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

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。

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.