利用miR-155过表达小鼠舌鳞癌模型研究miR-155靶向调控MMP13的作用机制

Tongue squamous cell carcinoma (TSCC) is one of the malignant cancers threatening human health. The existing chemical induced TSCC models have several disadvantages such as long induction time, low incidence of tumor, use of large doses of carcinogen, and more importantly, they do not exactly reflect the pathophysiological process of human TSCC. Although it has been shown that miR-155 is involved in the development of carcinoma, the mechanism by which miR-155 overexpression regulates TSCC is still unknown. We recently constructed a mouse model of TSCC with miR-155 overexpression, and found that stable cancer could be induced with high efficiency in a short period of time. Additionally, the model could largely mimic the development process of TSCC in human. However, the pathological and diagnostic criteria under molecular level and carcinogenic mechanism of this model will need to be elucidated. Interestingly, our preliminary study in combination with bioinformatics analysis showed that elevated expression of MMP13 was associated with overexpression of miR-155 in TSCC. Therefore, we hypothesize that miR-155 regulates the development of TSCC by activating MMP13. To evaluate this hypothesis, we propose to refine the mouse model of TSCC and conduct comparative studies with molecular diagnosis standard in human TSCC. Additionally, we will selectively inhibit the expression of miR-155 and/or MMP13 in cells and TSCC mice in which miR-155 and MMP13 are overexpressed. We plan to reveal the mechanism of the interactive relationship between miR-155 and MMP13 in the development of TSCC, using modern methods of quantitative detection for miR-155 and MMP13 expression, tumor proliferation, molecular and pathological diagnosis, etc. The success of the proposed research will facilitate our understanding of TSCC development and progression and ultimately identify potential targets for development of new therapeutics against TSCC.

舌鳞癌是威胁人类健康的恶性肿瘤之一。现有舌鳞癌模型诱导时间长、成癌率低、致癌剂用量大，尚不能很好反映人舌鳞癌发生的病理生理过程。miR-155可调控肿瘤发生。申请人前期利用miR-155过表达小鼠建立的舌鳞癌模型，造模时间短、成癌率高、模型稳定，与人舌鳞癌病理过程相似，但尚需完善该模型分子病理诊断标准及机制研究。我们前期结果结合生物信息学分析表明，过表达miR-155后MMP13异常升高，但miR-155是否通过激活MMP13调控舌鳞癌的发生？尚需实验验证。为此，本项目将结合人舌鳞癌分子病理诊断标准完善模型评价，并在miR-155过表达的细胞及小鼠舌鳞癌模型中靶向抑制miR-155和MMP13，通过检测miR-155和MMP13表达、肿瘤增殖和分子病理诊断等手段，明确miR-155和MMP13作用关系、靶向效应及共同调控舌鳞癌发生的机制，为舌鳞癌治疗新靶点提供科学依据。

口腔鳞状细胞癌是威胁人类健康的恶性肿瘤之一，也是口腔常见的肿瘤，其中舌鳞癌占90%以上。现有舌鳞癌模型动物模型有局限性，miR-155促进舌鳞癌的发生并上调MMP13的表达，担机制尚不明晰。本研究在原有工作基础上：（1）完善了比较病理学的研究内容：①通过第二代测序技术进行mRNA和piRNA表达谱分析，建立了包括Tmc5、Galnt6、Spedf、Mybl2、Muc5b、Six31、Pigr、Lamc2、Mmp13、Mal、Mamdc2在内的11个mRNA和11个新piRNAs的调控网络，将为舌鳞状细胞癌的诊断生物标志物和治疗靶点的开发提供了新的思路；②筛选出舌鳞癌患者癌组织和唾液中HGF、VEGF、PIGF、MMP-1、MMP-3、MMP-8、MMP-9、MMP-10、MMP-13、TIMP-2水平显著升高，利用生物信息学分析方法揭示了上述分子与头颈部肿瘤患者生存和癌症功能状态的相关性，表明上述分子可能是舌鳞癌基于唾液的无创诊断/预后标志物和治疗靶点；③本研究对现有常用的小鼠的舌癌模型制备方法进行了改良，建立了miR155过表达小鼠（FVB小鼠背景）舌鳞状细胞癌模型，这可为本研究团队和其他科研人员提供舌鳞癌用于病因学和药物研发的小鼠模型。（2）揭示miR-155对MMP13的调控作用研究方面：①分别利用MMPs 广谱抑制剂MMPsi(Ilomastat)、MMP13特异性抑制剂MMP13i和mir-155抑制剂处理CAL-27细胞，发现miR-155 i、MMP13 i和MMPs i联合处理CAL-27细胞3天的细胞抑制率达到78.5%；②利用GEO数据库中的临床样本测序数据，通过生物信息学分析手段找到mir-155调控MMP13升高的连接分子，在细胞水平验证发现miR-155可能通过靶向抑制CHRDL1调控MMP13致口腔舌鳞癌的发生，为靶向治疗提供了很好的科学指导意义。本研究成果发表学术论文4篇（其中3篇SCI收录论文，中国科技核心期刊1篇）、申请发明专利1项、。