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Hepatitis B virus X protein enhances hepatocarcinogenesis by depressing the targeting of NUSAP1 mRNA by miR-18b 被引量:6
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作者 Zhe Yang Jiong Li +6 位作者 guoxing feng Yuan Wang Guang Yang Yunxia Liu Shuqin Zhang Jinyan feng Xiaodong Zhang 《Cancer Biology & Medicine》 SCIE CAS CSCD 2019年第2期276-287,共12页
Objective: The aim of this study was to investigate the underlying mechanism whereby HBx modulates the targeting of NUSAP1 by miR-18b to enhance hepatocarcinogenesis.Methods: We employed an integrated approach of bioi... Objective: The aim of this study was to investigate the underlying mechanism whereby HBx modulates the targeting of NUSAP1 by miR-18b to enhance hepatocarcinogenesis.Methods: We employed an integrated approach of bioinformatics analysis and molecular experiments in hepatoma cells, HBV transgenic mice, and clinical liver cancer tissues to investigate the role of HBx-regulated miR-18b in the development of liver cancer.Results: In this study, we report that the HBx-mediated tumor suppressor miR-18b modulates hepatocarcinogenesis during the host-HBV interaction. The expression levels of miR-18b were lower in clinical HBV-positive liver cancer tissues and liver tissues of HBV-transgenic mice. Interestingly, HBx inhibited miR-18b expression by inducing the methylation of CpG islands in its promoter. Accordingly, we tested the hypothesis that HBx enhanced hepatocarcinogenesis by increasing the expression of target genes of miR-18b. Moreover, we identified nucleolar spindle-associated protein 1(NUSAP1) as one of the target genes of miR-18b.NUSAP1 was expressed at high levels in liver cancer tissues. Interestingly, HBx up-regulated NUSAP1 by suppressing miR-18b.Functionally, miR-18b significantly inhibited the proliferation of hepatoma cells by depressing NUSAP1 levels in vivo and in vitro.Conclusions: Thus, we conclude that the targeting of NUSAP1 mRNA by the tumor suppressor miR-18b is controlled by HBxmodulated promoter methylation during the host-virus interaction, leading to hepatocarcinogenesis. Our findings provide new insights into the mechanism by which HBx-mediated miRNAs modulate hepatocarcinogenesis. 展开更多
关键词 HEPATITIS B VIRUS HBX miR-18b NUSAP1 HCC
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组织基质金属蛋白酶抑制剂-2的表达纯化及活性鉴定 被引量:5
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作者 薛爱英 冯国兴 +1 位作者 朱长春 樊赛军 《生物工程学报》 CAS CSCD 北大核心 2020年第12期2868-2876,共9页
组织基质金属蛋白酶抑制剂-2(TIMP-2)抑制肿瘤迁移及侵袭。文中以人TIMP-2为研究对象,探索人TIMP-2蛋白的原核表达特征,并进行纯化及活性鉴定。以人肺癌A549细胞的总RNA反转录得到的cDNA为模板,克隆人TIMP-2基因,构建pET28a重组表达载体... 组织基质金属蛋白酶抑制剂-2(TIMP-2)抑制肿瘤迁移及侵袭。文中以人TIMP-2为研究对象,探索人TIMP-2蛋白的原核表达特征,并进行纯化及活性鉴定。以人肺癌A549细胞的总RNA反转录得到的cDNA为模板,克隆人TIMP-2基因,构建pET28a重组表达载体;经酶切检测和测序分析的重组表达载体pET28a-TIMP-2转入大肠杆菌Escherichia coli BL21(DE3)中,利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并对表达条件进行优化。经镍亲和柱纯化后,用Western blotting法鉴定融合蛋白His-TIMP-2,并用明胶酶谱法检测融合蛋白的活性。研究发现融合蛋白His-TIMP-2在E.coli BL21(DE3)中以包涵体的形式存在;在一定范围内,IPTG浓度对His-TIMP-2的表达量没有显著影响;而在该表达系统中,诱导温度和时间是关键参数,His-TIMP-2的表达量随诱导温度升高而增加;纯化并复性后的融合蛋白His-TIMP-2能有效抑制人肺癌A549细胞表达的基质金属蛋白酶的活性。具有活性的融合蛋白的获得为后续深入研究人TIMP-2的功能及机制奠定基础,并对肿瘤治疗具有重要意义。 展开更多
关键词 基质金属蛋白酶抑制剂-2 重组表达载体 包涵体 纯化 基质金属蛋白酶
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