miR-340在鳞状细胞癌侵袭中的作用及分子机制研究

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer in the world, with a 5-year survival rate of approximately 50 % due to its potent invasive capacity. Although it has been shown that the activation of Tiam 1/Rac1 pathway and overexpression of RhoA are involved in tumor invasion, more investigations are still required to elucidate the mechanisms regulating Tiam1/Rac1 and RhoA. Our previous study implicated that miR-340 was downregulated and could suppress the expression of RhoA, which is the target of miR-340. Resorting to bioinformatics methods, we found that Tiam1 is a potent target of miR-340. Additionally, convincing evidence demonstrated that transcription repressor E2F7 showed an abberantly increased expression in cSCC and potent binding sites with E2F7 were reported in miR-340 promoter region. Thus, we hypothesize that miR-340 suppression by E2F7 would induce augmentation of Tiam1 and RhoA expression, leading to an increse in the invasive properties of SCC. To verify our hypothesis, the study aim to testify the exsitence of E2F7-miR-340-Tiam1, RhoA pathway and its role in the regulation of SCC invasion in vivo and in vitro.

皮肤鳞癌侵袭性强，发生转移的皮肤鳞癌预后较差，一年生存率仅为44-56%。据文献报道，Tiam1/Rac1通路的激活和RhoA高表达与鳞状细胞癌的高侵袭性密切相关，但其上游调控机制有待阐明。我们的前期研究表明，miR-340在临床皮肤鳞癌组织中低表达，RhoA是其直接靶基因，而软件预测提示miR-340可能同时靶向抑制Tiam1的表达；同时，结合文献报道转录抑制因子E2F7在皮肤鳞癌中过表达的结论，我们运用生物信息学方法，发现miR-340启动子区存在多个E2F7的结合位点。据此我们提出，在皮肤鳞癌中E2F7转录抑制miR-340，解除了后者对其靶基因Tiam1和RhoA的抑制作用，从而促进皮肤鳞癌的侵袭。为验证这一假说，本项目拟在此基础上在细胞水平和体内模型中验证上述信号通路的存在及其在鳞状细胞癌侵袭中的作用，为深入认识鳞状细胞癌侵袭的分子机制以及miRNA在其中的调控作用提供实验依据。

皮肤鳞癌是世界上第二常见的肿瘤，且其发病率逐年上升。鳞癌细胞不可控的恶性增殖及转移是导致其死亡率上升的重要原因，特别是在器官移植的病人中鳞癌的转移率更高。研究表明miRNAs在多种肿瘤的生长和转移过程中发挥重要作用，但miRNAs参与鳞癌发生发展的具体机制尚不清楚。在本研究中我们取得了以下主要研究成果：miR-340在鳞癌组织及细胞系中均显著低表达。过表达miR-340可显著抑制鳞癌细胞迁移和侵袭，抑制内源性miR-340可促进鳞癌细胞迁移和侵袭过程。生物信息学分析发现miR-340与TGF-beta，PI3K-Akt，Wnt等信号通路相关，同时可调控细胞骨架蛋白，黏着斑等与细胞运动性相关的分子。双荧光素酶报告系统确定RhoA是miR-340新的靶基因，干涉RhoA可通过抑制MYPT1磷酸化抑制鳞癌细胞侵袭迁移过程，模拟miR-340 mimics的功能。干涉RhoA可回复由miR-340抑制所调控的鳞癌细胞迁移和侵袭，说明miR-340通过RhoA/ROCK1/MYPT1信号通路调控鳞癌细胞侵袭迁移。此外，鳞癌组织及细胞系中RhoA高表达，并与miR-340的表达水平呈负相关。最后，我们发现鳞癌中高表达的E2F7可降低miR-340的表达水平。上述结果表明皮肤鳞癌中E2F7抑制miR-340表达，解除了miR-340对其靶基因RhoA的抑制作用，从而激活ROCK1/MYPT1信号通路促进鳞癌的迁移和侵袭。以上成果明确了miR-340调控鳞癌侵袭转移的作用及分子机理，阐明了鳞癌中miR-340异常表达的分子机制。对于其它肿瘤的机制研究提供重要的理论参考，为肿瘤的治疗提供了新的策略。