信号素4D（Semaphorin4D，Sema4D）能在肿瘤缺氧情况下，与其受体神经丛素-B1（Plexin-B1）相互作用继而诱导肿瘤内血管新生，因而可能是VEGF途径受阻时的补救途径，维持肿瘤生长。在前期研究中发现，对病人结直肠癌病理切片分析、统计时显示，Sema4D的表达量在包括高分化、中分化、低分化的肿瘤中都非常高，可能是一个能指示肿瘤发展的分子靶标。为了研究Sema4D在肿瘤细胞中的作用机制，本研究通过建立缺氧模型，检测缺氧时肿瘤中Sema4D调控方式和反应机制，探究缺氧与Sema4D的相互关系。研究结果显示，缺氧条件下缺氧诱导因子（Hypoxia inducible factor，HIF-1α）能结合到Sema4D的启动子区缺氧调控元件上（hypoxia response elements，HREs），调高Sema4D转录水平。并且在不同细胞中，HIF-1α结合的HREs位点不同。通过对8株不同细胞Sema4D的启动子区序列的测序及二级结构预测，发现肿瘤细胞该区段序列有非常高的突变率，预示缺氧时在不同细胞内Sema4D的效应机制不同。通过生物软件预测、免疫共沉淀联合质谱检测方法，我们发现了Plexin-B1和Lck的蛋白相互作用。借助CRISPR Cas9建立高效的基因敲除体系，我们进一步探讨了Sema4D、Plexin-B1、Lck分子在结直肠癌中的作用机制。研究发现，Sema4D/Plexin-B1结合后能引发PI3K/Akt信号磷酸化水平提高，也能通过下游Lck这一桥梁信号分子提高Raf/Mek/Erk信号的活化水平。最终得出结论，Sema4D/Plexin-B1信号通路除了能促进肿瘤血管新生，还能能对肿瘤细胞本身的生长发展起促进作用。总的来说，肿瘤中高表达Sema4D原因之一是受到了HIF-1因子的调控，调控表达的Sema4D可以通过旁分泌诱导肿瘤血管新生、自分泌激活肿瘤细胞本身的信号两种途径影响肿瘤细胞的生长。

Sema4D might serve as a parallel option for tumor inducing angiogenesis particularly when VEGF pathway is disrupted, which in turn induces tumor development. Immunohistochemical analysis about 66 cases of human colorectal cancer (CRC) revealed high levels of Sema4D, and 84.85% of CRC samples exhibited moderate to strong Sema4D expression. As a result, Sema4D might turn out to be a biomarker of tumor, especially CRC. To investigate the role of Sema4D in tumor cells, hypoxia model was built, Dual-Glo Luciferase assay system was put into use.  Our results shows that hypoxia inducible factor-1(HIF-1)can combine with hypoxia response elements(HREs) within Sema4D promoter. 4 HREs were found within Sema4D promoter while HIF-1 bound different HRE in different cells. HIF-1 always bind with HRE4 in HUVEC cells, while bind with HRE2 in Caco-2 cells. 4 cancer cell lines (A549、Caco-2、CNE、RD) and 4 normal cell lines(BEAS-2B、HUVEC、MRC-5、293T ) were cultured, then their genomic DNA were isolated following by sequencing of Sema4D promoter gene. 3 mutations (T -471-C, A -600-G, C-862-T) were screen out by gene sequence blast. Secondary structures of Sema4D promoter sequences in these cells were predicted by RNA structure software. T -471-C and C-862-T would make huge structural change of secondary structure in Sema4D promoter. Both of these two mutation were discovered in 4 cancer cell lines. Meanwhile, A -600-G was only detected in CNE cell line, and it didn’t make big influence on secondary structure of Sema4D promoter gene. Therefore, we suggest that, in hypoxia state, regulatory mechanism of Sema4D were different in cancer cells from normal cells.

According to the ScanSite algorithm(scansite.mit.edu), we predicted that phosphorylated Plexin-B1 intracytoplasmic domain might be docking site of tyrosine kinase Lck domain SH2. Then, we investigated the intereaction between Plexin-B1 and Lck by co-immunoprecipitaion and MALDI-ToF mass spectrometry (MS). The result shows that immune coprecipitation sediments of anti-Plexin-B1contains Lck.To verified mechanism of Sema4D in Caco-2 cell, Sema4D, Plexin-B1, Lck gene of CRC cells were knock out by CRISPR/ Cas9 system. Meanwhile, Sema4D over expression model were established by transfected with proper lentivirus. Then, PI3k/Akt, Raf/Mek/Erk, phosphor- PI3k/Akt, phosphor- Raf/Mek/Erk were detected by western blot. The result showed that Sema4D/Plexin-B1 could upregulate both Lck-mediated Raf/Mek/Erk signaling pathway, and Lck-independent PI3k/Akt signaling pathway. As a conclusion, Sema4D can promote tumor growth by not only inducing angiogenesis, but also activatig incellular signaling pathway.