细胞核亚结构paraspeckle重塑肿瘤微环境影响免疫治疗的机制研究

肝细胞肝癌是一类严重威胁人类健康的疾病，针对肝癌的免疫治疗目前仍处于起步阶段。申报人前期发表的论文报道了一类由长链非编码RNA-NEAT1_2为骨架的细胞核内亚结构paraspeckle通过结合PRDX5 mRNA及GSTP1蛋白促进肝癌进展。而此类亚结构同肝癌免疫治疗效果不佳是否存在联系，尚不清楚。我们前期采用肝癌细胞系与抗PD1激活T细胞共孵育的方法，并对NEAT1_2和肝癌细胞存活率相关性分析，发现高表达NEAT1_2的肝癌细胞更容易耐受激活T细胞的杀伤。应用Crisper/Cas9技术和转录组分析，我们发现NEAT1_2可以特异性结合IFNGR1 mRNA抑制IFNGR1蛋白表达。本项目拟在体内外水平进一步探究paraspeckle结合IFNGR1 mRNA的分子机制及在肝癌耐受T细胞免疫杀伤中的作用，为临床改善肝癌免疫治疗效果提供新思路。

Hepatocellular carcinoma (HCC) is a serious threat to human health. Immune therapy about HCC is still in a development containing multiply problems including poor therapeutic effect. Our earlier published study reported a kind of nuclear substructure-paraspeckle mediated by lncRNA-NEAT1_2 promotes HCC progression through binding PRDX5 mRNA and GSTP1 protein. However, whether paraspeckle is related to the poor therapeutic effect of immune therapy on HCC is unknown. By incubating different kinds of HCC cell lines with PD1 antibody-mediated activated T cells, we screened the T cells killing-resistant HCC cell lines. Then, performing correlation analysis about NEAT1_2 expression of HCC cell lines and survival rate, we found a higher NEAT1_2 expression cell line attend to more T cells killing-resistant. To clarify the molecular mechanism involved in, we constructed HCC cell lines endogenous expressing M1 tag NEAT1_2. By co-IP using specific magnetic beads and subsequent mRNA-seq, we found NEAT1_2 binds to IFNGR1 mRNA suppressing IFNGR1 protein expression affecting the response capability to IFN-γ. Based on that, this study aims to further explore the molecular mechanism of paraspeckle binding to IFNGR1 mRNA and its detailed roles in immune therapy on HCC in vitro and in vivo, to provide new ideas for clinical treatment for HCC and new potential targets for the development of HCC drugs.