Laboratory of Neurovirology and Inflammation Biology, Centre for Cellular and Molecular Biology (CCMB), Council of Scientific and Industrial Research (CSIR), Uppal Road, Hyderabad 500007, India.
J Neuroinflammation. 2012 Jun 18;9:131. doi: 10.1186/1742-2094-9-131.
HIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion.
We were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA) expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study.
HIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor-associated factor 3 TRAF3) is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3) and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion.
HIV-1 Tat protein can modulate TRAF3 expression through miRNA mediated pathway and can change the downstream expression of IRF3 and IRF7. This study demonstrates a novel mechanism of HIV-1 Tat C protein-mediated perturbation of miRNA, resulting in dysregulation of cellular TRAF3.
已知 HIV-1 Tat 蛋白与神经炎症有关，而神经炎症是感染 HIV-1 的患者中近一半会出现的病症。HIV-1 Tat 可改变神经胶质的神经保护功能，导致中枢神经系统的神经毒性。已知 HIV-1 Tat 可从受感染的细胞中分泌出来，并通过旁分泌方式调节细胞基因表达，从而影响邻近的未受感染的细胞。
我们有兴趣研究外源性暴露于 HIV-1 Tat-C 蛋白是否会扰乱人小胶质细胞的 microRNA（miRNA）表达谱，从而导致蛋白表达改变。我们使用蛋白表达和纯化、miRNA 过表达、miRNA 敲低、转染、定点突变、实时 PCR、荧光素酶测定和 Western blot 技术进行研究。
HIV-1 Tat-C 处理人小胶质细胞导致 miR-32 的表达呈剂量依赖性增加。我们发现肿瘤坏死因子受体相关因子 3（TRAF3）是 miR-32 的直接靶标，CHME3 细胞中 miR-32 的过表达在 mRNA 和蛋白水平上均降低了 TRAF3。转染抗 miR-32 后 TRAF3 蛋白表达的恢复以及荧光素酶报告基因测定的结果提供了 TRAF3 受 miR-32 调控的直接证据。我们发现，干扰素调节因子 3（IRF3）和 IRF7 的调节受小胶质细胞中 TRAF3 蛋白的细胞水平控制，因为 miR-32 过表达和应用抗 miR-32 后，IRF3 和 IRF7 的表达水平呈相反的 TRAF3 表达水平调节。因此，我们的结果表明，在暴露于 HIV-1 Tat C 蛋白的人小胶质细胞中，一种新的 miRNA 介导的机制可以调节 TRAF3。这些结果可能有助于阐明 HIV-1 Tat C 蛋白以旁观者方式产生的有害神经炎症后果。
HIV-1 Tat 蛋白可以通过 miRNA 介导的途径调节 TRAF3 的表达，并可以改变下游的 IRF3 和 IRF7 的表达。本研究证明了 HIV-1 Tat C 蛋白介导的 miRNA 失调的新机制，导致细胞 TRAF3 失调。
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