SUZ12 ceRNAs参与miR-320a调控舌鳞癌tumor budding侵袭转移的分子机制

Previous study demonstrated that tumor budding, with more aggressive malignance, was correlated with invasion, metastasis and prognosis of tongue squamous cell carcinoma. But the regulation mechanisms during the malignant progression are not clear. Laser capture microdissection co-operated with miRNA microarray illustrated the characteristic miRNA expression profile of tumor budding cells, in which miR-320a etc. were downregulated. Further studies demonstrated that miR-320a could inhibit invasion and metastasis of tongue squamous cell carcinoma by targeting SUZ12. HTA2.0 transcriptome microarray showed that LncRNA H19, LINC00449, MMP2, FOSL1 were upregulated in tumor budding; Bioinformatics analysis indicate these molecules share the same miRNA response element of miR-320a with SUZ12. So we proposed that SUZ12 might interact with molecules of EMT, invasion and metastasis as ceRNA by competitive binding to miR-320a. In this project, we aim to screen SUZ12 ceRNAs which competitive binding with miR-320a, study the mechanism of them regulating invasion and metastasis of tumor budding, and finally evaluate their prognostic value for patients with tongue squamous cell carcinoma.

课题组前期研究证实tumor budding代表恶性程度更高的癌细胞亚群，与舌鳞癌侵袭转移、预后密切相关。但调控其恶性进展的分子机制尚不明确。激光捕获显微切割结合miRNA芯片显示tumor budding有特征性miRNA表达谱，其中miR-320a等下调。进一步研究证实miR-320a靶向SUZ12抑制舌鳞癌侵袭转移；转录组芯片显示tumor budding中LncRNA H19、LINC00449、MMP2、FOSL1等表达上调；生物信息学分析显示上述分子均与SUZ12对miR-320a有相同应答元件；提示SUZ12可能通过miR-320a与侵袭转移、EMT相关分子互为ceRNA。在此基础上，本课题拟筛选与SUZ12竞争性结合miR-320a的ceRNAs，研究其对tumor budding及侵袭转移的调控机制，结合tumor budding评价其在预测舌鳞癌患者预后转归中的价值。

本课题成功筛选到与SUZ12竞争性结合miR-320a的ceRNA，即Midkine（MDK）。体、内外实验发现其抑制舌鳞癌细胞的侵袭和转移，并与舌鳞癌患者恶性进展密切相关。初步建立了miR-320a、SUZ12、MDK及侵袭、转移相关分子的相互调控网络。目前取得的进展如下：.1.体外实验再次验证miR-320a抑制舌鳞癌细胞的迁移、侵袭，下调VIM和CTNNB1的表达。.2.在CAL-27中转染miR-320a-3p mimics，转录组测序检测表达谱变化。结合常用miRNA靶基因数据库（mirtarbase、TargetScan7、miRDB）进行分析，筛选具有三个以上交集靶基因。分析部分基因和SUZ12对于miR-320a的MRE，综合其在肿瘤恶性进展中作用及验证结果，选择MDK作为目标ceRNA。.3.体外实验研究结果显示干扰MDK可下调舌鳞癌细胞CAL-27和SCC9的增殖、迁移和侵袭，降低迁移、侵袭相关基因VIM、CTNNB1、MMP2、MMP9、N-cadherin、FOSL1的表达。.4.下调MDK建立稳定表达细胞株CAL27-shMDK，动物实验评估其对舌鳞癌细胞成瘤和转移的影响，发现MDK下调可降低舌鳞癌细胞转移能力。Western blot检测MDK干扰株MDK下游通路蛋白表达，发现PDK、AKT、pAKT表达均下降，说明MDK可能通过AKT信号通路，参与调控舌鳞癌的恶性进展。以上结果联合生物信息学分析，初步建立了miR-320a、SUZ12、MDK及侵袭、转移相关分子的相互调控网络。.5.舌鳞癌标本验证SUZ12、MDK及miR-320a之间的相关性，三者与患者临床病理参数和预后的关系。免疫组化染色显示MDK在舌鳞癌细胞的胞核和胞浆中表达，空间分布与Suz12在舌鳞癌组织中表达位置相似。生存曲线分析显示MDK低表达组的生存率高于MDK高表达组，MDK低表达同时tumor budding少的患者生存率高于MDK高表达同时tumor budding多的患者。多因素分析显示MDK是影响舌鳞癌患者预后的独立因素。.6.按照ITBCC(2016)推荐的评分系统，评价tumor budding在舌鳞状细胞癌中的意义和价值。结果表明ITBCC评分系统是一种简便、可靠、重复性好的舌鳞癌tumor budding检测方法，推荐纳入常规病理报告。