lncRNA-191对NK细胞抗肿瘤功能的影响和机制研究

Nature Killer (NK) cells play an important role in tumor immunosurveillance, whose anti-tumor ability is strictly correlated to the occurrence and development of tumor. Long non-coding RNA (lncRNA) has many biological functions, and also regulates immune cells. We firstly found that the expression of lncRNA-191 was obviously lower in NK cells from cancer patients comparing to healthy donors. The cytotoxicity and IFN-γ secretion of NK cells were down regulated through knockdown lncRNA-191 using CRISPR technique. These demonstrate that lncRNA-191 involves in the regulation of NK-mediated anti-tumor functions. Additionally, lncRNA-191 was found located in the nucleus, which suggested that lncRNA-191 might modulate the antitumor effects of NK cells via regulating the gene expression. Therefore, in our future work, the genes regulated by lncRNA-191 would be screened through reverse transcription-associated trap, and then confirm by knockdown or overexpress the gene. Furthermore, the mechanism of how lncRNA-191 regulates the target gene would be explored by double luciferase report system, truncated mutant, chromosome conformation capture (3c), and RNA 3C. Hence, these would disclose the reason why the anti-tumor functions of NK cells are decreased in tumor patients at lncRNA level, and provide theoretical basis for the cancer clinical treatment and new drug development.

NK细胞对肿瘤具有免疫监视作用,其抗肿瘤能力高低与肿瘤的发生、发展密切相关。lncRNA生物学功能丰富，对免疫细胞也有调节作用。我们首次发现肿瘤患者NK细胞中lncRNA-191的表达明显低于正常人；敲除lncRNA-191后，NK细胞对肿瘤细胞的杀伤作用和IFN-γ分泌能力显著降低，表明lncRNA-191能够调控NK细胞的抗肿瘤功能。由于lncRNA-191主要位于细胞核内，提示其可能通过调控基因的表达来影响NK细胞的抗肿瘤功能。因此，我们拟通过RNA相关捕获、基因敲除或过表达技术筛选受lncRNA-191调控的靶基因；采用双荧光素酶报告系统、截短突变、染色体构象捕获（3C）、RNA介导的3C等技术研究lncRNA-191调控靶基因的分子机制。本课题的完成将从lncRNA调控的角度找到肿瘤患者体内NK细胞抗肿瘤功能降低的原因，并为肿瘤治疗及新药研发提供理论依据。

长链非编码RNA（long non-coding RNA，lncRNA）在免疫系统的调节和自身免疫的发展中起着至关重要的作用。本研究发现了一个新的lncRNA——lncRNA-191，它能够提高NK细胞的抗肿瘤作用，进一步研究发现lncRNA-191可以同时通过抑制BTBD11分子的表达和促进Gab2分子的表达来提高NK细胞对肿瘤细胞的杀伤作用。主要结果如下：.1.明确了lncRNA-191能够提高NK细胞的抗肿瘤功能：在NK细胞中升高和降低lncRNA-191，通过calcein释放法和CD107a脱颗粒实验发现lncRNA-191与NK细胞的抗肿瘤作用正相关。.2.发现lncRNA-191能够调控BTBD11和Gab2的表达：在NK细胞中过表达lncRNA-191，采用RNA相关的捕获技术、高通量测序技术、qPCR技术和免疫印迹技术分析lncRNA-191调控的基因，发现lncRNA-191能够降低BTBD11分子的表达，同时能够上调Gab2分子的表达。.3.发现BTBD11分子能够抑制NK细胞的抗肿瘤功能：在NK细胞中升高和降低BTBD11分子，通过calcein释放法和CD107a脱颗粒实验发现BTBD11与NK细胞的抗肿瘤功能负相关。.4.发现Gab2分子能够提高NK细胞的抗肿瘤功能：在NK细胞中升高Gab2分子，通过calcein释放法和CD107a脱颗粒实验发现升高Gab2后，NK细胞的抗肿瘤功能也提高。.5.发现lncRNA-191能够通过表观遗传学修饰抑制BTBD11分子的表达：采用CHIP-qPCR技术，发现lncRNA-191能够降低BTBD11启动子区域组蛋白H3K4me3和H3K27ac3的修饰水平，抑制BTBD11的转录。.6.发现lncRNA-191能够通过表观遗传学修饰促进Gab2分子的表达：采用亚硫酸氢盐测序技术、CHIP-qPCR技术和免疫印迹技术，发现lncRNA-191能够降低Gab2启动子区域DNA的甲基化状态、提高组蛋白H3K4me3和H3K27ac3的修饰水平，促进Gab2的转录和蛋白的表达。. 本研究的顺利完成，从lncRNA层面阐述了NK细胞功能调控的新模式，开拓了lncRNA在免疫系统中的作用，拓展了人们对lncRNA的认识，能够为NK细胞的临床治疗及新药研究提供更加丰富的理论指导。