaxP cell line, which continuously express Tax, respectively.3. Comet assay:Measure the influence on DNA damage and cell growth in Bcl-3shRNA transfected cell.4. MTT assay:The MT2cells were transfected with Bcl-3shRNA plasmid, the effect on cell growth was detected;5. Luciferase reporter assay:Observe the influence on NF-κB activity after knockdown of Bcl-3by co-transfection of p NF-κB-luc plasmid and Bcl-3shRNA plasmid. Results1.The level of Bcl-3mRNA and protein in HTLV-1infected cell line was higher than that in uninfected cell line by real time PCR and western blot;2. The nuclear translocation of p65was observed in TaxP cell line by immunofluorescence, which continuously expresses Tax;3. The expression and nuclear translocation of Bcl-3was observed in Jurkat cell transfected with pCMV-Tax by immunofluorescence;4. Bcl-3shRNA can remarkably reduce Bcl-3mRNA and protein expression in MT2cells which were detected by real time PCR and western blot;5. The inhibition was observed in HTLV-1infected cell line transfected with Bcl-3shRNA by MTT;6. DNA damage was observed in HTLV-1infected cell line transfected with Bcl-3shRNA by Comet assay;7. The inhibition of NF-κB activation was observed in HTLV-1infected cell line transfected with Bcl-3shRNA by Luciferase reporter assay.Conclusion Knockdown of Bcl-3in HTLV-1cells can induce DNA damage, inhibition of cell growth and NF-κB activation.