DataSheet_1_Lnc-AIFM2-1 promotes HBV immune escape by acting as a ceRNA for miR-330-3p to regulate CD244 expression.docx

Chronic hepatitis B (CHB) virus infection is a major risk factor for cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) immune escape is regulated by the exhaustion of virus-specific CD8+ T cells, which is associated with abnormal expression of negative regulatory molecule CD244. However, the underlying mechanisms are unclear. To investigate the important roles of non-coding RNAs play in CD244 regulating HBV immune escape, we performed microarray analysis to determine the differential expression profiles of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in patients with CHB and patients with spontaneous clearance of HBV. Competing endogenous RNA (ceRNA) was analyzed by bioinformatics methods and confirmed by the dual-luciferase reporter assay. Furthermore, gene silencing and overexpression experiments were used to further identify the roles of lncRNA and miRNA in HBV immune escape through CD244 regulation. The results showed that the expression of CD244 on the surface of CD8+ T cells was significantly increased in CHB patients and in the co-culture system of T cells and HBV-infected HepAD38 cells, which was accompanied by the reduction of miR-330-3p and the elevation of lnc-AIFM2-1. The down-regulated miR-330-3p induced the apoptosis of T cells by lifting the inhibition of CD244, which was reversed by miR-330-3p mimic or CD244-siRNA. Lnc-AIFM2-1 promotes the accumulation of CD244, which is mediated by decreased miR-330-3p, and then reduced the clearance ability of CD8+ T cells to HBV through regulated CD244 expression. And the injury in the ability of CD8+ T cells to clear HBV can be reversed by lnc-AIFM2-1-siRNA, miR-330-3p mimic, or CD244-siRNA. Collectively, our findings indicate that lnc-AIFM2-1 on CD244 by acting as a ceRNA of miR-330-3p contributes to HBV immune escape, which may provide novel insights into the roles of interaction networks among lncRNA, miRNA, and mRNA in HBV immune escape, highlighting potential applications of lnc-AIFM2-1 and CD244 for diagnosis and treatment in CHB.

慢性乙型肝炎（Chronic hepatitis B, CHB）病毒感染是肝硬化与肝细胞癌（hepatocellular carcinoma, HCC）的主要危险因素。乙型肝炎病毒（hepatitis B virus, HBV）免疫逃逸受病毒特异性CD8+T细胞耗竭调控，该过程与负调控分子CD244的异常表达密切相关，但其潜在分子机制仍未阐明。为探究非编码RNA（non-coding RNAs）在CD244调控HBV免疫逃逸过程中的重要作用，本研究通过微阵列分析，明确了慢性乙型肝炎患者与HBV自发清除者体内长链非编码RNA（long non-coding RNAs, lncRNAs）、微小RNA（microRNAs, miRNAs）及mRNA的差异表达谱。本研究采用生物信息学方法分析内源竞争RNA（Competing endogenous RNA, ceRNA）调控网络，并通过双荧光素酶报告基因实验（dual-luciferase reporter assay）对其进行验证。此外，本研究还通过基因沉默与过表达实验，进一步明确了lncRNA与miRNA通过调控CD244在HBV免疫逃逸中发挥的生物学功能。研究结果显示，慢性乙型肝炎患者及T细胞与HBV感染的HepAD38细胞共培养体系中，CD8+T细胞表面CD244的表达水平显著升高，同时伴随miR-330-3p表达下调与lnc-AIFM2-1表达上调。下调的miR-330-3p通过解除对CD244的抑制作用诱导T细胞凋亡，该效应可被miR-330-3p模拟物或CD244-siRNA逆转。Lnc-AIFM2-1可通过降低miR-330-3p的表达水平介导CD244的积累，进而通过调控CD244的表达削弱CD8+T细胞对HBV的清除能力。而CD8+T细胞的HBV清除能力损伤可被lnc-AIFM2-1-siRNA、miR-330-3p模拟物或CD244-siRNA逆转。综上，本研究结果表明，lnc-AIFM2-1可作为miR-330-3p的内源竞争RNA靶向调控CD244，从而促进HBV免疫逃逸。该发现为阐明lncRNA、miRNA与mRNA之间的互作网络在HBV免疫逃逸中的作用提供了新的研究视角，同时也凸显了lnc-AIFM2-1与CD244在慢性乙型肝炎诊断与治疗中的潜在应用价值。