HIV感染疾病快速进展相关microRNA对T细胞功能调控的作用及机制研究

Multiple longitudinal studies have revealed dramatic variation in the outcome of untreated diseases following HIV-1 infection, ranging from rapid AIDS development in under a year to no progression decades later. The unique pathogenic development of HIV-infected patients with rapid progression remains less understood. microRNAs are small, highly conserved evolution no-coding RNAs which can repress the target gene expression at a post-transcriptional level. microRNAs play important roles in the regulation of T cell development and function. T cell responses in early HIV infection have been recognized as one of the most important factors in determining HIV disease progression. Although microRNAs have been shown to play a role in elite controlling of HIV-1 and differentiating multiply exposed uninfected individuals from LTNP and HIV-1-positive subjects, whether they contributed to rapid disease progression has not been reported to our knowledge. Our preliminary data showed 5 miRNAs (miR-31, -200c, -526a, -99a and -503) in rapid progressors (RPs) were significantly decreased compared to those in chronic progressors (CPs). The decreased expression of these miRNAs was associated with rapid disease progression of HIV infection with a 94% predictive value as measured by the area under the curve of ROC analysis. Pathway enrichment analysis showed that the upregulated predicted targets of the 5 signature miRNAs in RPs compared with that in CPs were primarily involved in pathways associated with apoptosis, cell cycle and DNA damage. Moreover, in vitro overexpression of miR-31 in primary human T cells promoted their survival. However, whether and how these microRNAs directly regulate the T cell function in HIV infection are not known. In this study, we will investigate the regulation of 5 microRNAs on T cell function and their roles in rapid disease progression by using the platforms of miRNAs overexpession/inhibition, T cell functional phenotype detection, target gene analysis and in vitro HIV infection. This study will help us to further understand the molecular mechanisms underlying the rapid disease progression and is essential for anti-HIV treatment, as well as therapeutic vaccine development.

HIV感染后疾病进展速度不同，部分感染者感染1-3年即进入艾滋病期，被称为快速进展者，具体机制尚不明确。microRNA对T细胞发育、分化和免疫应答具有全方位的调控作用，而T细胞对HIV急性感染的最初应答，已经被公认为影响HIV疾病进展的决定性因素。我们通过前期研究，发现miR-31等5种microRNA在HIV快速进展者外周血中明显低表达，可预测疾病进展；mRNA芯片检测及信号通路分析发现这组microRNA可能作用于细胞凋亡、细胞周期等基因；体外过表达miR-31可抑制T细胞凋亡。目前，这5种microRNA对于HIV感染T细胞功能的调控作用尚未见报道。我们拟通过miRNA过表达及抑制、T细胞功能表型分析、靶基因鉴定及体外HIV感染等平台，深入研究上述microRNA对T细胞功能的调控及机制，探讨其与HIV感染疾病进展的机制，为HIV的疫苗、免疫治疗及生物标志物的研究提供新靶标。

HIV感染人体后会导致严重的细胞免疫损伤，造成人体免疫机能缺陷，最终引发获得性免疫缺陷综合征。作为最重要的免疫效应细胞，T细胞功能在抗HIV感染免疫中发挥重要作用。但是T细胞免疫在HIV感染早期就出现了损伤，包括功能缺失，不能有效活化等，导致疾病进展，但其调控机制不明确。本研究首先过表达HIV感染疾病进展相关5个miRNA，对T细胞凋亡、细胞周期、增殖、免疫活化及细胞杀伤等功能进行了研究，筛选到了可有效调控T细胞有效活化的miR-31。我们的研究发现HIV感染者T细胞被TCR刺激后，有效活化指标CD69显著下降。为明确miR-31是否调控T细胞有效活化，我们在T细胞中加入miR-31 antagomir，发现抑制miR-31水平可显著降低CD4+、CD8+T细胞表面CD69的表达，抑制其有效活化；为明确其作用机制，我们通过靶基因分析，选择了4个miR-31可能作用的靶基因，过抑制及过表达实验证实miR-31可直接向Ras-MAPK信号通路中的负调控因子KSR2和DUSP7，从而影响T细胞有效活化；Ras-MAPK通路信号传导可磷酸化并激活ERK1/2蛋白，促进T细胞的有效活化，我们的进一步研究显示，miR-31过表达可以明显促进ERK磷酸化水平，证实miR-31可有效调控胞内信号Erk表达从而调控T细胞活化。最后，过表达miR-31可恢复CD4+、CD8+T细胞表面CD69的表达，有效改善HIV感染受损的T细胞反应能力。研究结果为明确HIV感染的免疫损伤机制及创新免疫干预提供重要信息。