The effect of miR-338-3p on HBx deletion-mutant (HBx-d382) mediated liver-cell proliferation through CyclinD1 regulation.
The effect of miR-338-3p on HBx deletion-mutant (HBx-d382) mediated liver-cell proliferation through CyclinD1 regulation.
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DOI：
10.1371/journal.pone.0043204
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Liu F
中科院分区：
文献类型：
--
作者：
Fu X;Tan D;Hou Z;Hu Z;Liu G;Ouyang Y;Liu F
B肝炎病毒（HBV）阳性肝癌患者存在HBV DNA整合和HBVX（HBx）基因缺失突变，HBx基因C端缺失突变与肝癌发生高度相关。我们的前期研究发现HBx-d382缺失突变体（在nt 382-400处缺失）可以下调miR-338- 3 p在HBx表达细胞中的表达。本研究的目的是检测miR-338- 3 p在HBx-d382介导的肝细胞增殖中的作用。我们通过Lipofectamine 2000转染建立了表达HBx的LO 2细胞。使用miR-NC作为对照miRNA，将miR-338- 3 p模拟物或抑制剂转染到LO 2/HBx-d382和LO 2/HBx细胞中。对潜在的miR-338- 3 p靶点的计算机模拟分析显示，miR-338- 3 p可以靶向细胞周期调控蛋白CyclinD 1。为了证实CyclinD 1受miR-338- 3 p负调控，我们构建了具有野生型和突变型CyclinD 1 -3′UTR靶位点的荧光素酶报告基因，用于miR-338- 3 p结合。我们通过实时荧光定量PCR和Western blot检测CyclinD 1的表达，并通过流式细胞仪细胞周期分析、Edu掺入和软琼脂集落形成检测细胞的增殖活性。HBx-d382在LO 2细胞中表现出增强的增殖和CyclinD 1表达。miR-338- 3 p表达抑制LO 2/HBx-d382细胞（和LO 2/HBx细胞）的细胞增殖，并且还负调节CyclinD 1蛋白表达。在CyclinD 1 -3′UTR区域的两个推定的miR-338- 3 p结合位点中，miR-338- 3 p对第二个结合位点（nt 2397-2403）的作用是抑制所必需的。miR-338- 3 p可通过与CyclinD 1 -3′UTR区域（主要位于nt 2397-2403）结合直接调控CyclinD 1的表达。miR-338- 3 p表达的下调是LO 2/HBx和LO 2/HBx-d382突变细胞中肝细胞增殖所必需的，尽管这种作用在LO 2/HBx-d382细胞中更明显。我们的研究阐明了一种新的机制，从一个新的miRNA调控的角度来看，HBx缺失突变体的倾向，以更快的速度比HBx诱导肝癌的发生。
Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation. We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony. HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition. miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.
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影响因子：
8
作者：
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通讯作者：
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DOI：
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影响因子：
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作者：
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