淋巴管生成关键基因sema4c功能性突变在诱导宫颈癌淋巴管生成和淋巴道转移中的作用机制研究

Lymph node metastasis is the independent prognostic factors of cervical cancer and its mechanism is unclear. preliminary studies of our laboratory found that SEMA4c could promote the tumor lymph node metastasis through promoting the tumor-associated macrophages integrating into lymphatic endothelial and changing the immune balance of the tumor microenvironment which had been confirmed in vitro. Our GWAS for lymph node metastasis of cervical cancer had confirmed that the lymph node metastasis of cervical cancer was closely related to genetic polymorhisms.The snp of key gene of lymphangiogenesis sema4c,rs185428881, 2316 C>T, could change the sema4c protein and mRNA expression,and there was significantlythe difference between lymph node metastasis group and non-metastasis group. so we concluded that sema4c snp played important roles in the sema4c activity and promoting lymph node metastasis. As a basis for that, we want to amplify and sequence the exon, promoter and enhancer of SEMA4C/PLEXB2 and further screen of the tag SNPs and study the snp function and expect to find genetic susceptibility loci of cervical cancer lymph node metastasis. We hope to find new ideas on cervical cancer lymph node metastasis in clinical treatment and prognostic evaluation.

淋巴结转移是宫颈癌预后的独立因素，其机制不清，本实验室前期研究发现SEMA4c可通过促进肿瘤相关巨噬细胞整合到肿瘤新生的淋巴内皮和改变肿瘤微环境中免疫平衡促进肿瘤的淋巴转移，并在体外得到验证，前期GWAS中发现宫颈癌淋巴结转移有一定遗传易感性，淋巴管生成关键基因sema4c rs185428881，2316 C>T 对sema4c蛋白及mRNA表达有显著影响，淋巴结转移组与非转移组有显著性差异，为此我们认为sema4c snp在sema4c表达、活性及促淋巴结转移中起到重要作用。申请者将依此为基础，旨在通过外显子、启动子及增强子扩增测序的方法对淋巴结转移相关基因SEMA4C/PLEXB2进行扩增测序，进一步筛查标签SNPs，并进行功能研究，通过生物信息学分析，以期待找到宫颈癌淋巴结转移遗传易感位点，为宫颈癌淋巴结转移临床治疗及预后评估提供新的思路。

淋巴结转移是宫颈癌预后的独立因素，其机制不清，本实验室前期研究发现SEMA4c可通过促进肿瘤相关巨噬细胞整合到肿瘤新生的淋巴内皮和改变肿瘤微环境中免疫平衡促进肿瘤的淋巴转移，并在体外得到验证，前期GWAS中发现宫颈癌淋巴结转移有一定遗传易感性，淋巴管生成关键基因sema4c rs185428881，2316 C>T 对sema4c蛋白及mRNA表达有显著影响，淋巴结转移组与非转移组有显著性差异，说明sema4c snp在sema4c表达、活性及促淋巴结转移中起到重要作用。对SEMA4C编码区、增强子、启动子扩增测序，分析对照组（无淋巴结转移宫颈癌妇女，无亲属关系）和宫颈癌淋巴结转移组（各1000例）SNPs测序结果，筛选出差异性SNPs。整合临床资料，找寻SEMA4C/PLEXB2遗传易感性与宫颈癌淋巴结转移的关联，确定SEMA4C/PLEXB2受体-配体遗传位点变异，分析变异分布方式和频率。并对相应SNP进行大样本验证（taqman探针法），并在分子和细胞水平对SEMA4C遗传变异进行体内、外功能验证。通过扩增测序筛查及整合临床资料，我们发现rs185428881是一个有意义的突变，生物信息学分析突变发生在2316位 C-A突变，氨基酸Pro-Hi；对此SNP进行了功能研究，成功构建了SEMA4C突变体细胞株，对细胞及临床病例研究结果显示突宫颈癌临床标本突变体mRNA和蛋白表达变化均下降，差异具有显著性；突变体细胞上清趋化血管内皮细胞HUVEC和巨噬细胞能力减弱，成管能力明显降低；但突变并不增加细胞的凋亡；临床标本突变组淋巴管密度明显低于非突变组，角膜微袋试验亦发现突变体成管能力显著下降。证实此突变在宫颈癌淋巴管转移中起保护作用。