HNF1α 调控的长链非编码 RNA HNF1A-AS1 通过增强 SHP-1 的磷酸酶活性逆转肝癌的恶性表型。
The HNF1α-regulated lncRNA HNF1A-AS1 reverses the malignancy of hepatocellular carcinoma by enhancing the phosphatase activity of SHP-1.
机构信息
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China.
Drug Discovery and Design Center, CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai, 201203, China.
出版信息
Mol Cancer. 2018 Feb 21;17(1):63. doi: 10.1186/s12943-018-0813-1.
BACKGROUND
Our previous study has demonstrated that hepatocyte nuclear factor 1α (HNF1α) exerts potent therapeutic effects on hepatocellular carcinoma (HCC). However, the molecular mechanisms by which HNF1α reverses HCC malignancy need to be further elucidated.
METHODS
lncRNA microarray was performed to identify the long noncoding RNAs (lncRNAs) regulated by HNF1α. Chromatin immunoprecipitation and luciferase reporter assays were applied to clarify the mechanism of the transcriptional regulation of HNF1α to HNF1A antisense RNA 1 (HNF1A-AS1). The effect of HNF1A-AS1 on HCC malignancy was evaluated in vitro and in vivo. RNA pulldown, RNA-binding protein immunoprecipitation and the Bio-Layer Interferometry assay were used to validate the interaction of HNF1A-AS1 and Src homology region 2 domain-containing phosphatase 1 (SHP-1).
RESULTS
HNF1α regulated the expression of a subset of lncRNAs in HCC cells. Among these lncRNAs, the expression levels of HNF1A-AS1 were notably correlated with HNF1α levels in HCC cells and human HCC tissues. HNF1α activated the transcription of HNF1A-AS1 by directly binding to its promoter region. HNF1A-AS1 inhibited the growth and the metastasis of HCC cells in vitro and in vivo. Moreover, knockdown of HNF1A-AS1 reversed the suppressive effects of HNF1α on the migration and invasion of HCC cells. Importantly, HNF1A-AS1 directly bound to the C-terminal of SHP-1 with a high binding affinity (KD = 59.57 ± 14.29 nM) and increased the phosphatase activity of SHP-1. Inhibition of SHP-1 enzymatic activity substantially reversed the HNF1α- or HNF1A-AS1-induced reduction on the metastatic property of HCC cells.
CONCLUSIONS
Our data revealed that HNF1A-AS1 is a direct transactivation target of HNF1α in HCC cells and involved in the anti-HCC effect of HNF1α. HNF1A-AS1 functions as phosphatase activator through the direct interaction with SHP-1. These findings suggest that regulation of the HNF1α/HNF1A-AS1/SHP-1 axis may have beneficial effects in the treatment of HCC.
背景
我们之前的研究表明，肝细胞核因子 1α（HNF1α）对肝细胞癌（HCC）具有强大的治疗作用。然而，HNF1α 逆转 HCC 恶性的分子机制仍需进一步阐明。
方法
采用 lncRNA 微阵列鉴定受 HNF1α 调控的长链非编码 RNA（lncRNA）。应用染色质免疫沉淀和荧光素酶报告基因检测阐明 HNF1α 对 HNF1A 反义 RNA 1（HNF1A-AS1）转录调控的机制。在体外和体内评估 HNF1A-AS1 对 HCC 恶性的影响。采用 RNA 下拉、RNA 结合蛋白免疫沉淀和生物层干涉测定验证 HNF1A-AS1 与 Src 同源区 2 域含磷酸酶 1（SHP-1）的相互作用。
结果
HNF1α 调控 HCC 细胞中一组 lncRNA 的表达。在这些 lncRNA 中，HNF1A-AS1 的表达水平与 HCC 细胞和人 HCC 组织中的 HNF1α 水平显著相关。HNF1α 通过直接结合其启动子区域激活 HNF1A-AS1 的转录。HNF1A-AS1 抑制 HCC 细胞的体外和体内生长和转移。此外，敲低 HNF1A-AS1 逆转了 HNF1α 对 HCC 细胞迁移和侵袭的抑制作用。重要的是，HNF1A-AS1 与 SHP-1 的 C 端具有高结合亲和力（KD=59.57±14.29 nM）并增加 SHP-1 的磷酸酶活性。抑制 SHP-1 酶活性显著逆转了 HNF1α 或 HNF1A-AS1 诱导的 HCC 细胞转移特性的降低。
结论
我们的数据表明，HNF1A-AS1 是 HCC 细胞中 HNF1α 的直接转录激活靶标，并参与 HNF1α 的抗 HCC 作用。HNF1A-AS1 通过与 SHP-1 的直接相互作用发挥磷酸酶激活剂的作用。这些发现表明，调节 HNF1α/HNF1A-AS1/SHP-1 轴可能对 HCC 的治疗有益。
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