Supplementary Material for: M6A RNA methylation-mediated dysregulation of AGAP2-AS1 promotes trastuzumab resistance of breast cancer

Introduction: Trastuzumab is commonly used in the treatment of human epidermal growth factor receptor-2 positive (HER-2+) breast cancer patients, but its efficacy is often limited by chemotherapy resistance. Recent studies indicate that long noncoding RNAs (lncRNAs) play important roles in tumor progression and response to therapy. However, the regulatory mechanism of lncRNAs in trastuzumab resistance is still unknown to date. Methods: qPCR was performed to detect the expression of related genes. Western blot and immunofluorescence assays were used for the evaluation of protein expression levels. A series of gain- or loss-functional assays confirmed the function of AGAP2-AS1 in trastuzumab resistance, both in vitro and in vivo. RNA immunoprecipitation and pulldown analysis was conducted to verify the interaction between METTL3/YTHDF2 and lncRNA AGAP2-AS1. , Results: AGAP2-AS1 was upregulated in trastuzumab-resistant cells and SKBR-3R-generated xenograft in nude mice. Silence of AGAP2-AS1 significantly decreased trastuzumab-induced cell cytotoxicity both in vitro and in vivo. The m6A methylation of AGAP2-AS1 was found to be reduced in trastuzumab resistant cells compared to parental cells. In addition, METTL3 increased the m6A methylation of AGAP2-AS1, which finally induced the suppression of AGAP2-AS1 expression. Moreover, YTHDF2 was essential for METTL3-mediated m6A methylation of AGAP2-AS1. Functionally, AGAP2-AS1 regulated trastuzumab resistance via inducing autophagy and increasing ATG5 protein level. Conclusion: Taken together, we proved that METTL3/YTHDF2-mediated m6A methylation indued the increased expression AGAP2-AS1, which could promote the trastuzumab resistance of breast cancer. In addition, AGAP2-AS1 also regulates trastuzumab resistance via inducing autophagy. Therefore, AGAP2-AS1 may be promising predictive biomarker and therapeutic target breast cancer patients.

引言：曲妥珠单抗（Trastuzumab）是治疗人表皮生长因子受体2阳性（human epidermal growth factor receptor-2 positive, HER-2+）乳腺癌患者的常用药物，但其疗效常受化疗耐药的限制。近期研究表明，长链非编码RNA（long noncoding RNAs, lncRNAs）在肿瘤进展及治疗应答中发挥重要作用，但截至目前，lncRNAs在曲妥珠单抗耐药中的调控机制仍未明确。方法：采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）检测相关基因的表达水平；通过蛋白质印迹（Western blot）与免疫荧光实验评估蛋白表达量；借助一系列功能获得与功能缺失实验，在体内外验证AGAP2-AS1在曲妥珠单抗耐药中的作用；采用RNA免疫沉淀与RNA pulldown分析验证METTL3、YTHDF2与lncRNA AGAP2-AS1之间的相互作用。结果：AGAP2-AS1在曲妥珠单抗耐药细胞及SKBR-3R细胞诱导的裸鼠异种移植瘤中表达上调。敲低AGAP2-AS1可显著降低曲妥珠单抗诱导的细胞毒性，该效应在体内外实验中均得到证实。与亲本细胞相比，曲妥珠单抗耐药细胞中AGAP2-AS1的m6A甲基化水平降低。此外，METTL3可增强AGAP2-AS1的m6A甲基化水平，最终抑制AGAP2-AS1的表达；且YTHDF2是METTL3介导AGAP2-AS1 m6A甲基化的必需因子。功能实验显示，AGAP2-AS1通过诱导自噬并上调ATG5蛋白水平调控曲妥珠单抗耐药。结论：综上，本研究证实METTL3/YTHDF2介导的m6A甲基化通过上调AGAP2-AS1的表达，促进乳腺癌细胞对曲妥珠单抗的耐药性；此外，AGAP2-AS1还可通过诱导自噬调控曲妥珠单抗耐药。因此，AGAP2-AS1有望成为乳腺癌患者的潜在预测生物标志物与治疗靶点。