Abstract: Objective:To observe the changes of the cells of human hepatocellular carcinoma (HepG2) using RNA for silencing the expression of COMMD7 gene, and investigate related mechanism of COMMD7 gene promoting HepG2 proliferation.
Methods:COMMD7 gene shRNA was designed and constructed into COMMD7shRNA plasmid. HepG2 cells were divided into the HepG2 group, controlshRNA group (empty vectors were infected) and COMMD7shRNA group (positive vectors were infected) . Cells shapes were observed by fluorescence microscope after infecting. The expression of COMMD7 and expression and phosphorylation of extracellular regulated protein kinase1/2 (ERK1/2) and MEK1/2 protein were measured by Western blot. The cell vitality was measured by cholecystokinin octapeptide (CCK-8), and the apoptosis of cell was detected by flow cytometry. The measurement data with normal distribution were presented as ±s. The comparisons among groups were evaluated with the oneway ANOVA, and pairwise comparison was analyzed by the LSDt test.
Results:The cells were oval or spindle shapes and displayed green fluorescent after infected successfully. The results of Western blot showed that the relative quantitative expression of COMMD7 protein in the HepG2 group, controlshRNA group and COMMD7shRNA group were 0.90±0.18, 1.03±0.05 and 0.23±0.03, respectively, with a significant difference among the 3 groups (F=152.08, P<0. 05), and the expression of COMMD7 protein in the COMMD7shRNA group was significantly lower than those in the other 2 groups (t=20.74, 21.16, P<0.05). The results of CCK-8 showed that the scores of the HepG2 vitality in the HepG2 group, controlshRNA group and COMMD7shRNA group were 1.193±0.024, 1.225±0.034 and 1.147±0.021, respectively, with a significant difference among the 3 groups (F=6.90, P<0. 05), and the HepG2 vitality in the COMMD7shRNA group was significantly lower than those in the other 2 groups (t=3.53, 3.69, P<0.05). The results of flow cytometry showed that the apoptosis rate of HepG2 in the HepG2 group, controlshRNA group and COMMD7shRNA group were 6.1%±0.3%, 7.8%±0.5% and 20.9%±1.4%，showing a significant difference among the 3 groups (F=270.80, P<0. 05), and the apoptosis rate of HepG2 in the COMMD7shRNA group was significant higher than those in the other 2 groups (t=21.77, 19.36, P<0.05). The results of Western blot showed that the relative quantitative expression of phosphorylation (p) ERK1/2 and pMEK1/2 in the HepG2 group, controlshRNA group and COMMD7shRNA group were 0.932±0.046, 0.945±0.017, 0.553±0.052 and 0.452±0.031, 0.468±0.027, 0.263±0.022，respectively, showing significant differences among the 3 groups (F=93.61, 49.16, P<0.05)，and the relative quantitative expression of pERK1/2 and pMEK1/2 in the COMMD7shRNA group were significantly lower than those in the other 2 groups (t=11.94, 12.17, 9.33, 8.65, P<0.05).
ConclusionsCOMMD7 gene can promote HepG2 proliferation via activating ERK/mitogenactivated protein kinase (MAPK) signaling pathway, and its mechanism may be promoting the phosphorylation of expression of ERK1/2 and MEK1/2.