| 韩 刚,曹 羽,张 云,张言言,张 旭,胡 建,龚航军,刘宁宁,贾 茹.METTL3介导EZH2 m6A修饰在结直肠癌5-氟尿嘧啶耐药中的作用机制研究[J].,2022,(3):418-422 |
| METTL3介导EZH2 m6A修饰在结直肠癌5-氟尿嘧啶耐药中的作用机制研究 |
| METTL3 Contributes to 5-Fu Resistance via EZH2 m6A Modification in Colorectal Cancer |
| 投稿时间：2021-04-28 修订日期：2021-05-24 |
| DOI：10.13241/j.cnki.pmb.2022.03.004 |
| 中文关键词: RNA甲基化 METTL3 EZH2 结直肠癌 5-氟尿嘧啶 耐药 |
| 英文关键词: RNA methylation METTL3 EZH2 Colorectal cancer 5-FU Resistance |
| 基金项目:上海中医药大学预算内项目资助（2019LK019）；国家自然科学基金面上项目（81973651） |
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| 中文摘要: |
| 摘要 目的：探讨甲基转移酶样蛋白3（METTL3）在5-氟尿嘧啶耐药中的作用机制。方法：大剂量间歇诱导法建立5-FU耐药细胞株。在耐药组细胞中分别敲减METTL3及EZH2抑制GSK343处理。qPCR及Western blot检测METTL3和EZH2表达。CCK-8检测各组细胞增殖情况。m6A甲基化RNA免疫沉淀技术分析EZH2 mRNA m6A修饰修饰情况。结果：各药物浓度处理下的耐药组细胞增殖活性与未处理细胞无显著差异（P>0.05）而原代细胞组肠癌细胞较未处理细胞在0.5-10 μg/mL处理下细胞增殖活性显著减低（P<0.01）。耐药组细胞与原代细胞组相比，METTL3及EZH2表达水平显著升高（P<0.01）。耐药组细胞METTL3敲减后或GSK343处理后，在0.5 μg/mL-10 μg/mL浓度间下的细胞增殖活性与0 μg/mL处理细胞增殖活性相比显著减低（P<0.05）。耐药组细胞METTL3敲减细胞的EZH2表达与对照细胞比，显著下调（P<0.01）。m6A甲基化RNA免疫沉淀实验显示耐药组细胞METTL3敲减细胞的EZH2 mRNA m6A修饰水平（m6A富集度为6361.95±67.47%），较未敲减细胞修饰水平（396.30±57.74）显著减低（P<0.01）。结论：METTL3在肠癌细胞5-FU耐药抵抗中起到关键作用，靶向抑制METTL3有望成为缓解肠癌耐药的重要分子靶点。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the role of METTL3 in 5-fluorouracil resistance. Methods: High-dose intermittent induction method was used to establish 5-FU resistant cell lines. Knockdown of METTL3 or EZH2 inhibitor GSK343 treatment were performed in drug-resistant cells. qPCR and Western blot were used to detect the expression of METTL3 and EZH2. CCK-8 was used to detect cell proliferation in each group. The m6A methylated RNA immunoprecipitation was used to analyze the modification of EZH2 mRNA m6A. Results: There was no significant difference in the proliferation activity of the drug-resistant group and the untreated cells under the treatment of various drug concentrations (P>0.05), however the primary cell group of intestinal cancer cells had a significant decreased proliferation activity compared to untreated cells under 0.5-10 μg/mL. Compared with the primary cell group, the expression levels of METTL3 and EZH2 in the drug-resistant group were significantly higher (P<0.01). After knockdown of METL3 or GSK343 in the drug-resistant group, the proliferation activity of cells at a concentration of 0.5 μg/mL-10 μg/mL was significantly lower than that of cells treated with 0 μg/mL (P<0.05). Compared with the control cells, the expression of EZH2 in the METTL3 knockdown cells of the drug resistance group was significantly down-regulated(P<0.01). The m6A methylated RNA immunoprecipitation experiment showed that the EZH2 mRNA m6A modification level (m6A enrichment of 6361.95±67.47%) of the METL3 knockdown cells in the drug-resistant group was significantly lower than that of the non-knockdown cells (396.30±57.74)(P<0.01). Conclusion: METL3 plays a key role in 5-FU resistance of colorectal cancer cells. Targeted inhibition of METL3 is expected to become an important molecular target for alleviating resistance of colorectal cancer. |
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