黄长山,余伟,王谦,叶柯,谢毅.过表达miR-29c靶向抑制AKT2增强肝癌HepG2细胞放射敏感性的实验研究[J].中华放射医学与防护杂志,2019,39(3):185-191
过表达miR-29c靶向抑制AKT2增强肝癌HepG2细胞放射敏感性的实验研究
Overexpressing miR-29c targeting AKT2 enhances the radiosensitivity of human hepatocellular carcinoma cell line HepG2
投稿时间:2018-08-06  
DOI:10.3760/cma.j.issn.0254-5098.2019.03.005
中文关键词:  肝癌  miR-29c  AKT2  放射敏感性
英文关键词:Liver cancer  miR-29c  AKT2  Radiosensitivity
基金项目:
作者单位
黄长山 河南省肿瘤医院肝胆胰脾外科, 郑州 450008 
余伟 河南省肿瘤医院肝胆胰脾外科, 郑州 450008 
王谦 河南省肿瘤医院肝胆胰脾外科, 郑州 450008 
叶柯 河南省肿瘤医院放疗科, 郑州 450008 
谢毅 河南省人民医院胃肠外科, 郑州 450008 
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中文摘要:
      目的 探讨miR-29c靶向AKT2对肝癌细胞HepG2放射敏感性的影响。方法 RT-PCR检测人正常肝THLE-3细胞和肝癌HepG2细胞中miR-29c表达。给予不同剂量(0、2、4、6和8 Gy)的X射线照射后,RT-PCR检测HepG2细胞中miR-29c表达变化。经生物信息学预测并采用双荧光素酶报告基因实验和Western blot检测miR-29c与AKT2的靶向关系。采用脂质体2000将miR-29c mimic/AKT2基因重组质粒和miR-29c inhibitor/慢病毒载体AKT2 shRNA转染至HepG2细胞中,并给予不同剂量X射线照射后,克隆形成实验和MTT实验检测miR-29/AKT2对HepG2细胞存活率和细胞活力的影响。结果 与THLE-3细胞相比,HepG2细胞中miR-29c明显降低,差异有统计学意义(t=17.816,P<0.05);HepG2经2、4、6和8 Gy X射线照射后,细胞存活率较THLE-3细胞显著降低(t=4.541、6.823、7.218、9.363,P<0.05),HepG2细胞中miR-29c表达显著下降(t=5.599、9.262、10.470、10.873,P<0.05)。miR-29c过表达可降低HepG2细胞存活率和细胞活力(t存活率=4.307、7.668、7.668、6.894,P<0.05;t细胞活力=3.443、8.116、13.434,P<0.05);反之,抑制miR-29c表达则升高HepG2细胞存活率和细胞活力(t=4.003、6.713、7.141,P<0.05;t细胞活力=4.282、5.113,P<0.05)。双荧光素酶报告基因实验表明,AKT2是miR-29c的靶基因,Western blot检测结果显示,miR-29c可负向调控AKT2蛋白表达。沉默AKT2后,HepG2细胞的存活分数及细胞存活率趋势与miR-29c过表达相一致;反之,AKT2过表达则与抑制miR-29c表达相一致。结论 miR-29c可通过靶向AKT2增加肝癌细胞HepG2放射敏感性。
英文摘要:
      Objective To investigate the effect of miR-29c on radiosensitivity of hepatoma HepG2 cells by targeting AKT2 gene. Methods The expression of miR-29c in human normal hepatocytes THLE-3 and hepatoma cell HepG2 was detected by RT-PCR. The relationship between miR-29c and AKT2 were predicted by predicted by informative analysis and verified by dual luciferase reporter gene test and Western blot. miR-29c mimic/AKT2 gene recombinant plasmid and miR-29c inhibitor/lentivirus vector AKT2 shRNA were transfected into HepG2 cells by Liposome 2000. The cells were irradiated with different doses (0, 2, 4, 6 and 8 Gy) of X-rays, and the effects of miR-29/AKT2 on the survival and cell viability of HepG2 cells were detected by cloning and MTT assays. Results Compared with THLE-3 cells, the expression of miR-29c in HepG2 cells was significantly lower (t=17.816, P<0.05). After 2, 4, 6 and 8 Gy X-ray irradiation, the survival of HepG2 cells was significantly lower than that of THLE-3 cells (t=4.541, 6.823, 7.218, 9.363, P<0.05), and the expression of miR-29c in HepG2 cells was significantly decreased (t=5.599, 9.262, 10.470, 10.873, P<0.05). The survival and viability of HepG2 cells were decreased by miR-29c overexpression (tsurvival rate=4.307, 7.668, 7.668, 6.894, P<0.05; tcell viability=3.443, 8.116, 13.434, P<0.05) but they were increased by miR-29c inhibition (tsurvival rate=4.003, 6.713, 7.141, P<0.05; tcell viability=4.282, 5.113, P<0.05). Double luciferase reporter gene experiments showed that AKT2 was the target gene of miR-29c since the expression of AKT2 was negatively regulated by miR-29c. After the silence of AKT2 or overexpression of AKT2, the survival and viability of HepG2 cells were consistent with the overexpression of miR-29c or the inhibition of miR-29c, respectively. Conclusions MiR-29c increases the radiosensitivity of hepatoma cell HepG2 by targeting AKT2.
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