The Apoptosis Induced By Pig11 Protein In Human Hepatocellular Carcinoma Hepg2 Cells And The Preliminary Discussion Of Its Effect Mechanism

Objective: To construct the human PIG11 gene miRNA expressing vector pcDNA?6.2-GW/EmGFPmiR and obtain a HepG2 cell line low expressing PIG11 gene. Explore the effect of PIG11 gene expression on HepG2 cell line apoptosis with HepG2 cell line high expressing PIG11 gene which was constructed before by our research group. ROS and the expressions of Survivin, Caspase3, Caspase9 were researched to get the information of apoptotic mechanism induced by PIG11.Methods: Construct four miRNA expression plasmid and transform them to the Competent bacteria DH5αfor DNA sequencing analysis. Transfecting these vectors into HepG2 cell by lipofectamineTM2000. Then we achieved PIG11 gene low expression HepG2 cell line after BSD screening. The expression of PIG11 cells was detected by RT-PCR and PIG11 protein was analyzed by Western Blot and then choose the best interference group. So the groups for following experiments include HepG2, pLXSN-HepG, pLXSN-PIG11-HepG2, miR-PIG11-HepG2, miR-NC-HepG2. The cell growth was detected by MTT method and Plate Cloning. Using the flow cytometer scans (FACS) to check the cell apoptosis. Intracellular content of reactive oxygen species (ROS) was measured by 2′, 7′-Dichlorofluorescin diacetate (DCFH-DA).The expression of Survivin, Caspase3, Caspase9 was checked by Western Blot .Results: Constructed miRNA vector pcDNA?6.2-GW/EmGFPmiR, which the same as PIG11 cDNA in GenBank report. Obtained stable HepG2 cell lines after transfaction. It showed that PIG11 mRNA and PIG11 protein of the fourth group were both down- regulated(p<0.01).The growth rate of pLXSN-PIG11-HepG2 was lower(p<0.01) and that of miR-PIG11-HepG2 was higher(p<0.01).The apoptosis ratio of HepG2,pLXSN-HepG2,pLXSN-PIG11-HepG2,miR-PIG11-HepG2,miR-NC-HepG2 is 5.72%±0.81,5.34%±0.60,34.83%±2.29,1.34%±0.71,5.10%±0.40. The apoptosis ratio of pLXSN-PIG11-HepG2 was higher while that of miR-PIG11-HepG2 was the lower(p<0.01).It showed that intracellular content of ROS was 5.52±0.97, 4.92±0.71, 15.71±0.82, 2.39±0.22 and 5.12±0.15 in control, pLXSN-HepG2, pLXSN-PIG11-HepG2, miR-PIG11-HepG2 and miR-NC-HepG2. The intracellular content of ROS was higher in pLXSN-PIG11-HepG2(p<0.05), while that of miR-PIG11-HepG2 was lower(p<0.05). Pretreatment with N-acetylcystine (NAC), PIG11- induced apoptosis was blocked markedly. Intracellular ROS level was decreased by CsA. So the ROS maybe generated partly from mitochondria in the process.The expression of Caspase3 and Caspase9 in pLXSN-PIG11-HepG2 cell was higher (p<0.01)with lower of miR-PIG11-HepG2(p<0.01), and the expression of Survivin was the opposite(p<0.01).Conclusion: These findings suggest that the expression of PIG11 induce HepG2 cells apoptosis. ROS, high expression of caspase3 and caspase9 and low expression of survivin maybe play an important role in the regulation of apoptosis.