Department of Radiotherapy, Second Affiliated Hospital of Xi'an Jiaotong University, No. 57, Siwu Road, Xi'an City, Shaanxi, 710004, China.
Biol Direct. 2024 Aug 19;19(1):68. doi: 10.1186/s13062-024-00511-z.
Cancer-associated fibroblasts (CAFs) have been reported that can affect cancer cell proliferation, metastasis, ferroptosis, and immune escape. METTL3-mediated N6-methyladenine (m6A) modification is involved in the tumorigenesis of colorectal cancer (CRC). Herein, we investigated whether METTL3-dependent m6A in CAFs-derived exosomes (exo) affected CRC progression.
qRT-PCR and western blotting analyses detected levels of mRNAs and proteins. Cell proliferation and metastasis were evaluated using MTT, colony formation, transwell, and wound healing assays, respectively. Cell ferroptosis was assessed by detecting cell viability and the levels of Fe+, reactive oxygen species, and glutathione after erastin treatment. Exosomes were isolated from CAFs by ultracentrifugation. The m6A modification profile was determined by methylated RNA immunoprecipitation assay and the interaction between METTL3 and ACSL3 (acyl-CoA synthetase 3) was verified using dual-luciferase reporter assay. Animal models were established for in vivo analysis.
CAFs promoted CRC cell proliferation and metastasis, and suppressed cell ferroptosis. METTL3 was enriched in CAFs and was packaged into exosomes. The m6A modification and METTL3 expression were increased in CRC samples. Knockdown of METTL3 in CAFs-exo suppressed CRC cell proliferation and metastasis, and induced cell ferroptosis. Mechanistically, METTL3 induced ACSL3 m6A modification and stabilized its expression. The anticancer effects mediated by METTL3-silenced CAFs-exo could be rescued by ACSL3 overexpression. Moreover, in vivo assay also showed that CAFs-exo with decreased METTL3 could hinder CRC growth and metastasis in mice models.
CAFs promoted the proliferation and metastasis, and restrained the ferroptosis in CRC by exosomal METTL3-elicited ACSL3 m6A modification.
癌症相关成纤维细胞（CAFs）已被报道可影响癌细胞增殖、转移、铁死亡和免疫逃逸。METTL3 介导的 N6-甲基腺苷（m6A）修饰参与结直肠癌（CRC）的发生。在此，我们研究了 CAFs 来源的细胞外囊泡（exo）中 METTL3 依赖性 m6A 是否影响 CRC 进展。
qRT-PCR 和 Western blot 分析检测 mRNAs 和蛋白水平。分别通过 MTT、集落形成、Transwell 和划痕愈合实验评估细胞增殖和转移。用 erastin 处理后，通过检测细胞活力和 Fe+、活性氧和谷胱甘肽水平来评估细胞铁死亡。通过超速离心从 CAFs 中分离 exo。通过甲基化 RNA 免疫沉淀测定法确定 m6A 修饰谱，并通过双荧光素酶报告基因测定法验证 METTL3 和 ACSL3（酰基辅酶 A 合成酶 3）之间的相互作用。建立动物模型进行体内分析。
CAFs 促进 CRC 细胞增殖和转移，并抑制细胞铁死亡。METTL3 在 CAFs 中富集并被包装到 exo 中。CRC 样本中 m6A 修饰和 METTL3 表达增加。在 CAFs-exo 中敲低 METTL3 抑制 CRC 细胞增殖和转移，并诱导细胞铁死亡。机制上，METTL3 诱导 ACSL3 m6A 修饰并稳定其表达。ACSL3 过表达可挽救由 METTL3 沉默的 CAFs-exo 介导的抗癌作用。此外，体内实验也表明，METTL3 减少的 CAFs-exo 可阻碍小鼠模型中 CRC 的生长和转移。
CAFs 通过 exosomal METTL3 诱导的 ACSL3 m6A 修饰促进 CRC 中的增殖和转移，并抑制铁死亡。
