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

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0.5-10μg/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0.5μg/mL-10μg/mL浓度间下的细胞增殖活性与0μg/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。

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 0.5-10μg/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 0.5μg/mL-10μg/mL was significantly lower than that of cells treated with 0μg/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.