LC3基因沉默对小鼠肝癌细胞Heap1-6凋亡影响的研究

Effect of silencing LC3 on apoptosis of mouse hepatocellular carcinoma Heap1-6 cells

目的建立小鼠LC3基因沉默的Heap1-6稳定表达细胞系,探讨其对衣霉素诱导的肝癌细胞凋亡的影响。方法设计并合成一段针对小鼠LC3基因的shRNA及不针对任何基因的shRNA作为阴性对照,将它们退火后连接构建重组载体,转化扩增及酶切鉴定之后将重组质粒与病毒包装、包膜质粒共同转染293T,收集病毒上清转染Heap1-6细胞,嘌呤霉素筛选10 d,获得稳定的细胞株。以导入LC3基因shRNA的Heap1-6为实验组(Heap1-6 sh LC3),以导入shcoo2的Heap1-6为对照组(Heap1-6 shctrl)。衣霉素处理实验组和对照组的细胞,Western Blot检测LC3Ⅱ、c-Caspase3、Caspase9蛋白的表达,流式细胞术检测细胞凋亡。Western Blot条带灰度值之间两组比较采用独立样本的t检验。结果成功构建了pLKO.1-sh LC3重组慢病毒载体,与野生型的Heap1-6相比,LC3基因沉默之后,LC3Ⅱ蛋白表达水平降低了62.9﹪(P<0.01);野生型的Heap1-6和LC3基因沉默之后的Heap1-6 sh LC3都经Tm处理12 h之后,后者LC3Ⅱ蛋白表达水平降低了58.6﹪(P<0.01)。与对照组Heap1-6 shctrl相比,衣霉素作用12 h后实验组Heap1-6 sh LC3 c-Caspase3增加了37.7﹪(P=0.007),Caspase9增加了37.1﹪(P=0.023));衣霉素作用24 h后sh LC3组c-Caspase3增加了12.6﹪(P=0.04),Caspase9增加了14.3﹪(P=0.043)。药物干预12 h和24 h后,Heap1-6 sh LC3组比对照组Heap1-6 shcoo2凋亡比例分别增加22.8﹪和18.6﹪。结论成功建立小鼠LC3基因沉默的Heap1-6稳定表达细胞系,LC3基因沉默促进衣霉素诱导的小鼠肝癌细胞Heap1-6的凋亡。

Objective To establish a stable hepatocellular carcinoma Heap1-6 cell line expressing shRNA against mouse LC3 and to study apoptosis of Heap1-6 cells treated with tunicamycin. Methods shRNA targeting LC3 gene and negative shRNA were designed and synthesized, pLKO.1-TRC-shRNA LC3 vector and negative vector were constructed. After amplification and identification, the recombinant lentivirus vectors were transfected into 293T cells with packaging and envelope plasmids. The supernatant of 293T ceils transfected with recombinant vector was collected, and Heap1-6 cells were transfected with plasmids, and treated with puromycin for ten days to acquire a cell line with stable expression of shRNA against mouse LC3. Western blot analysis was used to detect the expression level of LC3 Ⅱ, cleaved-caspase3, and caspase9 protein respectively. Apoptotic cells were measured by flow cytometry. Results We successfully constructed pLKO. 1-shLC3 lentivirus vector. Before treated by tunicamycin, the level of LC3 Ⅱ in the Heap1-6 shLC3 cells was decreased by 62.9 % compared with that in the WT Heap1-6 cells （P = 0.0001 ）. After being treated by tunicamycin for 12 h, the level of LC3 Ⅱ in Heap1-6 shLC3 cells was decreased by 58.6 % compared with that in the WT Heapl-6 cells P = 0.0003 ）. Compared with the control group （Heap1-6 cells transfected with negative shRNA vector）, the level of cleaved-caspase3 and caspase9 in the shLC3 group was increased by 37.7 % and 37.1% respectively under tunicamycin for 12 h （P = 0.007, 0.023 ）. And the level of the same two proteins in the shRNA group was elevated by 12.6 % and 14.3 % compared with those of Heap1-6 shctrl cells respectively （P = 0.040, 0.043）. The ratio of apoptotic cells of the experiment group was increased by 22.8 % and 18.6 % compared with that of the control treated with ttmicamycin for 12 h and 24 h, respectively. Conclusion LC3 knockdown could promote apoptosis of mouse hepatocellular carcinoma Heap 1-6 cell line induced by tunicamycin.