miRNA-561-5p调控CX3CL1表达介导NK细胞浸润在肝癌侵袭转移中的作用及机制

Tumor microenvironment plays an important role in metastasis of hepatocellular carcinoma (HCC). In our previous study, through small RNA sequencing, we found that miR-561-5p was associated with tumor metastasis. Further, miR-561-5p significantly promoted the invasion of HCC cells and suppressed the luciferase activity of CX3CL1 containing a wild-type 3'-UTR. Interestingly, CX3CL1 secreted by HCC cells has a powerful ability for recruiting natural killer (NK) cells in a dose-dependent manner. In this study, we firstly plan to confirm how miR-561-5p affected the metastasis of HCC through the regulating the expression of CX3CL1 by in vitro and in vivo study. In addition, we will explore the effects of CX3CL1 expression on the important signaling pathways related to HCC progression, including ERK1/2, MAPK and so on. Secondly, we will determine the key chemokine/cytokine CX3CL1 inducing NK cells recruitment which were involved in the metastasis and recurrence of HCC. Co-transfection and orthotopic transplantation model will be used to analyze the role of miR-561-5p on CX3CL1-induced NK infiltration. Finally, using tissue microarrays (TMAs) and fluorescent in situ hybridization (FISH) in HCC samples, we will determine the relationship between expressions of miRNA-561-5p and CX3CL1 and NK infiltration, and evaluate their prognostic significance. Through the above research, we will determine the effect and mechanism of miRNA-561-5p-introduced CX3CL1 in recruiting regulatory NK cells infiltration in the HCC microenvironment, and provide experimental evidence for exploring the new intervention targets on the metastasis and recurrence of HCC.

肿瘤微环境在肝癌转移中发挥重要作用。前期工作中，通过小RNA测序和荧光素酶实验发现miR-561-5p促进肝癌转移，与其调控靶基因CX3CL1表达密切相关；体外趋化实验证实CX3CL1是肝癌细胞趋化自然杀伤细胞(NK)，并诱导其癌内浸润的关键因子。本课题拟通过体外细胞功能实验和体内小鼠肝癌成瘤实验，明确miR-561-5p调控CX3CL1表达在肝癌侵袭转移中的作用，并观察CX3CL1表达对ERK1/2、MAPK等肝癌相关主要信号通路的影响；同时应用磷酸化蛋白芯片和免疫印迹分析CX3CL1诱导NK细胞浸润的关键分子；通过共转染和原位移植瘤实验分析miRNA-561-5p在CX3CL1介导NK细胞浸润中的作用；最后在肝癌组织样本中进行免疫组化和原位杂交检测，观察miR-561-5p、CX3CL1及NK细胞表达和定位的关联性，并对其预后价值进行评价。本研究为肝癌治疗中新的干预靶点提供实验依据。

通过microRNA测序检测有或无肺转移的肝癌患者肝癌及癌旁组织和肝癌细胞microRNA表达，发现miR-561-5p与肝癌细胞的肺转移能力密切相关。它在体内可促进肝癌增殖和肺转移，但体外对肝癌细胞的运动侵袭能力未见明显影响。生物信息学和趋化因子表达谱检测提示CX3CL1为miR-561-5p的潜在靶标。miR-561-5p通过抑制CX3CL1表达，减少CX3CR1+NK细胞的浸润和细胞毒性，促进肝癌恶化。CX3CR1+NK细胞呈现较强的抗肿瘤转移活性，该作用与CX3CR1+NK细胞内STAT3信号通路活化相关。临床上，miR-561-5p与CX3CL1表达水平、CX3CR1+NK细胞数量呈显著负相关。高表达miR-561-5p, 低表达CX3CL1,CX3CR1+ NK细胞数减少提示肝癌患者不良预后。该成果以通讯作者，发表在Theranostics杂志（2019年）（IF=11.6）。此外，我们体内外实验证实RocA通过抑制肝癌细胞自噬、增强NK细胞颗粒酶的释放以增强杀伤肝肿瘤细胞的效果。我们还发现MTDH可通过β-catenin/LEF-1信号通路上调PD-L1转录活性，MTDH反义寡核苷酸（ASO)可增加PD-1单抗反应；HNRNPM抑制可促进CD8+ T细胞活化，其ASO能促进CD8+ T细胞浸润增强PD-1免疫治疗。研究成果以通讯作者，分别发表在“Cancer Lett”（2022年，IF=9.8）和“Cell Mol Gastroenterol Hepatol”（2022年，IF=8.8）。我们构建了与TP53突变谱、免疫细胞特征和肝癌患者预后密切相关的“表观遗传调节因子得分”；建立了由6个基因组成的患者预后预测价值的干性标签；发现m6A RNA调节基因的突变和拷贝数变异与肝癌患者的不良预后密切相关。研究成果以通讯作者，发表在“Front Immunol”（2022年，IF=8.8），“Cancer Lett”（2021年，IF=9.8）等。.在本项目资助下，课题负责人以通讯作者发表与本课题相关的论文12篇（均为SCI收录），获得专利5项。2019年荣获华夏医学科技奖一等奖和第三十一届上海市优秀发明选拔赛优秀发明金奖。作为大会秘书长，组织5次大型全国学术会议。共培养研究生6人，其中,已毕业博士和硕士生分别为3人和3人。