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

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。

Overexpressing miR-29c targeting AKT2 enhances the radiosensitivity of human hepatocellular carcinoma cell line HepG2

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 (t_{survival rate}=4.307, 7.668, 7.668, 6.894, P<0.05; t_{cell viability}=3.443, 8.116, 13.434, P<0.05) but they were increased by miR-29c inhibition (t_{survival rate}=4.003, 6.713, 7.141, P<0.05; t_{cell 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.