lncRNA NKCLR3调控NK细胞抗肿瘤活性的表观遗传学机制

Nature killer (NK) cells play an important role in tumor immunosurveillance, whose quantity and function is strictly correlated to the survival and prognosis of hepatocellular carcinoma(HCC). Long non-coding RNA (lncRNA) has many biological functions, and also regulates immune cells. We firstly found that the expression of lncRNA NKCLR3 was obviously higher in NK cells from hepatocellular carcinoma patients comparing to healthy donors. The cytotoxicity and IFN-γ secretion of NK cells were up regulated through knockdown NKCLR3, reminding us that NKCLR3 has a regulatory role on NK cell cytotoxicity. Further experiments proved that NKCLR3 was located in the nucleus, suggesting that NKCLR3 might modulate the antitumor effects of NK cells via regulating the gene expression. Therefore, in this project, the genes regulated by NKCLR3 would be screened through reverse transcription-associated trap, and then confirmed by knockdown or overexpression the gene. Furthermore, the mechanism by which NKCLR3 regulates the target gene would be explored by chromosome conformation capture (3c), and RNA 3C. Also qPCR was used to detect the differential expression of NKCLR3 in peripheral blood NK cells of healthy donors and different stages of HCC patients, and then analyze the correlation between NKCLR3 expression and the prognosis of HCC. If this research should be completed, it is hoped to disclose the reason why the anti-tumor functions of NK cells are decreased in hepatocellular carcinoma patients at lncRNA level, provide new target to improve the antitumor activity of NK cells in hepatocellular carcinoma patients, and offer a new perspective and theoretical basis for the cancer immunotherapy.

NK细胞对肿瘤具有免疫监视作用，其数量和功能与肝癌患者的生存和预后明显相关。lncRNA生物学功能丰富，对免疫细胞有调控作用。我们首次发现lncRNA NKCLR3在肝癌患者NK细胞中的表达明显高于健康人，敲除NKCLR3后，NK细胞对肿瘤细胞的杀伤作用和IFN-γ分泌能力显著提高，表明NKCLR3能够调控NK细胞的抗肿瘤活性。NKCLR3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细胞的功能。因此，我们拟通过RAT、基因敲除或过表达等筛选受NKCLR3调控的靶基因；采用3C、R3C等揭示NKCLR3调控靶基因的分子机制；利用qPCR分析NKCLR3在正常人和不同分期肝癌患者NK细胞中的差异性表达及与预后的相关性。本课题的完成将从lncRNA调控的角度解释肝癌患者NK细胞抗肿瘤功能降低的原因，为提高NK细胞的抗肿瘤活性提供新的靶点，为肿瘤的免疫治疗提供全新的视角及理论基础。

长链非编码RNA（long non-coding RNA，lncRNA）在自身免疫的发展和免疫系统的调控中起着重要的作用。本研究发现了一个新的lncRNA——NKCLR3，它能够通过抑制NCR1基因的表达来抑制NK细胞的抗肿瘤活性。主要结果如下：.1.明确了NKCLR3能够抑制NK细胞的抗肿瘤功能：肝癌患者NK细胞对肿瘤细胞的杀伤作用低于健康对照组，而NKCLR3的表达明显高于健康对照组；电转siRNA敲除NK-92MI细胞及肝癌患者NK细胞中的NKCLR3，NK 细胞的抗肿瘤活性明显提高。说明NKCLR3与NK细胞的抗肿瘤作用负相关。.2.发现了NKCLR3能够调控NCR1的表达：通过核质分离 PCR和 RNA FISH 确定NKCLR3主要存在于胞核中；在NK-92MI细胞中敲除或过表达NKCLR3，采用RNA相关捕获实验、高通量测序、qPCR等技术分析NKCLR3调控的基因，发现NKCLR3能够降低NCR1基因的表达。.3. 探讨了NKCLR3调控NCR1基因的分子机制：采用Luciferase双荧光素酶报告实验，提示NKCLR3可抑制NCR1启动子的活性；利用CHIP-qPCR技术、亚硫酸氢盐测序技术，发现NKCLR3能够降低NCR1启动子区域组蛋白H3K4me3和H3K27ac3的修饰水平，提高 NCR1启动子区域 DNA甲基化状态，抑制NCR1的转录。.4. 分析了NKCLR3表达水平与肝癌分期的相关性：肝癌患者巴塞罗那分期越晚，NKCLR3的表达越高，NK细胞对肿瘤细胞的杀伤作用越弱。.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，为提高肿瘤患者NK细胞的抗肿瘤活性提供新的靶点，并为肿瘤的免疫治疗提供全新的思路及理论基础。