RUNX3介导的CD49d表达增加NK细胞活性影响肝癌侵袭转移的作用机制

NK细胞在肝癌进展中发挥重要作用。我们前期发现，抑制CD49d表达可减少NK细胞杀伤活性，并促进肝癌细胞恶化，通过荧光素酶实验证实RUNX3可调控CD49d表达，于是，我们提出RUNX3介导的CD49d表达增加NK细胞活性影响肝癌进展的假说。本课题拟通过干预NK细胞中CD49d表达，观察其生长凋亡、运动和杀伤活力的变化，同时明确CD49d通过其配体paxillin影响肝癌生长和侵袭转移的作用及机制；通过共转染RUNX3和CD49d过表达质粒或shRNA，进行体内外功能分析，明确NK细胞中RUNX3调控CD49d表达影响肝癌侵袭转移的作用；应用染色质免疫沉淀、荧光素报告基因分析RUNX3表达对CD49d转录调控的影响；最后，在肝癌组织中进行免疫组化实验观察RUNX3、CD49d、paxillin和NK细胞表达和定位的关联性，对其预后价值进行评估，本研究为肝癌治疗中新的干预靶点提供新思路。

Natural killer (NK) cells play an important role in metastasis of hepatocellular carcinoma (HCC). In our previous study, we found that CD49d expression was lower in NK cells purified from HCC tissues, compared with the adjacent non-tumor tissues. Furthermore, it was found that inhibition of CD49d expression in NK cells could reduce its cytolytic activity when co-cultured with different HCC cell lines, and promote invasive ability of HCC cell lines in vitro; luciferase assays confirmed that CD49d expression could be regulated by RUNX3. This was a novel finding of the impact of CD49d in NK cells on cancer progression. This study will establish in vivo and in vitro model of human HCC immune micro-environment. Using the model, we will investigate whether CD49d is involved in regulation of survival, migration and cytolytic activity of NK cells. In addition, through short hairpin RNA (shRNA) interference or up-regulation of CD49 expression in NK cells, we will determine the role of NK cells in HCC growth and invasion. At the same time, we will observe the role of its ligand paxillin on the involvement of CD49d in regulation of HCC progression. We will co-transfect shRNA or over-expression plasmids of CD49 and RUNX3 in NK cells, then co-culture these NK cells with different HCC cell lines and assess their roles in tumor invasion and in vivo metastasis. Furthermore, chromatin immunoprecipitation (ChIP) and luciferase assays will be used to analyze whether RUNX3 is involved in the transcriptional regulation of CD49 expression in NK cells. Finally, using tissue microarrays (TMAs) in HCC samples, we will determine the relationship between expressions of RUNX3, CD49, paxillin and NK infiltration, and evaluate their prognostic significance. Based on the above research, we will determine the effect of RUNX3 -introduced CD49 in the cytolytic activity of NK cells and the roles of these NK cells on HCC invasion and metastasis. We will provide experimental evidence for exploring the new intervention targets on the metastasis and recurrence of HCC.