Department of Surgical Oncology, General Hospital of Ningxia Medical University, No. 804, Shengli Street, Xingqing District, Yinchuan, 750004, Ningxia Hui Autonomous Region, China.
Ningxia Medical University, Yinchuan, 750004, China.
Mol Med. 2023 Aug 1;29(1):103. doi: 10.1186/s10020-023-00696-5.
Cancers aggressively reorganize collagen in their microenvironment, leading to the evasion of tumor cells from immune surveillance. However, the biological significance and molecular mechanism of collagen alignment in breast cancer (BC) have not been well established.
In this study, BC-related RNA-Seq data were obtained from the TCGA database to analyze the correlation between DDR1 and immune cells. Mouse BC cells EO771 were selected for in vitro validation, and dual-luciferase experiments were conducted to examine the effect of TFAP2A on DDR1 promoter transcription activity. ChIP experiments were performed to assess TFAP2A enrichment on the DDR1 promoter, while Me-RIP experiments were conducted to detect TFAP2A mRNA m6A modification levels, and PAR-CLIP experiments were conducted to determine VIRMA's binding to TFAP2A mRNA and RIP experiments to investigate HNRNPC's recognition of m6A modification on TFAP2A mRNA. Additionally, an in vivo mouse BC transplant model and the micro-physiological system was constructed for validation, and Masson staining was used to assess collagen fiber arrangement. Immunohistochemistry was conducted to identify the number of CD8-positive cells in mouse BC tumors and Collagen IV content in ECM, while CD8 + T cell migration experiments were performed to measure CD8 + T cell migration.
Bioinformatics analysis showed that DDR1 was highly expressed in BC and negatively correlated with the proportion of anti-tumor immune cell infiltration. In vitro cell experiments indicated that VIRMA, HNRNPC, TFAP2A, and DDR1 were highly expressed in BC cells. In addition, HNRNPC promoted TFAP2A expression and, therefore, DDR1 transcription by recognizing the m6A modification of TFAP2A mRNA by VIRMA. In vivo animal experiments further confirmed that VIRMA and HNRNPC enhanced the TFAP2A/DDR1 axis, promoting collagen fiber alignment, reducing anti-tumor immune cell infiltration, and promoting immune escape in BC.
This study demonstrated that HNRNPC promoted DDR1 transcription by recognizing VIRMA-unveiled m6A modification of TFAP2A mRNA, which enhanced collagen fiber alignment and ultimately resulted in the reduction of anti-tumor immune cell infiltration and promotion of immune escape in BC.
癌症在其微环境中积极重组胶原蛋白，导致肿瘤细胞逃避免疫监视。然而，胶原蛋白在乳腺癌（BC）中的排列的生物学意义和分子机制尚未得到很好的建立。
本研究从 TCGA 数据库中获取与 BC 相关的 RNA-Seq 数据，以分析 DDR1 与免疫细胞之间的相关性。选择体外 EO771 小鼠 BC 细胞进行验证，进行双荧光素酶实验以检查 TFAP2A 对 DDR1 启动子转录活性的影响。进行 ChIP 实验以评估 TFAP2A 在 DDR1 启动子上的富集，进行 Me-RIP 实验以检测 TFAP2A mRNA m6A 修饰水平，进行 PAR-CLIP 实验以确定 VIRMA 与 TFAP2A mRNA 的结合，并进行 RIP 实验以研究 HNRNPC 对 TFAP2A mRNA 上 m6A 修饰的识别。此外，构建了体内小鼠 BC 移植模型和微生理系统进行验证，并通过 Masson 染色评估胶原蛋白纤维排列。进行免疫组织化学以鉴定小鼠 BC 肿瘤中 CD8 阳性细胞的数量和 ECM 中 Collagen IV 的含量，同时进行 CD8+T 细胞迁移实验以测量 CD8+T 细胞的迁移。
生物信息学分析表明，DDR1 在 BC 中表达较高，与抗肿瘤免疫细胞浸润比例呈负相关。体外细胞实验表明，VIRMA、HNRNPC、TFAP2A 和 DDR1 在 BC 细胞中高表达。此外，HNRNPC 通过识别 VIRMA 揭示的 TFAP2A mRNA 的 m6A 修饰促进 TFAP2A 表达，从而促进 DDR1 转录。体内动物实验进一步证实，VIRMA 和 HNRNPC 增强了 TFAP2A/DDR1 轴，促进胶原蛋白纤维排列，减少抗肿瘤免疫细胞浸润，并促进 BC 中的免疫逃避。
本研究表明，HNRNPC 通过识别 VIRMA 揭示的 TFAP2A mRNA 的 m6A 修饰促进 DDR1 转录，增强胶原蛋白纤维排列，最终导致抗肿瘤免疫细胞浸润减少和免疫逃避促进在 BC 中。
