Role of Cav-1 in HIV-1 Tat-Induced Dysfunction of Tight Junctions and Aβ-Transferring Proteins

Abstract

Objective. To evaluate the role of caveolin-1 (Cav-1) in HIV-1 Tat-induced dysfunction of tight junction and amyloid β-peptide- (Aβ-) transferring proteins. Methods. A Cav-1 shRNA interference target sequence was cloned into the lentiviral vector pHBLV-U6-Scramble-ZsGreen-Puro and verified by double enzyme digestion and DNA sequencing. Human cerebral microvascular endothelium (HBEC-5i) cells were transduced with viral particles made in 293T cells by transfection with lentiviral packaging plasmids. HBEC-5i cells transduced with Cav-1 shRNA or Ctr shRNA were exposed to HIV-1 Tat for 24 h, and the protein and mRNA levels of the tight junction protein occludin, Aβ-transferring protein, receptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein- (LRP-) 1, and RhoA were evaluated with Western blot and real-time reverse transcription polymerase chain reaction (qRT-PCR) assays, respectively. Results. After sequencing, an RNA interference recombinant lentivirus expressing a vector targeting Cav-1 was successfully established. The recombined lentiviral particles were made by using 293T cells to package the recombined lentiviral vector. A stable monoclonal cell line with strong GFP expression was acquired with a Cav-1 knockdown rate of 85.7%. The occludin protein and mRNA levels in the Ctr shRNA group were decreased with HIV-1 Tat exposure but were upregulated in the Cav-1 shRNA group. The HIV-1 Tat-induced alterations of RAGE and LRP-1 protein and mRNA levels in the Ctr shRNA group were attenuated in the Cav-1 shRNA group. The RhoA protein levels in the Ctr shRNA group were upregulated by HIV-1 Tat exposure but were downregulated in the Cav-1 shRNA group. Conclusion. These results show that HIV-1 Tat-induced downregulation of occludin and LRP-1 and upregulation of RAGE and RhoA may result in the accumulation of Aβ in the brain. Silencing the Cav-1 gene with shRNA plays a key role in the protection against HIV-1 Tat-induced dysfunction of the blood-brain barrier and Aβ accumulation.

Occludin, caveolin‐1, and Alix form a multi‐protein complex and regulate HIV‐1 infection of brain pericytes

“…Other studies, however, have suggested that cav‐1 may serve as an effective target for protecting against HIV‐1‐related disruption of the BBB 51 . Silencing the cav‐1 gene has also been described as having a key role in protecting against HIV‐1 by defending against HIV‐1 Tat‐induced downregulation of ocln 52 …”

“…51 Silencing the cav-1 gene has also been described as having a key role in protecting against HIV-1 by defending against HIV-1 Tat-induced downregulation of ocln. 52 Modification of the cav-1-ocln-Alix complex by overexpression of ocln also resulted in a prominent decrease in p24 levels in HIV-1-infected pericyte cultures ( Figure 6). These findings are consistent with our reports 25 in which we characterized ocln as a novel NADH oxidase that inhibits HIV-1 transcription by controlling SIRT-1 expression and activation in human brain pericytes.…”

Long non‑coding RNA CASC2 suppresses pancreatic cancer cell growth and progression by regulating the miR‑24/MUC6 axis

“…Morphological changes of pancreatic cancer cells were assessed by light microscopy. The sequences were as follows: miR-24 mimics, sense 5 '-UGG CUC AGU UCA GCA GGA ACA G-3', antisense 5'-GUU CCU GCU GAA CUG AGC CAU U-3'; mimics NC, sense 5'-UUC UCC GAA CGU GUC ACG UTT-3' , antisense 5'-ACG UGA CAC GUU CGG AGA ATT-3'; miR-24 inhibitor, 5'-CUG UUC CUG CUG AAC UGA GCC A-3'; inhibitor NC, 5'-CAG UAC UUU UGU GUA GUA CAA-3'. For in vivo studies, AsPC-1 cells were transduced with lentivirus (LV)-CASC2 (LV5-EF1a-GFP/Puro vector; Shanghai GenePharma Co., Ltd.) and LV-miR-24 (LV3-pGLV-h1-GFP-puro vector; Shanghai GenePharma Co., Ltd.), or LV-NC vectors (LV-CASC2-NC and LV-miR-24 NC; Shanghai GenePharma Co., Ltd.) as previously described (22). Briefly, AsPC-1 cells (5x10 5 per well) were plated in 6-well plates for 24 h; the medium was then replaced with fresh medium containing 8 µg/ml polybrene.…”

Systemic exosomal miR-193b-3p delivery attenuates neuroinflammation in early brain injury after subarachnoid hemorrhage in mice

“…The cells (2 × 10 5 per well in 6-well plates) were transfected with LipoFiter™ Liposomal Transfection Reagent (Hanbio Biotechnology, China), as per the protocol provided by the lentivirus manufacturer. Retroviral infection was performed as previously described [26].…”