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.%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8Gy 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.
