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肝癌发病隐匿、发展迅速、恶性程度高, 术后易复发和转移,5年生存率低,且近些年的发病率和死亡率明显上升[1]。肝癌是一个多基因参与、多因素作用和多阶段发病的疾病,其发生发展机制尚未明确[2]。有研究[3-4]发现,抑癌基因、癌基因、信号通路等参与了肿瘤形成,如RTEN基因、p53基因、Wnt信号通路等。因此,研究引起肝癌发生发展的分子机制对于其诊疗尤为重要。SOX7是含高迁移率族蛋白(HMG)结构域的SOX家族成员之一,可与β-catenin竞争结合TCF/LEF,从而抑制Wnt/β-catenin信号通路激活[5]。有研究[6-7]报道,肝癌、肺癌等多种肿瘤中SOX7呈现低表达,其表达与肿瘤进展明显相关。miR-935可通过靶向抑制SOX7促进肝癌细胞增殖和迁移[8];miR-184可靶向抑制SOX7促进肝癌细胞增殖,加速细胞周期进程[9]。关于SOX7对肝癌细胞凋亡影响及机制尚未明确。因此,本研究将pcDNA3.1-SOX7重组质粒转染人肝癌细胞,旨在观察过表达SOX7对肝癌细胞凋亡影响,并进一步研究其对细胞活性氧(ROS)水平及Wnt/β-catenin信号通路影响, 以期为肝癌治疗提供理论基础。
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pcDNA3.1-SOX7重组质粒转染Huh-7细胞48 h,Western blotting检测结果显示,pcDNA3.1-SOX7组SOX7表达明显高于空白组(P < 0.05),Vector组SOX7表达与空白组差异无统计学意义(P>0.05)(见图 1、表 1)。
分组 SOX7蛋白相对表达量 空白组 0.072±0.008 Vector组 0.065±0.007 pcDNA3.1-SOX7组 0.572±0.042* F 405.25 P < 0.01 MS组内 0.001 与空白组比较*P < 0.05 表 1 pcDNA3.1-SOX7重组质粒转染Huh-7细胞后SOX7蛋白表达情况(x±s)
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pcDNA3.1-SOX7重组质粒转染Huh-7细胞24、48和72 h,MTT检测结果显示,与空白组比较,pcDNA3.1-SOX7组在3个时间点的细胞活力均明显降低(P < 0.05)(见表 2)。
分组 24 h 48 h 72 h 空白组 0.488±0.045 0.712±0.059 0.978±0.072 Vector组 0.501±0.042 0.687±0.051 0.954±0.078 pcDNA3.1-SOX7组 0.311±0.028* 0.502±0.043* 0.697±0.056* F 22.17 14.93 15.16 P < 0.01 < 0.01 < 0.01 MS组内 0.002 0.003 0.005 与空白组比较*P < 0.05 表 2 过表达SOX7对Huh-7细胞活力的影响(x±s)
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pcDNA3.1-SOX7重组质粒转染Huh-7细胞48 h,Annexin V-FITC/PI双染法检测细胞凋亡结果显示,与空白组比较,pcDNA3.1-SOX7组细胞凋亡率及ROS含量均明显升高(P < 0.05)(见图 2、表 3)。
分组 凋亡率/% 荧光强度 空白组 4.15±0.46 51.25±5.06 Vector组 4.26±0.51 52.88±5.39 pcDNA3.1-SOX7组 25.54±1.62* 114.9±8.42* F 441.06 94.39 P < 0.01 < 0.01 MS组内 1.032 41.851 与空白组比较*P < 0.05 表 3 过表达SOX7对Huh-7细胞凋亡的影响(x±s)
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Western blotting检测各组细胞β-catenin、cyclin D1和cleaved-caspase 3蛋白表达,与空白组比较,pcDNA3.1-SOX7组β-catenin和cyclin D1明显降低,cleaved-caspase 3表达明显升高(P < 0.05)(见图 3、表 4)。
分组 β-catenin cyclin D1 cleaved-caspase 3 空白组 0.815±0.069 0.271±0.032 0.026±0.004 Vector组 0.832±0.072 0.294±0.035 0.030±0.005 pcDNA3.1-SOX7组 0.356±0.039* 0.150±0.018* 0.104±0.010* F 57.24 20.93 123.15 P < 0.01 < 0.01 < 0.01 MS组内 0.004 0.001 0.001 与空白组比较*P < 0.05 表 4 β-catenin、cyclin D1和cleaved-caspase 3的蛋白相对表达量情况(x±s)
SOX7基因对肝癌细胞凋亡、氧化应激及Wnt/β-catenin信号通路的影响
Effect of SOX7 gene on the apoptosis, oxidative stress and Wnt/β-catenin signaling pathway in liver cancer cells
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摘要:
目的探讨过表达SOX7对肝癌细胞凋亡、活性氧(ROS)水平及Wnt/β-catenin信号通路的影响。 方法人肝癌Huh-7细胞分为空白组、空质粒pcDNA3.1组(Vector组)和pcDNA3.1-SOX7重组质粒组(pcDNA3.1-SOX7组)。转染48 h,Annexin V-FITC/PI双染法细胞凋亡试剂盒、DCFH-DA法及Western blotting分别检测细胞凋亡率、ROS含量及SOX7、β-catenin、cyclin D1和cleaved-caspase 3蛋白表达。MTT法检测pcDNA3.1转染24 h、48 h和72 h的细胞活力。 结果pcDNA3.1-SOX7重组质粒组SOX7蛋白表达明显高于空白组(P < 0.05)。与空白组比较,pcDNA3.1-SOX7组细胞活力及β-catenin和cyclin D1蛋白表达均明显降低,细胞凋亡率、ROS含量及cleaved-caspase 3蛋白表达明显升高(P < 0.05)。 结论过表达SOX7可诱导肝癌细胞凋亡,凋亡机制与细胞内ROS水平提高及Wnt/β-catenin信号通路抑制有关。 -
关键词:
- 肝肿瘤 /
- SOX7基因 /
- 氧化应激 /
- Wnt/β-catenin信号通路 /
- 细胞凋亡
Abstract:ObjectiveTo investigate the effects of overexpression of SOX7 gene on apoptosis, ractive oxygen species (ROS) level and Wnt/β-catenin signaling pathway in hepatocellular carcinoma (HCC) cells. MethodsThe human hepatoma Huh-7 cells were divided into the blank group, blank plasmid pcDNA3.1 group (vector group) and pcDNA3.1-SOX7 recombinant plasmid group (pcDNA3.1-SOX7 group).The apoptosis rate, ROS content, and expression levels of SOX7, β-catenin, cyclin D1 and cleaved-caspase 3 protein were detected by Annexin V-FITC/PI double staining, DCFH-DA assay and Western blotting at 48 h after transfection, respectively.The cell viability was detected using MTT assay after 24 h, 48 h and 72 h of pcDNA3.1 transfecting. ResultsThe expression level of SOX7 protein in pcDNA3.1-SOX7 recombinant plasmid group was significantly higher than that in blank group (P < 0.05).Compared with the blank group, the cell viability and expression levels of β-catenin and cyclin D1 protein in pcDNA3.1-SOX7 group significantly decreased, while the apoptosis rate, ROS content and cleaved-caspase 3 protein expression level significantly increased (P < 0.05). ConclusionsThe overexpression of SOX7 can induce the apoptosis of HCC cells.The mechanism of apoptosis is related to the increasing of ROS level and inhibition of Wnt/β-catenin signaling pathway. -
Key words:
- liver neoplasms /
- SOX7 gene /
- oxidative stress /
- Wnt/β-catenin signaling pathway /
- cells apoptosis
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表 1 pcDNA3.1-SOX7重组质粒转染Huh-7细胞后SOX7蛋白表达情况(x±s)
分组 SOX7蛋白相对表达量 空白组 0.072±0.008 Vector组 0.065±0.007 pcDNA3.1-SOX7组 0.572±0.042* F 405.25 P < 0.01 MS组内 0.001 与空白组比较*P < 0.05 表 2 过表达SOX7对Huh-7细胞活力的影响(x±s)
分组 24 h 48 h 72 h 空白组 0.488±0.045 0.712±0.059 0.978±0.072 Vector组 0.501±0.042 0.687±0.051 0.954±0.078 pcDNA3.1-SOX7组 0.311±0.028* 0.502±0.043* 0.697±0.056* F 22.17 14.93 15.16 P < 0.01 < 0.01 < 0.01 MS组内 0.002 0.003 0.005 与空白组比较*P < 0.05 表 3 过表达SOX7对Huh-7细胞凋亡的影响(x±s)
分组 凋亡率/% 荧光强度 空白组 4.15±0.46 51.25±5.06 Vector组 4.26±0.51 52.88±5.39 pcDNA3.1-SOX7组 25.54±1.62* 114.9±8.42* F 441.06 94.39 P < 0.01 < 0.01 MS组内 1.032 41.851 与空白组比较*P < 0.05 表 4 β-catenin、cyclin D1和cleaved-caspase 3的蛋白相对表达量情况(x±s)
分组 β-catenin cyclin D1 cleaved-caspase 3 空白组 0.815±0.069 0.271±0.032 0.026±0.004 Vector组 0.832±0.072 0.294±0.035 0.030±0.005 pcDNA3.1-SOX7组 0.356±0.039* 0.150±0.018* 0.104±0.010* F 57.24 20.93 123.15 P < 0.01 < 0.01 < 0.01 MS组内 0.004 0.001 0.001 与空白组比较*P < 0.05 -
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