The Mechanism Of Caveolin-1 Gene Silencing By ShRNA On The Destruction Of The Blood-brain Barrier Induced By HIV-1 TAT And The Aβ Transporter

Objectives:To construct a human cerebral microvascular endothelium cell line to stable knock-down Cav-1 by transfected with shRNA Cav-1recombinant lentivirus vectors.Methods:Cav-1 shRNA interfere target sequence was cloned into the lentiviral vector pHBLV-U6-Scramble-ZsGreen-Puro,and verified by using the method of double enzyme digestion and DNA sequencling.293T cells were transfected with the lentiviral packaging plasmids and the virus particles were collected.The virus titer was tested by using whole dilution method.Human cerebral microvascular endothelium cells were transfected with the virus particles.2 stable monoclonal cell lines with GFP strong expression were chosen for testing the Cav-1 inhibition rate by fluorescence quantitative PCR(Q-PCR).Results:After sequencing,RNA interference recombinant lentiviral expression vector targeting Cav-1 was established successfully.The recombined lentiviral particles were made by using 293T cells packaged with recombined lentiviral vector.The virus titer was 2×10~8TU/mL.2 stable monoclonal cell lines with GFP strong expression were acquired with the Cav-1inhibition rate85.7%and 81.4%,respectively.Conclusion:Human cerebral microvascular endothelium cells were constructed to stable knock-down Cav-1 by infection with shRNA Cav-1recombinant lentivirus vectors.These cell lines provide a good stool for further study of the relationship between HIV-associated neurocognitive dysfunction(HAND)and Cav-1 gene.Objective: To evaluate the role of Cav-1-sh RNA in HIV-1 Tat-induced dysfunction of blood-brain barrier(BBB)and amyloid accumulations.Methods: Cav-1 expression was silenced by transfected of HBEC-5i with Cav-1 sh RNA and Ctr sh RNA were exposed to HIV-1 Tat for 24 h,the viability of cells was tested by CCK8 assay.HBEC-5i transfected with cav-1 sh RNA and Ctr sh RNA were exposed to HIV-1 Tat for 24 h,the Rho A,occludin,RAGE,LRP-1 protein and m RNA levels were evaluated with Western blot or Real-time reverse transcription polymerase chain reaction(q RT-PCR)assay respectively.Also,HIV-1 Tat induced alteration of immunoreactivity of RAGE and LRP-1was detected by Immunofluorescence(IF).Results: The concentrations of HIV-1 Tat under 1μg/m L had no significant effect on the Cav-1 sh RNA cell and Ctr sh RNA cell viability(P > 0.05)as determined by the CCK8 assay.Compared with the Ctr sh RNA,the Occludin protein and m RNA levels was decreased with HIV-1 Tat exposure,however,upregulated with HIV-1 Tat and Cav-1 sh RNA group.Compared with the Ctr sh RNA,the RAGE protein/m RNA levels,and stronger immunoreactivity was increased with HIV-1 Tat exposure,however,downregulated with HIV-1 Tat and Cav-1 sh RNA group.Comparedwith the Ctr sh RNA,the LRP-1protein/m RNA levels,and immunoreactivity was decreased with HIV-1 Tat exposure,however,upregulated with HIV-1 Tat and Cav-1 sh RNA group.Compared with the Ctr sh RNA,the Rho A protein level was upregulated with HIV-1 Tat exposure,however,downregulated with HIV-1 Tat and Cav-1 sh RNA group.Conclusion: These results show that HIV-1 Tat-induced downregulation of Occludin and LRP-1 andupregulation of Rho A and RAGE,may result in accumulation of Aβ in the brain.Silencing Cav-1 gene with sh RNA plays a key role on the protection against HIV-1 Tat-induced dysfunction blood-brain barrier and Aβ accumulations.