肿瘤相关成纤维细胞表达COL6A3基因对结直肠癌恶性潜能的影响及临床意义

Cancer-associated fibroblasts (CAFs) can form a favorable microenvironment for cancer cells, playing an important role in cancer cell growth, invasion and metastasis. The effects of extracellular matrix secreted by CAFs on tumor development are still unclear. We previously have constructed a gene co-expression network to reveal functional modules in colorectal cancer (CRC), and found that the hub gene of extracellular matrix module COL6A3 was significantly associated with patients survival, recurrence and AJCC staging. COL6A3 was primarily expressed in CAFs and was expressed higher in primary CRC than in normal mucosa. Therefore, we hypothesis that CAFs may influence cancer malignant potential through secretion of COL6A3. To testify the hypothesis, we will treat CRC cancer cell line with conditioned medium collected from COL6A3-/- CAFs, then observe apoptosis, proliferation, invasion and metastasis of cancer cell; evaluate the proliferation rate change and chemotherapy resistance after incubation with ETP (active C5 terminal peptide spliced from COL6A3) medium. Further, a mixture of CAFs and CRC cancer cells will be injected subcutaneouly or into the spleen, to assess the effects of COL6A3 expression on tumor sphere formation or metastasis in vivo. Clinical samples will be collected for immunohistochemistry and ELISA validation. Our results will provide a new therapy target for CRC treatment.

肿瘤相关成纤维细胞分泌的细胞外基质在肿瘤发生发展过程中的作用不清。我们通过构建基因共表达网络揭示结直肠癌相关的功能模块，发现细胞外基质模块中的关键分子COL6A3表达和病人生存、复发及AJCC分期相关。COL6A3在原发性结直肠癌中高表达，细胞水平分析发现其主要在肿瘤相关成纤维细胞中表达。因此，我们推测肿瘤相关成纤维细胞可能通过分泌COL6A3影响肿瘤恶性潜能。为证明该假说，我们拟敲除肿瘤相关成纤维细胞的COL6A3基因，观察其增殖、侵袭和迁移能力改变；以其条件培养基处理结直肠癌肿瘤细胞株，观察增殖、侵袭、迁移和耐药能力改变。COL6A3 C5端活性片段ETP处理肿瘤细胞和成纤维细胞，检测其增殖能力变化。肿瘤相关成纤维细胞和肿瘤细胞混合注射到裸鼠皮下/脾内，观察COL6A3表达改变对肿瘤细胞体内成瘤/转移能力影响。临床样本进行免疫组化和ELISA验证。为结直肠癌治疗提供新靶标。

随着人民收入提高和生活水平改善，生活习惯的改变，近年来结直肠癌呈增长趋势并趋于年轻化。前期通过构建基因共表达网络揭示结直肠癌相关的功能模块，发现细胞外基质模块中的关键分子col6a3表达和病人生存、复发及AJCC分期相关。COL6A3在原发性结直肠癌中高表达。为验证其功能，敲除肿瘤细胞系SW480的col6a3基因，观察其增殖、侵袭和迁移能力降低。利用CRISPR/Cas9技术构建2株col6a3基因ETP片段突变细胞株20和28；28号突变体比20号突变体的增殖、侵袭、转移能力下降更多；20号突变体比28号突变体的克隆形成能力下降更多，其凋亡细胞数也更多；另外，发现来自肿瘤组织的基因芯片的间质和免疫打分与病人预后相关，基于基因共表达模块的药物重定位策略可以找到潜在的新治疗药物。该结果为结直肠癌治疗提供新靶标奠定基础。