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Abstract：
BACKGROUND: Regorafenib, a multi-targeted kinase inhibitor, has been used in the treatment of Hepatocellular carcinoma (HCC). The purpose of this study is to investigate the mechanism of Regorafenib in HCC.
METHODS: Regorafenib\'s impact on the sensitivity of HCC cells was assessed using CCK8. Differential gene expression analysis was performed by conducting mRNA sequencing after treatment with Regorafenib. The m6A methylation status of CHOP and differential expression of m6A methylation-related proteins were assessed by RIP and Western Blot. To explore the molecular mechanisms involved in the therapeutic effects of Regorafenib in HCC and the impact of METTL14 and CHOP on Regorafenib treatment, we employed shRNA/overexpression approaches to transfect METTL14 and CHOP genes, as well as conducted in vivo experiments.
RESULTS: Treatment with Regorafenib led to a notable decrease in viability and proliferation of SK-Hep-1 and HCC-LM3 cells. The expression level of CHOP was upregulated after Regorafenib intervention, and CHOP underwent m6A methylation. Among the m6A methylation-related proteins, METTL14 exhibited the most significant downregulation. Mechanistic studies revealed that Regorafenib regulated the cell cycle arrest in HCC through METTL14-mediated modulation of CHOP, and the METTL14/CHOP axis affected the sensitivity of HCC to Regorafenib. In vivo, CHOP enhanced the anticancer effect of Regorafenib.
CONCLUSIONS: The inhibition of HCC development by Regorafenib is attributed to its modulation of m6A expression of CHOP, mediated by METTL14, and the METTL14/CHOP axis enhances the sensitivity of HCC to Regorafenib. These findings provide insights into the treatment of HCC and the issue of drug resistance to Regorafenib.
METHODS: Regorafenib\'s impact on the sensitivity of HCC cells was assessed using CCK8. Differential gene expression analysis was performed by conducting mRNA sequencing after treatment with Regorafenib. The m6A methylation status of CHOP and differential expression of m6A methylation-related proteins were assessed by RIP and Western Blot. To explore the molecular mechanisms involved in the therapeutic effects of Regorafenib in HCC and the impact of METTL14 and CHOP on Regorafenib treatment, we employed shRNA/overexpression approaches to transfect METTL14 and CHOP genes, as well as conducted in vivo experiments.
RESULTS: Treatment with Regorafenib led to a notable decrease in viability and proliferation of SK-Hep-1 and HCC-LM3 cells. The expression level of CHOP was upregulated after Regorafenib intervention, and CHOP underwent m6A methylation. Among the m6A methylation-related proteins, METTL14 exhibited the most significant downregulation. Mechanistic studies revealed that Regorafenib regulated the cell cycle arrest in HCC through METTL14-mediated modulation of CHOP, and the METTL14/CHOP axis affected the sensitivity of HCC to Regorafenib. In vivo, CHOP enhanced the anticancer effect of Regorafenib.
CONCLUSIONS: The inhibition of HCC development by Regorafenib is attributed to its modulation of m6A expression of CHOP, mediated by METTL14, and the METTL14/CHOP axis enhances the sensitivity of HCC to Regorafenib. These findings provide insights into the treatment of HCC and the issue of drug resistance to Regorafenib.
摘要：
背景：Regorafenib，一种多靶点激酶抑制剂，已用于治疗肝细胞癌（HCC）。目的探讨Regorafenib在肝癌中的作用机制。
方法：使用CCK8评估Regorafenib对HCC细胞敏感性的影响。在用Regorafenib处理后,通过进行mRNA测序进行差异基因表达分析。通过RIP和WesternBlot评估CHOP的m6A甲基化状态和m6A甲基化相关蛋白的差异表达。探讨Regorafenib治疗HCC的分子机制以及METTL14和CHOP对Regorafenib治疗的影响。我们采用shRNA/过表达方法转染METTL14和CHOP基因，以及进行体内实验。
结果：用Regorafenib处理导致SK-Hep-1和HCC-LM3细胞的活力和增殖显著降低。瑞戈非尼干预后CHOP的表达水平上调，CHOP进行了m6A甲基化。在m6A甲基化相关蛋白中，METTL14表现出最显著的下调。机制研究表明，Regorafenib通过METTL14介导的CHOP调节肝癌细胞周期阻滞，METTL14/CHOP轴影响HCC对雷戈拉非尼的敏感性。在体内,CHOP增强了雷戈拉非尼的抗癌作用。
结论：Regorafenib对HCC发展的抑制作用归因于其对CHOP的m6A表达的调节，由METTL14介导，METTL14/CHOP轴增强HCC对雷戈拉非尼的敏感性。这些发现为HCC的治疗和对Regorafenib的耐药性问题提供了见解。
方法：使用CCK8评估Regorafenib对HCC细胞敏感性的影响。在用Regorafenib处理后,通过进行mRNA测序进行差异基因表达分析。通过RIP和WesternBlot评估CHOP的m6A甲基化状态和m6A甲基化相关蛋白的差异表达。探讨Regorafenib治疗HCC的分子机制以及METTL14和CHOP对Regorafenib治疗的影响。我们采用shRNA/过表达方法转染METTL14和CHOP基因，以及进行体内实验。
结果：用Regorafenib处理导致SK-Hep-1和HCC-LM3细胞的活力和增殖显著降低。瑞戈非尼干预后CHOP的表达水平上调，CHOP进行了m6A甲基化。在m6A甲基化相关蛋白中，METTL14表现出最显著的下调。机制研究表明，Regorafenib通过METTL14介导的CHOP调节肝癌细胞周期阻滞，METTL14/CHOP轴影响HCC对雷戈拉非尼的敏感性。在体内,CHOP增强了雷戈拉非尼的抗癌作用。
结论：Regorafenib对HCC发展的抑制作用归因于其对CHOP的m6A表达的调节，由METTL14介导，METTL14/CHOP轴增强HCC对雷戈拉非尼的敏感性。这些发现为HCC的治疗和对Regorafenib的耐药性问题提供了见解。
