hnRNPA2B1 通过 m6A RNA 甲基化方式下调 PCK1 mRNA 促进肝细胞癌的发生和发展。
hnRNPA2B1 promotes the occurrence and progression of hepatocellular carcinoma by downregulating PCK1 mRNA via a m6A RNA methylation manner.
机构信息
State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Department of Ultrasound, Fudan University Shanghai Cancer Center, Shanghai, China.
出版信息
J Transl Med. 2023 Nov 28;21(1):861. doi: 10.1186/s12967-023-04704-4.
BACKGROUND
N6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few types of tumors, its role in hepatocellular carcinoma (HCC) and the underlying molecular mechanism remains unclear.
METHODS
Multiple public databases were used to analyze the expression of hnRNPA2B1 in HCC and its correlation with survival prognosis. We employed a CRISPR-Cas9 sgRNA editing strategy to knockout hnRNPA2B1 expression in HCC cells. The biological function of hnRNPA2B1 in vitro in HCC cells was measured by CCK8, colony formation, migration, and invasion assay. The tumorigenic function of hnRNPA2B1 in vivo was determined by a subcutaneous tumor formation experiment and a HCC mouse model via tail injection of several plasmids into the mouse within 5s-7s. RNA binding protein immunoprecipitation (RIP) experiment using hnRNPA2B1 was performed to test the target genes of hnRNPA2B1 and methylated RNA immunoprecipitation (MeRIP) assay was performed to explore the m6A methylated mRNA of target genes.
RESULTS
hnRNPA2B1 highly expressed in HCC tissues, correlated with high grades and poor prognosis. Its knockout reduced HCC cell proliferation, migration, and invasion in vitro, while overexpression promoted these processes. hnRNPA2B1-knockout cells inhibited tumor formation in graft experiments. In HCC mice, endogenous knockout attenuated hepatocarcinogenesis. RNA-seq showed downregulated gluconeogenesis with high hnRNPA2B1 expression. hnRNPA2B1 negatively correlated with PCK1, a key enzyme. RIP assay revealed hnRNPA2B1 binding to PCK1 mRNA. hnRNPA2B1 knockout increased m6A-methylation of PCK1 mRNA. Interestingly, PCK1 knockout partially counteracted tumor inhibition by hnRNPA2B1 knockout in mice.
CONCLUSION
Our study indicated that hnRNPA2B1 is highly expressed in HCC and correlated with a poor prognosis. hnRNPA2B1 promotes the tumorigenesis and progression of HCC both in vitro and in vivo. Moreover, hnRNPA2B1 downregulates the expression of PCK1 mRNA via a m6A methylation manner. More importantly, the ability of hnRNPA2B1 to induce tumorigenesis and progression in HCC is dependent on its ability to decrease the expression of PCK1. Therefore, this study suggested that hnRNPA2B1 might be a diagnostic marker of poor prognosis of HCC and a potential therapeutic target for HCC patients.
背景
N6-甲基腺嘌呤（m6A）是最普遍的 RNA 修饰。尽管 hnRNPA2B1 作为 m6A 修饰的阅读器，已被报道在几种类型的肿瘤中促进肿瘤发生，但它在肝细胞癌（HCC）中的作用及其潜在的分子机制尚不清楚。
方法
使用多个公共数据库分析 hnRNPA2B1 在 HCC 中的表达及其与生存预后的相关性。我们采用 CRISPR-Cas9 sgRNA 编辑策略敲除 HCC 细胞中的 hnRNPA2B1 表达。通过 CCK8、集落形成、迁移和侵袭试验测定 hnRNPA2B1 在 HCC 细胞中的体外生物学功能。通过皮下肿瘤形成实验和通过尾静脉在 5-7 秒内向小鼠内注射几种质粒的 HCC 小鼠模型，测定 hnRNPA2B1 的体内致瘤功能。进行 hnRNPA2B1 的 RNA 结合蛋白免疫沉淀（RIP）实验以测试 hnRNPA2B1 的靶基因，并进行甲基化 RNA 免疫沉淀（MeRIP）实验以探索靶基因的 m6A 甲基化 mRNA。
结果
hnRNPA2B1 在 HCC 组织中高表达，与高分级和预后不良相关。其敲除降低了 HCC 细胞的增殖、迁移和侵袭能力，而过表达则促进了这些过程。hnRNPA2B1 敲除细胞抑制了移植实验中的肿瘤形成。在 HCC 小鼠中，内源性敲除可减弱肝癌发生。RNA-seq 显示高 hnRNPA2B1 表达时糖异生下调。hnRNPA2B1 与关键酶 PCK1 呈负相关。RIP 实验显示 hnRNPA2B1 与 PCK1 mRNA 结合。hnRNPA2B1 敲除增加了 PCK1 mRNA 的 m6A 甲基化。有趣的是，PCK1 敲除部分抵消了 hnRNPA2B1 敲除在小鼠中对肿瘤抑制的作用。
结论
我们的研究表明，hnRNPA2B1 在 HCC 中高表达，与预后不良相关。hnRNPA2B1 促进 HCC 的体内外肿瘤发生和进展。此外，hnRNPA2B1 通过 m6A 甲基化方式下调 PCK1 mRNA 的表达。更重要的是，hnRNPA2B1 在 HCC 中诱导肿瘤发生和进展的能力取决于其降低 PCK1 表达的能力。因此，本研究提示 hnRNPA2B1 可能是 HCC 预后不良的诊断标志物和 HCC 患者的潜在治疗靶点。
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