To establish a stable hepatocellular carcinoma Heap1-6 cell line expressing shRNA against mouseLC3and to study apoptosis of Heap1-6 cells treated with tunicamycin.

shRNA targetingLC3gene 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 cells 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 mouseLC3. Western blot analysis was used to detect the expression level of LC3Ⅱ, cleaved-caspase3, and caspase9 protein respectively. Apoptotic cells were measured by flow cytometry.

We successfully constructed pLKO.1-shLC3 lentivirus vector. Before treated by tunicamycin, the level of LC3Ⅱin 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 LC3Ⅱin Heap1-6 shLC3 cells was decreased by 58.6﹪compared with that in the WT Heap1-6 cellsP= 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 tunicamycin for 12 h and 24 h, respectively.

LC3knockdown could promote apoptosis of mouse hepatocellular carcinoma Heap1-6 cell line induced by tunicamycin.