MiRNA expression variation in primary NK cells treated with TGF-β1. Homo sapiens

Human NK cells activity against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. In order to identify possible mechanisms mediating chemokine receptors modulation, we analyzed the miRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3’ untranslated region (3’UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3’UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p down-regulates the surface expression of CX3CR1. Finally we showed that Neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX3CR1 expression. Overall design: Real-time PCR-based miRNA expression profiling. Primary resting NK cells from three donors were treated for 24h with 5 or 40 ng/ml TGF-β1 or were left untreated.

人类自然杀伤细胞（Natural Killer cells, NK）对癌细胞的杀伤活性可被转化生长因子-β1（Transforming Growth Factor-β1, TGF-β1）深度抑制，而TGF-β1是一种在肿瘤微环境中释放并激活的免疫调节细胞因子。此外，我们此前的研究数据显示，TGF-β1可改变NK细胞的趋化因子受体谱。具体而言，它会降低CX3CR1的表达，而CX3CR1可引导这些免疫效应细胞迁移至包括肿瘤部位在内的外周组织。
为了阐明介导趋化因子受体调控的潜在机制，我们分析了经TGF-β1处理的原代NK细胞的miRNA表达谱。该分析筛选出微小RNA-27a-5p（microRNA-27a-5p, miR-27a-5p）可作为CX3CR1的潜在调控因子。我们通过两种不同的实验方法证实了miR-27a-5p与CX3CR1 mRNA的3’非翻译区（3’ untranslated region, 3’UTR）存在功能性结合：其一为基于携带CX3CR1 3’UTR的报告基因载体的荧光素酶报告实验，其二为使用miR-27a-5p抑制剂转染原代NK细胞的实验。
我们同时证实，TGF-β1介导的miR-27a-5p表达上调，源于miR-23a-27a-24-2基因簇的诱导激活。此外，我们证实miR-27a-5p可下调CX3CR1的细胞表面表达水平。最后我们发现，神经母细胞瘤细胞可诱导静息NK细胞的CX3CR1表达下调，且该过程伴随miR-27a-5p表达的显著升高。
综上，本研究明确了miR-27a-5p是TGF-β1诱导的CX3CR1表达关键调控因子。
实验整体设计：基于实时荧光定量PCR的miRNA表达谱分析。将来自3名健康供体的静息NK细胞分为三组：分别用5 ng/ml、40 ng/ml的TGF-β1处理24小时，以及不做处理的对照组。