RNAm6A甲基化关键酶YTHDF2介导肿瘤巨噬细胞TLR4免疫，调控肝细胞癌放疗后复发及侵袭转移的机制研究

Radiotherapy (RT) is recommended for patients with local advanced hepatic cell carcinoma (HCC). However, HCC recurrence or metastasis post-RT are common RT failure reasons. The reversible RNA methylation at the 6 position of adenosine (m6A) (mRNA m6A) always serves as a “beacon” for DNA damage repairs, and can dynamically modify TLR4 -dependent immune. In our previous studies, we showed that TLR4-dependent immune could promote invasion and metastasis of HCC post-RT. RT may incentive tumor macrophage (Mac) (a nonparenchymal cell with TLR4 on membrane surface), change the microenvironment and then influence HCC invasion or metastasis. Moreover, analysis of TCGA database and 118 cases of HCC data in our hospital showed that patients with lower YTHDF2，a kind key protein of reversible mRNA m6A, expression in HCC might survive longer post-operation; and detected by the second generation gene sequencing, TLR4 immunity was associated with YTHDF2 expression level in Mac with 6Gy irradiation. Herein, we hypothesize that mechanisms of HCC radiosensitivity change, HCC invasion or metastasis post-RT are as follows: YTHDF2 regulate the reversible mRNAm6A of TLR4-dependent immune response in tumor Mac; and microenvironment may serve as a critical medium between them. Therefore, in this project, using cellular experiments in vitro, animal experiments in vivo and clinical verification, we will explore novel targets，from new perspectives of microenvironment characteristic protein and reversible mRNAm6A of TLR4-dependent immune, to predict or intervene HCC radiosensitivity, invasion or metastasis post-RT!

肝细胞肝癌（HCC）放射治疗（RT）后复发转移是常见的放疗失败原因。RNAm6A甲基化的可逆修饰在DNA损伤修复中起“灯塔性”作用，并可介导TLR4免疫。我们前期研究示：HCC放疗中，TLR4免疫可促进侵袭转移；刺激肿瘤巨噬细胞（Mac）（胞膜表达TLR4的间质细胞），亦可影响微环境，促进侵袭转移；分析TCGA数据库和我院118例HCC资料则发现：癌组织m6A关键蛋白 YTHDF2表达下调的HCC患者术后生存时间长；而二代基因测序检测6Gy照射的Mac示：YTHDF2与TLR4免疫相关。故我们推测：YTHDF2介导TLR4免疫mRNAm6A可逆修饰是调控HCC放射敏感性及RT后复发转移的重要机制；微环境为关键媒介。项目中我们拟采用体外细胞、体内动物实验与临床验证相结合，期望从TLR4免疫mRNAm6A可逆修饰及微环境特征蛋白等新角度，探索预测或干预HCC放射敏感性及RT后复发转移的新靶点!

放射治疗（RT）是肝细胞癌（HCC）的有效治疗方法，但不少患者仍死于RT后复发或转移。TLR4免疫可调控HCC侵袭转移，影响肿瘤RT疗效；而RNAm6A甲基化的可逆修饰在DNA损伤修复中起“灯塔性”作用，并可介导TLR4免疫。课题组在本项目中，首先，建立并获得了6Gy放疗后存活THP1、97H、7721细胞株，发现：下调TLR4免疫，可以抑制THP细胞产生免疫炎症因子（如GM-CSF等）; m6A通路蛋白（YTHDF2等）参与了RT中免疫微环境的调控，影响RT后肝癌细胞侵袭转移；建立裸鼠肝HCC高转移模型和低转移放疗模型，抑制TLR4免疫通路，可抑制放疗后肿瘤生长。进一步，创造性地建立具有辐射耐受性的肝癌97H-RT(18Gy)和免疫功能性细胞THP1-RT(18Gy)细胞株，建立免疫缺陷小鼠重现人肝细胞癌放疗后肿瘤免疫微环境的动物模型。构建RNAi，沉默97H及THP1细胞Ythdf2基因。体外细胞侵袭实验发现沉默肝癌细胞与免疫细胞Ythdf2基因，与诱导肝癌细胞与免疫细胞放疗抵抗，具有相同的抑制侵袭转移的能力。最后，行RNA-SQ基因组测序及m6A测序分析等筛选出：肿瘤免疫微环境中受m6A甲基化调控的相关基因（如LOXL4、VCAM1、ICAM5、TIFAB等），TLR4等免疫细胞相关炎症因子（如GM-CSF等）作为防治HCC放疗后复发转移的潜在靶点。这将有助于改变HCC放疗时，临床长期无有效防治其放疗后复发转移方法的状况。