神经生长因子受体P75NGFR调控TIMP2/MMP25平衡逆转EMT抑制结直肠癌侵袭转移的机制研究

Nerve growth factor receptor (P75NGFR) plays an important regulatory role in tumor development. In our previous studies, we found that silence expression caused by P75NGFR methylation in colorectal cancer promotes tumor metastasis, while overexpression of P75NGFR inhibits the invasion and metastasis of colorectal cancer cells. In order to figure out the mechanism how P75NGFR regulates the invasion and metastasis of colorectal cancer, we detected mRNA expression in colorectal cancer cells which transfected with empty vector and P75NGFR overexpression plasmid. The results of microarray analysis showed that TIMP2 was one of the most up-regulated biomarker and MMP25 was the most down-regulated one. The expression balance between TIMP2 and MMP25 gene was crucial for the epithelial mesenchymal transition (EMT). Based on the previous studies, we hypothesized that P75NGFR inhibit the invasion and metastasis of colorectal cancer by regulating of the balance between the TIMP2 and MMP25 expression which will cause the reversal of EMT. The following questions will be figured out and studies will be performed to prove the hypothesis. P75NGFR high/low expression cell lines will be conducted to test its effect on the invasion and metastasis of colorectal cancer. The mechanism why P75NGFR can regulate the metastasis of colorectal cancer via regulating the balance of TIMP2/MMP25 on the process of EMT will be explored. Transcription factors c-jun and c-fos, which were screened out from the microarray chip, will be used to detect the differences in AP-1 (which is the promoter site of both TIMP2 and MMP25) binding ability using the luciferase reporter gene system under different P75NGFR expression levels. We will reveal the mechanism of P75NGFR reversing EMT to inhibit the invasion and metastasis of colorectal cancer by regulating the balance of TIMP2/MMP25 in vitro and in vivo. This study will explore the knowledge of the molecular mechanisms of invasion and metastasis of colorectal cancer and will be beneficial for us to search the new biomarkers of metastasis.

神经生长因子受体P75NGFR在肿瘤中起重要调控作用，我们发现过表达P75NGFR可抑制结直肠癌细胞的转移。为探讨其抑制机制，对P75NGFR过表达结直肠癌细胞进行表达谱芯片检测，结果显示TIMP2和MMP25是上下调最明显的靶基因，两者的平衡是调控EMT引起肿瘤转移的重要机制。我们通过构建P75NGFR高/低表达细胞株，检测其对结直肠癌侵袭转移的影响；探索P75NGFR通过TIMP2/MMP25调节EMT抑制结直肠癌转移的机制；通过芯片探寻转录因子c-jun及c-fos；荧光素酶报告基因检测不同P75NGFR表达水平下转录因子与TIMP2及MMP25启动子区域AP-1位点结合能力差异及对两者转录激活的调控；通过体内外实验揭示P75NGFR调控TIMP2/MMP25平衡逆转EMT降低结直肠癌侵袭转移的分子机制。本研究对补充结直肠癌侵袭转移的分子机制，探寻新的复发转移预测标志物有临床意义。

本研究利用我院结直肠患者丰富完善的组织、血液标本和随访资料，探索P75NGFR调节 TIMP2/MMP25 的表达平衡来抑制上皮间质转化EMT，降低结直肠癌细胞侵袭及转移能力。检测结直肠癌肿瘤组织和细胞中验证P75NGFR与MMP25表达负相关，与TIMP2的表达正相关，抑制结直肠癌细胞系中MMP25的表达，并可预测结直肠癌术后复发转移。通过体内外实验验证P75NGFR 可在小鼠体内抑制结直肠癌细胞肝转移瘤形成；同时分子机制实验P75NGFR通过抑制结直肠癌细胞EMT来抑制其侵袭转移，是通过增强c-jun的磷酸化激活，从而增强TIMP2与MMP25的结合抑制来实现的。本研究揭示了P75NGFR抑制结直肠癌侵袭转移的分子机制，并证实其可作为预测复发转移的新型分子标志物。相关成果已总结成文，投稿发表中。