Shi Zhitian, Wu Xuesong, Ke Yang, Wang Lin
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming City, 650101, Yunnan Province, China.
Department of Gastroenterological Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming City, 650101, Yunnan Province, China.
Dig Dis Sci. 2016 Mar;61(3):785-94. doi: 10.1007/s10620-015-3927-y. Epub 2015 Oct 31.
There is increasing evidence that histidine triad nucleotide-binding protein 1 (HINT1) is a novel tumor suppressor. In the present study, we investigated the mechanism by which HINT1 promotes the stability of inhibitor of NF-κB α (IκBα) in the cytoplasm of hepatocellular carcinoma (HCC) cells, which was observed in our previous study (Wang et al. in Int J Cancer 124:1526-1534, 2009).
We examined HINT1 and IκBα expression in HCC cell lines and determined the effect of HINT1 overexpression and knockdown on IκBα protein and mRNA expression in these cell lines. Then, ubiquitination assays were performed to investigate the effects of HINT1 expression plasmid transfection on IκBα ubiquitination. Next, the interaction between HINT1 and β-TrCP was investigated in immunoprecipitation and immunofluorescence assays.
Our data showed that increased HINT1 expression in HepG2 and SMMC7702 cells markedly increased IκBα protein levels, while decreased HINT1 expression markedly decreased them. Overexpression or knockdown of HINT1 did not alter the transcription of IκBα, but HINT1 inhibited proteasomal IκBα degradation and reduced its ubiquitination levels. This inhibition might occur because HINT1 is a component of the SCF(β-TrCP) E3 ligase, which is responsible for IκBα ubiquitination and degradation.
This study provides new evidence that HINT1 is a regulator of IκBα through SCF(β-TrCP) E3 ligase. These findings help to clarify the mechanism underlying the anticancer effects of HINT1.
越来越多的证据表明，组氨酸三联体核苷酸结合蛋白1（HINT1）是一种新型肿瘤抑制因子。在本研究中，我们探讨了HINT1促进核因子κBα抑制因子（IκBα）在肝癌（HCC）细胞胞质中稳定性的机制，这一现象在我们之前的研究中已被观察到（Wang等人，《国际癌症杂志》，2009年，第124卷，第1526 - 1534页）。
我们检测了HCC细胞系中HINT1和IκBα的表达，并确定了HINT1过表达和敲低对这些细胞系中IκBα蛋白和mRNA表达的影响。然后，进行泛素化分析以研究HINT1表达质粒转染对IκBα泛素化的影响。接下来，在免疫沉淀和免疫荧光分析中研究HINT1与β - 转导素重复序列包含蛋白（β-TrCP）之间的相互作用。
我们的数据显示，HepG2和SMMC7702细胞中HINTi表达增加显著提高了IκBα蛋白水平，而HINT1表达降低则显著降低了IκBα蛋白水平。HINT1的过表达或敲低并未改变IκBα的转录，但HINT1抑制了蛋白酶体介导的IκBα降解并降低了其泛素化水平。这种抑制可能是因为HINT1是SCF(β-TrCP) E3连接酶的一个组分，该连接酶负责IκBα的泛素化和降解。
本研究提供了新的证据，表明HINT1是通过SCF(β-TrCP) E3连接酶对IκBα进行调控的。这些发现有助于阐明HINT1抗癌作用的潜在机制。
